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Sample records for catalytic rna molecular

  1. Cations and hydration in catalytic RNA: Molecular dynamics of the hepatitis delta virus ribozyme

    Czech Academy of Sciences Publication Activity Database

    Krasovská, Maryna V.; Šefčíková, J.; Réblová, Kamila; Schneider, Bohdan; Walter, N.G.; Šponer, Jiří

    2006-01-01

    Roč. 91, č. 2 (2006), s. 626-638 ISSN 0006-3495 R&D Projects: GA ČR(CZ) GA203/05/0388; GA ČR(CZ) GA203/05/0009; GA AV ČR(CZ) 1QS500040581; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z40550506 Keywords : molecular dynamics * cations * hydration Subject RIV: BO - Biophysics Impact factor: 4.757, year: 2006

  2. Molecular catalytic coal liquid conversion

    Energy Technology Data Exchange (ETDEWEB)

    Stock, L.M.; Yang, Shiyong [Univ. of Chicago, IL (United States)

    1995-12-31

    This research, which is relevant to the development of new catalytic systems for the improvement of the quality of coal liquids by the addition of dihydrogen, is divided into two tasks. Task 1 centers on the activation of dihydrogen by molecular basic reagents such as hydroxide ion to convert it into a reactive adduct (OH{center_dot}H{sub 2}){sup {minus}} that can reduce organic molecules. Such species should be robust withstanding severe conditions and chemical poisons. Task 2 is focused on an entirely different approach that exploits molecular catalysts, derived from organometallic compounds that are capable of reducing monocyclic aromatic compounds under very mild conditions. Accomplishments and conclusions are discussed.

  3. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, Joseph Albert [Univ. of California, Berkeley, CA (United States)

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 121±s are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  4. Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiao; Yang, Hanjing; Arutiunian, Vagan; Fang, Yao; Besse, Guillaume; Morimoto, Cherie; Zirkle, Brett; Chen, Xiaojiang S. (USC)

    2017-05-30

    The catalytic activity of human cytidine deaminase APOBEC3B (A3B) has been correlated with kataegic mutational patterns within multiple cancer types. The molecular basis of how the N-terminal non-catalytic CD1 regulates the catalytic activity and consequently, biological function of A3B remains relatively unknown. Here, we report the crystal structure of a soluble human A3B-CD1 variant and delineate several structural elements of CD1 involved in molecular assembly, nucleic acid interactions and catalytic regulation of A3B. We show that (i) A3B expressed in human cells exists in hypoactive high-molecular-weight (HMW) complexes, which can be activated without apparent dissociation into low-molecular-weight (LMW) species after RNase A treatment. (ii) Multiple surface hydrophobic residues of CD1 mediate the HMW complex assembly and affect the catalytic activity, including one tryptophan residue W127 that likely acts through regulating nucleic acid binding. (iii) One of the highly positively charged surfaces on CD1 is involved in RNA-dependent attenuation of A3B catalysis. (iv) Surface hydrophobic residues of CD1 are involved in heterogeneous nuclear ribonucleoproteins (hnRNPs) binding to A3B. The structural and biochemical insights described here suggest that unique structural features on CD1 regulate the molecular assembly and catalytic activity of A3B through distinct mechanisms.

  5. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  6. Catalytic metal ions and enzymatic processing of DNA and RNA.

    Science.gov (United States)

    Palermo, Giulia; Cavalli, Andrea; Klein, Michael L; Alfonso-Prieto, Mercedes; Dal Peraro, Matteo; De Vivo, Marco

    2015-02-17

    CONSPECTUS: Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resolution X-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the experimental data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by Steitz and Steitz ( Proc. Natl. Acad. Sci. U.S.A. 1993 , 90 ( 14 ), 6498 - 6502 ). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby second metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymatic transition state. Nevertheless, static X-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymatic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. In this Account, we discuss the role of multiscale molecular simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymatic targets for drug discovery, namely, ribonuclease H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles molecular simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, our analysis of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical molecular dynamics simulations coupled with enhanced sampling techniques have contributed to describe

  7. Molecular Components of Catalytic Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Somorjai, Gabor A.; Park, Jeong Y.

    2008-07-02

    Selectivity, that is, to produce one molecule out of many other thermodynamically feasible product molecules, is the key concept to develop 'clean manufacturing' processes that do not produce byproducts (green chemistry). Small differences in potential energy barriers for elementary reaction steps control which reaction channel is more likely to yield the desired product molecule (selectivity), instead of the overall activation energy for the reaction that controls turnover rates (activity). Recent studies have demonstrated the atomic- or molecular-level tailoring of parameters such as the surface structures of active sites that give rise to nanoparticle size and shape dependence of turnover rates and reaction selectivities. Here, we highlight seven molecular components that influence reaction selectivities. These include: surface structure, adsorbate-induced restructuring, adsorbate mobility, reaction intermediates, surface composition, charge transport, and oxidation states for model metal single crystal and colloid nanoparticle catalysts. We show examples of their functioning and describe in-situ instruments that permit us to investigate their roles in surface reactions.

  8. Slow molecular recognition by RNA.

    Science.gov (United States)

    Gleitsman, Kristin R; Sengupta, Raghuvir N; Herschlag, Daniel

    2017-12-01

    Molecular recognition is central to biological processes, function, and specificity. Proteins associate with ligands with a wide range of association rate constants, with maximal values matching the theoretical limit set by the rate of diffusional collision. As less is known about RNA association, we compiled association rate constants for all RNA/ligand complexes that we could find in the literature. Like proteins, RNAs exhibit a wide range of association rate constants. However, the fastest RNA association rates are considerably slower than those of the fastest protein associations and fall well below the diffusional limit. The apparently general observation of slow association with RNAs has implications for evolution and for modern-day biology. Our compilation highlights a quantitative molecular property that can contribute to biological understanding and underscores our need to develop a deeper physical understanding of molecular recognition events. © 2017 Gleitsman et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. Domain motions of Argonaute, the catalytic engine of RNA interference

    Directory of Open Access Journals (Sweden)

    Wall Michael E

    2007-11-01

    Full Text Available Abstract Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

  10. Programmable formation of catalytic RNA triangles and squares by assembling modular RNA enzymes.

    Science.gov (United States)

    Oi, Hiroki; Fujita, Daisuke; Suzuki, Yuki; Sugiyama, Hiroshi; Endo, Masayuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

    2017-05-01

    RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM). © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  11. Quasispecies-like behavior observed in catalytic RNA populations evolving in a test tube

    Directory of Open Access Journals (Sweden)

    Lehman Niles

    2010-03-01

    Full Text Available Abstract Background During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous. Results Using continuous in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen with a catalytic RNA. Extensive genotypic sampling from two replicate lineages was gathered and phylogenetic networks were constructed to elucidate the structure of the evolving RNA populations. A common distribution was found in which a mutant sequence was present at high frequency, surrounded by a cloud of mutant with lower frequencies. This is a typical distribution of quasispecies. Most of the mutants in these clouds were connected by short Hamming distance values, indicating their close relatedness. Conclusions The quasispecies nature of mutant RNA clouds facilitates the recovery of genotypes under pressure of being removed from the population by random drift. The empirical populations therefore evolved a genotypic resiliency despite a high mutation rate by adopting the characteristics of quasispecies, implying that primordial RNA pools could have used this strategy to avoid extinction.

  12. Quasispecies-like behavior observed in catalytic RNA populations evolving in a test tube.

    Science.gov (United States)

    Díaz Arenas, Carolina; Lehman, Niles

    2010-03-23

    During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous. Using continuous in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen with a catalytic RNA. Extensive genotypic sampling from two replicate lineages was gathered and phylogenetic networks were constructed to elucidate the structure of the evolving RNA populations. A common distribution was found in which a mutant sequence was present at high frequency, surrounded by a cloud of mutant with lower frequencies. This is a typical distribution of quasispecies. Most of the mutants in these clouds were connected by short Hamming distance values, indicating their close relatedness. The quasispecies nature of mutant RNA clouds facilitates the recovery of genotypes under pressure of being removed from the population by random drift. The empirical populations therefore evolved a genotypic resiliency despite a high mutation rate by adopting the characteristics of quasispecies, implying that primordial RNA pools could have used this strategy to avoid extinction.

  13. Exploring RNA structure by integrative molecular modelling

    DEFF Research Database (Denmark)

    Masquida, Benoît; Beckert, Bertrand; Jossinet, Fabrice

    2010-01-01

    on interactive all-atom molecular modelling relies on the observation that specific structural motifs are recurrently found in RNA sequences. Once identified by a combination of comparative sequence analysis and biochemical data, the motifs composing the secondary structure of a given RNA can be extruded...

  14. Molecular dynamics simulations of RNA motifs

    Czech Academy of Sciences Publication Activity Database

    Csaszar, K.; Špačková, Naďa; Šponer, Jiří; Leontis, N. B.

    2002-01-01

    Roč. 223, - (2002), s. 154 ISSN 0065-7727. [Annual Meeting of the American Chemistry Society /223./. 07.04.2002-11.04.2002, Orlando ] Institutional research plan: CEZ:AV0Z5004920 Keywords : molecular dynamics * RNA * hydration Subject RIV: BO - Biophysics

  15. DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

    Science.gov (United States)

    Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

    2018-01-01

    Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Extraction of low molecular weight RNA from Citrus trifolita tissues ...

    African Journals Online (AJOL)

    Jane

    2010-12-20

    Dec 20, 2010 ... reaction (RT-PCR) and Northern blotting. Key words: Citrus, low molecular weight RNA, trizol reagent, 4 M LiCl, microRNAs. INTRODUCTION. Low molecular weight RNA (LMW RNA) consists of several components with microRNAs (miRNAs) being among the important ones. miRNAs are endogenous 19-.

  17. Molecular Self-Assembly Strategy for Generating Catalytic Hybrid Polypeptides.

    Science.gov (United States)

    Maeda, Yoshiaki; Fang, Justin; Ikezoe, Yasuhiro; Pike, Douglas H; Nanda, Vikas; Matsui, Hiroshi

    2016-01-01

    Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.

  18. Catalytic

    Directory of Open Access Journals (Sweden)

    S.A. Hanafi

    2014-03-01

    Full Text Available A series of dealuminated Y-zeolites impregnated by 0.5 wt% Pt catalysts promoted by different amounts of Ni, Pd or Cr (0.3 and 0.6 wt% were prepared and characterized as hydrocracking catalysts. The physicochemical and structural characterization of the solid catalysts were investigated and reported through N2 physisorption, XRD, TGA-DSC, FT-IR and TEM techniques. Solid catalysts surface acidities were investigated through FT-IR spectroscopy aided by pyridine adsorption. The solid catalytic activities were evaluated through hydroconversion of n-hexane and n-heptane employing micro-catalytic pulse technique directly connected to a gas chromatograph analyzer. The thermal stability of the solids was also investigated up to 800 °C. Crystallinity studies using the XRD technique of all modified samples proved analogous to the parent Y-zeolite, exhibiting nearly an amorphous and microcrystalline character of the second metal oxides. Disclosure of bimetallic catalysts crystalline characterization, through XRD, was not viable. The nitrogen adsorption–desorption isotherms for all samples concluded type I adsorption isotherms, without any hysteresis loop, indicating that the entire pore system is composed of micropores. TEM micrographs of the solid catalysts demonstrate well-dispersed Pt, Ni and Cr nanoparticles having sizes of 2–4 nm and 7–8 nm, respectively. The catalytic activity results indicate that the bimetallic (0.5Pt–0.3Cr/D18H–Y catalyst is the most active towards n-hexane and n-heptane isomerization while (0.5Pt–0.6Ni/D18H–Y catalyst can be designed as most suitable as a cracking catalyst.

  19. Mechanism of microRNA-target interaction: molecular dynamics simulations and thermodynamics analysis.

    Science.gov (United States)

    Wang, Yonghua; Li, Yan; Ma, Zhi; Yang, Wei; Ai, Chunzhi

    2010-07-29

    MicroRNAs (miRNAs) are endogenously produced approximately 21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5'-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg(2+)) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago.

  20. tRNA--the golden standard in molecular biology.

    Science.gov (United States)

    Barciszewska, Mirosława Z; Perrigue, Patrick M; Barciszewski, Jan

    2016-01-01

    Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology.

  1. Catalytic Reforming of Lignin-Derived Bio-Oil Over a Nanoporous Molecular Sieve Silicoaluminophosphate-11.

    Science.gov (United States)

    Park, Y K; Kang, Hyeon Koo; Jang, Hansaem; Suh, Dong Jin; Park, Sung Hoon

    2016-05-01

    Catalytic pyrolysis of lignin, a major constituent of biomass, was performed. A nanoporous molecular sieve silicoaluminophosphate-11 (SAPO-11) was selected as catalyst. Thermogravimetric analysis showed that 500 degrees C was the optimal pyrolysis temperature. Pyrolyzer-gas chromatography/mass spectroscopy was used to investigate the pyrolysis product distribution. Production of phenolics, the dominant product from the pyrolysis of lignin, was promoted by the increase in the catalyst dose. In particular, low-molecular-mass phenolics were produced more over SAPO-11, while high-molecular-mass phenolics and double-bond-containing phenolics were produced less. The fraction of aromatic compounds, including benzene, toluene, xylene, and ethylbenzene, was also increased by catalytic reforming. The catalytic effects were more pronounced when the catalyst/biomass ratio was increased. The enhanced production of aromatic compounds by an acidic catalyst obtained in this study is in good agreement with the results of previous studies.

  2. Heterogeneous Molecular Catalysis of Electrochemical Reactions: Volcano Plots and Catalytic Tafel Plots.

    Science.gov (United States)

    Costentin, Cyrille; Savéant, Jean-Michel

    2017-06-14

    We analyze here, in the framework of heterogeneous molecular catalysis, the reasons for the occurrence or nonoccurrence of volcanoes upon plotting the kinetics of the catalytic reaction versus the stabilization free energy of the primary intermediate of the catalytic process. As in the case of homogeneous molecular catalysis or catalysis by surface-active metallic sites, a strong motivation of such studies relates to modern energy challenges, particularly those involving small molecules, such as water, hydrogen, oxygen, proton, and carbon dioxide. This motivation is particularly pertinent for what concerns heterogeneous molecular catalysis, since it is commonly preferred to homogeneous molecular catalysis by the same molecules if only for chemical separation purposes and electrolytic cell architecture. As with the two other catalysis modes, the main drawback of the volcano plot approach is the basic assumption that the kinetic responses depend on a single descriptor, viz., the stabilization free energy of the primary intermediate. More comprehensive approaches, investigating the responses to the maximal number of experimental factors, and conveniently expressed as catalytic Tafel plots, should clearly be preferred. This is more so in the case of heterogeneous molecular catalysis in that additional transport factors in the supporting film may additionally affect the current-potential responses. This is attested by the noteworthy presence of maxima in catalytic Tafel plots as well as their dependence upon the cyclic voltammetric scan rate.

  3. A non-catalytic role for inositol 1,3,4,5,6-pentakisphosphate 2-kinase in the synthesis of ribosomal RNA.

    Science.gov (United States)

    Brehm, Maria A; Wundenberg, Torsten; Williams, Jason; Mayr, Georg W; Shears, Stephen B

    2013-01-15

    Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.

  4. Molecular catalytic hydrogenation of aromatic hydrocarbons and hydrotreating of coal liquids.

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shiyong; Stock, L.M.

    1996-05-01

    This report presents the results of research on the development of new catalytic pathways for the hydrogenation of multiring aromatic hydrocarbons and the hydrotreating of coal liquids at The University of Chicago under DOE Contract No. DE-AC22-91PC91056. The work, which is described in three parts, is primarily concerned with the research on the development of new catalytic systems for the hydrogenation of aromatic hydrocarbons and for the improvement of the quality of coal liquids by the addition of dihydrogen. Part A discusses the activation of dihydrogen by very basic molecular reagents to form adducts that can facilitate the reduction of multiring aromatic hydrocarbons. Part B examines the hydrotreating of coal liquids catalyzed by the same base-activated dihydrogen complexes. Part C concerns studies of molecular organometallic catalysts for the hydrogenation of monocyclic aromatic hydrocarbons under mild conditions.

  5. Are viroids molecular fossils of the RNA world?

    International Nuclear Information System (INIS)

    Chela Flores, J.

    1993-07-01

    We discuss a difficulty that may be raised against the Diener hypothesis that viroids may be interpreted as molecular fossils of the RNA world. We provide a possible way of removing such a difficulty (hence supporting the Diener hypothesis). Our reasoning entails further work by plant pathologists on a proposed search for a well defined molecular process. It is shown that such process would be of biological and evolutionary significance. (author). 23 refs

  6. Unraveling Mg2+-RNA binding with atomistic molecular dynamics.

    Science.gov (United States)

    Cunha, Richard A; Bussi, Giovanni

    2017-05-01

    Interaction with divalent cations is of paramount importance for RNA structural stability and function. We report here a detailed molecular dynamics study of all the possible binding sites for Mg 2+ on an RNA duplex, including both direct (inner sphere) and indirect (outer sphere) binding. In order to tackle sampling issues, we develop a modified version of bias-exchange metadynamics, which allows us to simultaneously compute affinities with previously unreported statistical accuracy. Results correctly reproduce trends observed in crystallographic databases. Based on this, we simulate a carefully chosen set of models that allows us to quantify the effects of competition with monovalent cations, RNA flexibility, and RNA hybridization. Our simulations reproduce the decrease and increase of Mg 2+ affinity due to ion competition and hybridization, respectively, and predict that RNA flexibility has a site-dependent effect. This suggests a nontrivial interplay between RNA conformational entropy and divalent cation binding. © 2017 Cunha and Bussi; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. Synthesis and catalytic performance of ZSM-5/MCM-41 composite molecular sieve from palygorskite

    Science.gov (United States)

    Jiang, Jinlong; Wu, Mei; Yang, Yong; Duanmu, Chuansong; Chen, Jing; Gu, Xu

    2017-10-01

    ZSM-5/MCM-41 composite molecular sieve has been hydrothermally synthesized through a two-step crystallization process using palygorskite (PAL) as silicon and aluminum source. The products were characterized by various means and their catalytic properties for acetalization of cyclohexanone and esterification of acetic acid and n-butanol were also investigated. In the first step ZSM-5 zeolite could be formed from the acid-treated PAL after hydrothermal treatment using tetrapropylammonium bromide as template. XRD patterns, N2 adsorption and desorption data, and TEM images show that the composite obtained in the secondary step had a well-ordered mesoporous MCM-41 phase and a microporous ZSM-5 zeolite phase. Compared with ZSM-5, ZSM-5/MCM-41 composite possessed more total acid amount, weak acid sites and large pore structure due to the formation of MCM-41 and exhibited higher catalytic activity for the acetalization and esterification reaction.

  8. Prebiotic RNA Network Formation: A Taxonomy of Molecular Cooperation.

    Science.gov (United States)

    Mathis, Cole; Ramprasad, Sanjay N; Walker, Sara Imari; Lehman, Niles

    2017-10-16

    Cooperation is essential for evolution of biological complexity. Recent work has shown game theoretic arguments, commonly used to model biological cooperation, can also illuminate the dynamics of chemical systems. Here we investigate the types of cooperation possible in a real RNA system based on the Azoarcus ribozyme, by constructing a taxonomy of possible cooperative groups. We construct a computational model of this system to investigate the features of the real system promoting cooperation. We find triplet interactions among genotypes are intrinsically biased towards cooperation due to the particular distribution of catalytic rate constants measured empirically in the real system. For other distributions cooperation is less favored. We discuss implications for understanding cooperation as a driver of complexification in the origin of life.

  9. Fast, clash-free RNA conformational morphing using molecular junctions.

    Science.gov (United States)

    Héliou, Amélie; Budday, Dominik; Fonseca, Rasmus; van den Bedem, Henry

    2017-07-15

    Non-coding ribonucleic acids (ncRNA) are functional RNA molecules that are not translated into protein. They are extremely dynamic, adopting diverse conformational substates, which enables them to modulate their interaction with a large number of other molecules. The flexibility of ncRNA provides a challenge for probing their complex 3D conformational landscape, both experimentally and computationally. Despite their conformational diversity, ncRNAs mostly preserve their secondary structure throughout the dynamic ensemble. Here we present a kinematics-based procedure to morph an RNA molecule between conformational substates, while avoiding inter-atomic clashes. We represent an RNA as a kinematic linkage, with fixed groups of atoms as rigid bodies and rotatable bonds as degrees of freedom. Our procedure maintains RNA secondary structure by treating hydrogen bonds between base pairs as constraints. The constraints define a lower-dimensional, secondary-structure constraint manifold in conformation space, where motions are largely governed by molecular junctions of unpaired nucleotides. On a large benchmark set, we show that our morphing procedure compares favorably to peer algorithms, and can approach goal conformations to within a low all-atom RMSD by directing fewer than 1% of its atoms. Our results suggest that molecular junctions can modulate 3D structural rearrangements, while secondary structure elements guide large parts of the molecule along the transition to the correct final conformation. The source code, binaries and data are available at https://simtk.org/home/kgs . amelie.heliou@polytechnique.edu or vdbedem@stanford.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  10. Ribosomal RNA Kink-turn motif - A flexible molecular hinge

    Czech Academy of Sciences Publication Activity Database

    Rázga, F.; Špačková, Naďa; Réblová, K.; Koča, J.; Leontis, N. B.; Šponer, Jiří

    2004-01-01

    Roč. 22, č. 2 (2004), s. 183-193 ISSN 0739-1102 R&D Projects: GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF; National Institutes of Health(US) 2R15 GM55898 Institutional research plan: CEZ:AV0Z5004920 Keywords : ribosomal RNA * K-turn * molecular dynamics Subject RIV: BO - Biophysics Impact factor: 1.113, year: 2004

  11. Small-Pore Molecular Sieves SAPO-34 with Chabazite Structure: Theoretical Study of Silicon Incorporation and Interrelated Catalytic Activity

    Science.gov (United States)

    Wang, Hong; Lewis, James; Liu, Zhongmin

    2011-03-01

    The catalytic conversion of methonal to olefin (MTO) has attracted attention both in industrial and academic fields. Strong evidence shows that small-pore molecular sieves with certain amount silicon incorporated (SAPO) present promising high catalytic activity in MTO conversion. Using DFT, we study the structural and electronic properties of chabazite SAPO-34. Although there are extensively experimental results show that silicon incorporation does not change the overall structure as the original AlPO structure, local structural changes are still created by silicon substitution, which probably accounted for the high catalytic activity. It is noted that the catalytic activity of SAPO-34 presents increasing trend along with the silicon incorporation amount increasing and maintain a flat peak even with more silicon incorporated. Hence, there is an optimal silicon incorporation amount which possibly yields the highest catalytic MTO conversion.

  12. Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide.

    Science.gov (United States)

    Liu, Yuchen; Dos Santos, Patricia C; Zhu, Xiang; Orlando, Ron; Dean, Dennis R; Söll, Dieter; Yuan, Jing

    2012-02-17

    Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

  13. Molecular determinants of nucleolar translocation of RNA helicase A

    International Nuclear Information System (INIS)

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-01-01

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

  14. Catalytic mechanism and inhibition of tRNA (Uracil-5-)methyltransferase: evidence for covalent catalysis

    International Nuclear Information System (INIS)

    Santi, D.V.; Hardy, L.W.

    1987-01-01

    tRNA (Ura-5-) methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5- 3 H] Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5- 3 H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analog having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggest that a mechanism for the RNA effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes

  15. Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study

    DEFF Research Database (Denmark)

    da Graça Thrige, D; Buur, J R; Jørgensen, Flemming Steen

    1997-01-01

    phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state...... cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between...... the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic...

  16. Molecular dynamics characterization of five pathogenic factor X mutants associated with decreased catalytic activity

    KAUST Repository

    Abdel-Azeim, Safwat

    2014-11-11

    Factor X (FX) is one of the major players in the blood coagulation cascade. Upon activation to FXa, it converts prothrombin to thrombin, which in turn converts fibrinogen into fibrin (blood clots). FXa deficiency causes hemostasis defects, such as intracranial bleeding, hemathrosis, and gastrointestinal blood loss. Herein, we have analyzed a pool of pathogenic mutations, located in the FXa catalytic domain and directly associated with defects in enzyme catalytic activity. Using chymotrypsinogen numbering, they correspond to D102N, T135M, V160A, G184S, and G197D. Molecular dynamics simulations were performed for 1.68 μs on the wild-type and mutated forms of FXa. Overall, our analysis shows that four of the five mutants considered, D102N, T135M, V160A, and G184S, have rigidities higher than those of the wild type, in terms of both overall protein motion and, specifically, subpocket S4 flexibility, while S1 is rather insensitive to the mutation. This acquired rigidity can clearly impact the substrate recognition of the mutants.

  17. Controllable molecular motors engineered from myosin and RNA

    Science.gov (United States)

    Omabegho, Tosan; Gurel, Pinar S.; Cheng, Clarence Y.; Kim, Laura Y.; Ruijgrok, Paul V.; Das, Rhiju; Alushin, Gregory M.; Bryant, Zev

    2018-01-01

    Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3-5 and modified natural protein motors6-10 have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors11-15. Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s-1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

  18. Quantum mechanics/molecular mechanics study of the catalytic cycle of water splitting in photosystem II.

    Science.gov (United States)

    Sproviero, Eduardo M; Gascón, José A; McEvoy, James P; Brudvig, Gary W; Batista, Victor S

    2008-03-19

    This paper investigates the mechanism of water splitting in photosystem II (PSII) as described by chemically sensible models of the oxygen-evolving complex (OEC) in the S0-S4 states. The reaction is the paradigm for engineering direct solar fuel production systems since it is driven by solar light and the catalyst involves inexpensive and abundant metals (calcium and manganese). Molecular models of the OEC Mn3CaO4Mn catalytic cluster are constructed by explicitly considering the perturbational influence of the surrounding protein environment according to state-of-the-art quantum mechanics/molecular mechanics (QM/MM) hybrid methods, in conjunction with the X-ray diffraction (XRD) structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The resulting models are validated through direct comparisons with high-resolution extended X-ray absorption fine structure spectroscopic data. Structures of the S3, S4, and S0 states include an additional mu-oxo bridge between Mn(3) and Mn(4), not present in XRD structures, found to be essential for the deprotonation of substrate water molecules. The structures of reaction intermediates suggest a detailed mechanism of dioxygen evolution based on changes in oxidization and protonation states and structural rearrangements of the oxomanganese cluster and surrounding water molecules. The catalytic reaction is consistent with substrate water molecules coordinated as terminal ligands to Mn(4) and calcium and requires the formation of an oxyl radical by deprotonation of the substrate water molecule ligated to Mn(4) and the accumulation of four oxidizing equivalents. The oxyl radical is susceptible to nucleophilic attack by a substrate water molecule initially coordinated to calcium and activated by two basic species, including CP43-R357 and the mu-oxo bridge between Mn(3) and Mn(4). The reaction is concerted with water ligand exchange, swapping the activated water by a water molecule in the second coordination shell of

  19. Enzyme catalytic amplification of miRNA-155 detection with graphene quantum dot-based electrochemical biosensor.

    Science.gov (United States)

    Hu, Tianxing; Zhang, Le; Wen, Wei; Zhang, Xiuhua; Wang, Shengfu

    2016-03-15

    A specific and sensitive method was developed for quantitative detection of miRNA by integrating horseradish peroxidase (HRP)-assisted catalytic reaction with a simple electrochemical RNA biosensor. The electrochemical biosensor was constructed by a double-stranded DNA structure. The structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture DNA), assembled on the gold electrode surface, with target DNA and aminated indicator probe (NH2-DNA). After the construction of the double-stranded DNA structure, the activated carboxyl groups of graphene quantum dots (GQDs) assembled on NH2-DNA. GQDs were used as a new platform for HRP immobilization through noncovalent assembly. HRP modified biosensor can effectively catalyze the hydrogen peroxide (H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), accompanied by a change from colorless to blue in solution color and an increased electrochemical current signal. Due to GQDs and enzyme catalysis, the proposed biosensor could sensitively detect miRNA-155 from 1 fM to 100 pM with a detection limit of 0.14 fM. High performance of the biosensor is attributed to the large surface-to-volume ratio, excellent compatibility of GQDs. For these advantages, the proposed method holds great potential for analysis of other interesting tumor makers. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    International Nuclear Information System (INIS)

    Kvissel, Anne-Katrine; Orstavik, Sigurd; Eikvar, Sissel; Brede, Gaute; Jahnsen, Tore; Collas, Philippe; Akusjaervi, Goeran; Skalhegg, Bjorn Steen

    2007-01-01

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism

  1. Redox status affects the catalytic activity of glutamyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Katz, Assaf; Banerjee, Rajat; de Armas, Merly

    2010-01-01

    Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles...... in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that...... inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme....

  2. Molecular and catalytic properties of angiotensin converting enzyme-I from bovine seminal plasma.

    Science.gov (United States)

    Sharma, M; Singh, U S

    1988-07-01

    Angiotensin converting enzyme [EC 3.4.15.1] was shown to exist in two distinct forms in bovine seminal plasma. The higher molecular weight form of the enzyme (angiotensin convering enzyme I) was purified to homogeneity by Sephadex G-200 gel filtration, and DEAE-Sepharose, blue Sepharose, and concanavalin A-Sepharose column chromatography. Final recovery of the enzyme was 9.0. The molecular weight of the enzyme was estimated to be 8 x 10(5) by the gel filtration method. A value of 4.6 x 10(5) was obtained for the reduced and denatured enzyme by dodecylsulfate polyacrylamide gel electrophoresis. The Stokes' radius, diffusion coefficient, and intrinsic viscosity of the purified enzyme were determined to be 95 A, 2.3 x 10(-7) cm2/s, and 6.76 ml/g. The enzyme had a specific activity of 105.12 mumol/min/mg protein for hippurylhistidylleucine. The Km value for hippurylhistidylleucine was found to be 20 mM. Studies with EDTA suggest that metal ions which are tightly bound are required for its activity. The enzyme was inhibited by some heavy metal ions but did not required sulfhydryl groups for its activity. Trypsin treatment of the urea-denatured enzyme produced a catalytically active fragment with an Mr of 30,000. Chemical hydrolysis of the native enzyme did not produce any active fragment.

  3. DNA nanotechnology for nucleic acid analysis: multifunctional molecular DNA machine for RNA detection.

    Science.gov (United States)

    Cox, A J; Bengtson, H N; Rohde, K H; Kolpashchikov, D M

    2016-12-06

    The Nobel prize in chemistry in 2016 was awarded for 'the design and synthesis of molecular machines'. Here we designed and assembled a molecular machine for the detection of specific RNA molecules. An association of several DNA strands, named multifunctional DNA machine for RNA analysis (MDMR1), was designed to (i) unwind RNA with the help of RNA-binding arms, (ii) selectively recognize a targeted RNA fragment, (iii) attract a signal-producing substrate and (iv) amplify the fluorescent signal by catalysis. MDMR1 enabled detection of 16S rRNA at concentrations ∼24 times lower than that by a traditional deoxyribozyme probe.

  4. Molecular beacon biosensors for DNA/RNA analysis

    Science.gov (United States)

    Fang, Xiaohong; Schuster, Sheldon; Liu, Xiaojing; Correll, Tiffany; Zhang, Peng; Tapec, Ruby; Santra, Swadeshmukul; Qhobosheanne, Monde; Lou, Jane H.; Tan, Weihong

    2000-03-01

    We have developed a variety of novel DNA biosensors using a new class of oligonucleotide probe, molecular beacon (MB). MB has the fluorescence signal transduction mechanism built within the molecules. It can report the presence of specific nucleic acids with high sensitivity and excellent selectivity. Biotinylated MBs have been designed and synthesized for immobilization onto silica surface through avidin-biotin binding. The effect of the avidin-biotin bridge on the MB hybridization has been studied. Our result shows that using streptavidin has less effect than using avidin in MB hybridization. Two kinds of fiber optical DNA sensors have been prepared and characterized: a fiber optic evanescent wave sensor and a submicrometer optical fiber sensor. The sensors are rapid,stable, highly selective, reproducible and regenerable. They have been applied to detect specific DNA and mRNA sequences and to the study of the DNA hybridization kinetics. Silica nanoparticles have also been used for MB immobilization in order to prepare a large quantity of nanometer sized DNA/RNA biosensors.

  5. Topological and thermodynamic factors that influence the evolution of small networks of catalytic RNA species.

    Science.gov (United States)

    Yeates, Jessica A M; Nghe, Philippe; Lehman, Niles

    2017-07-01

    An RNA-directed recombination reaction can result in a network of interacting RNA species. It is now becoming increasingly apparent that such networks could have been an important feature of the RNA world during the nascent evolution of life on the Earth. However, the means by which such small RNA networks assimilate other available genotypes in the environment to grow and evolve into the more complex networks that are thought to have existed in the prebiotic milieu are not known. Here, we used the ability of fragments of the Azoarcus group I intron ribozyme to covalently self-assemble via genotype-selfish and genotype-cooperative interactions into full-length ribozymes to investigate the dynamics of small (three- and four-membered) networks. We focused on the influence of a three-membered core network on the incorporation of additional nodes, and on the degree and direction of connectivity as single new nodes are added to this core. We confirmed experimentally the predictions that additional links to a core should enhance overall network growth rates, but that the directionality of the link (a "giver" or a "receiver") impacts the growth of the core itself. Additionally, we used a simple mathematical model based on the first-order effects of lower-level interactions to predict the growth of more complex networks, and find that such a model can, to a first approximation, predict the ordinal rankings of nodes once a steady-state distribution has been reached. © 2017 Yeates et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  6. Aplicação catalítica de peneiras moleculares básicas micro e mesoporosas Catalytic applications of basic micro and mesoporous molecular sieves

    Directory of Open Access Journals (Sweden)

    Leandro Martins

    2006-04-01

    Full Text Available Catalysis by solid acids has received much attention due to its importance in petroleum refining and petrochemical processes. Relatively few studies have focused on catalysis by bases and even les on using basic molecular sieves. This paper deals with the potential application of micro and mesoporous molecular sieves in base catalysis reactions. The paper is divided in two parts, the first one dedicated to the design of the catalysts and the second to some relevant examples of catalytic reactions, which find a huge field of applications essentially in the synthesis of fine chemicals. Here, recent developments in catalysis by basic molecular sieves and the perspectives of applications in correlated catalytic processes are described.

  7. The de novo cytosine methyltransferase DRM2 requires intact UBA domains and a catalytically mutated paralog DRM3 during RNA-directed DNA methylation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ian R Henderson

    2010-10-01

    Full Text Available Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS rearranged methyltransferase2 (DRM2 is required for establishment of small interfering RNA (siRNA directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA-directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term domains rearranged methyltransferase3 (DRM3. Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA-directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA-directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA-directed DNA methylation systems consist of a combination of ancestral and convergent features.

  8. Extraction of low molecular weight RNA from Citrus trifolita tissues ...

    African Journals Online (AJOL)

    We employed a simple and quick method involving trizol for total RNA extraction from citrus tissues, then generation of LMW RNA using 4M LiCl, which have been successfully utilized in studies in our laboratory. Compared with traditional methods, this method is less expensive and produced high RNA yields while avoiding ...

  9. [Synergetic effects of silicon carbide and molecular sieve loaded catalyst on microwave assisted catalytic oxidation of toluene].

    Science.gov (United States)

    Wang, Xiao-Hui; Bo, Long-Li; Liu, Hai-Nan; Zhang, Hao; Sun, Jian-Yu; Yang, Li; Cai, Li-Dong

    2013-06-01

    Molecular sieve loaded catalyst was prepared by impregnation method, microwave-absorbing material silicon carbide and the catalyst were investigated for catalytic oxidation of toluene by microwave irradiation. Research work examined effects of silicon carbide and molecular sieve loading Cu-V catalyst's mixture ratio as well as mixed approach changes on degradation of toluene, and characteristics of catalyst were measured through scanning electron microscope, specific surface area test and X-ray diffraction analysis. The result showed that the fixed bed reactor had advantages of both thermal storage property and low-temperature catalytic oxidation when 20% silicon carbide was filled at the bottom of the reactor, and this could effectively improve the utilization of microwave energy as well as catalytic oxidation efficiency of toluene. Under microwave power of 75 W and 47 W, complete-combustion temperatures of molecular sieve loaded Cu-V catalyst and Cu-V-Ce catalyst to toluene were 325 degrees C and 160 degrees C, respectively. Characteristics of the catalysts showed that mixture of rare-earth element Ce increased the dispersion of active components in the surface of catalyst, micropore structure of catalyst effectively guaranteed high adsorption capacity for toluene, while amorphous phase of Cu and V oxides increased the activity of catalyst greatly.

  10. Exponential growth for self-reproduction in a catalytic reaction network: relevance of a minority molecular species and crowdedness

    Science.gov (United States)

    Kamimura, Atsushi; Kaneko, Kunihiko

    2018-03-01

    Explanation of exponential growth in self-reproduction is an important step toward elucidation of the origins of life because optimization of the growth potential across rounds of selection is necessary for Darwinian evolution. To produce another copy with approximately the same composition, the exponential growth rates for all components have to be equal. How such balanced growth is achieved, however, is not a trivial question, because this kind of growth requires orchestrated replication of the components in stochastic and nonlinear catalytic reactions. By considering a mutually catalyzing reaction in two- and three-dimensional lattices, as represented by a cellular automaton model, we show that self-reproduction with exponential growth is possible only when the replication and degradation of one molecular species is much slower than those of the others, i.e., when there is a minority molecule. Here, the synergetic effect of molecular discreteness and crowding is necessary to produce the exponential growth. Otherwise, the growth curves show superexponential growth because of nonlinearity of the catalytic reactions or subexponential growth due to replication inhibition by overcrowding of molecules. Our study emphasizes that the minority molecular species in a catalytic reaction network is necessary for exponential growth at the primitive stage of life.

  11. Atomistic mechanism of microRNA translation upregulation via molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Wei Ye

    Full Text Available MicroRNAs are endogenous 23-25 nt RNAs that play important gene-regulatory roles in animals and plants. Recently, miR369-3 was found to upregulate translation of TNFα mRNA in quiescent (G0 mammalian cell lines. Knock down and immunofluorescence experiments suggest that microRNA-protein complexes (with FXR1 and AGO2 are necessary for the translation upregulation. However the molecular mechanism of microRNA translation activation is poorly understood. In this study we constructed the microRNA-mRNA-AGO2-FXR1 quadruple complex by bioinformatics and molecular modeling, followed with all atom molecular dynamics simulations in explicit solvent to investigate the interaction mechanisms for the complex. A combined analysis of experimental and computational data suggests that AGO2-FXR1 complex relocalize microRNA:mRNA duplex to polysomes in G0. The two strands of dsRNA are then separated upon binding of AGO2 and FXR1. Finally, polysomes may improve the translation efficiency of mRNA. The mutation research confirms the stability of microRNA-mRNA-FXR1 and illustrates importance of key residue of Ile304. This possible mechanism can shed more light on the microRNA-dependent upregulation of translation.

  12. Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA.

    Science.gov (United States)

    Mickiewicz, Agnieszka; Sarzyńska, Joanna; Miłostan, Maciej; Kurzyńska-Kokorniak, Anna; Rybarczyk, Agnieszka; Łukasiak, Piotr; Kuliński, Tadeusz; Figlerowicz, Marek; Błażewicz, Jacek

    2017-02-01

    Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

    Science.gov (United States)

    Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

    2008-08-22

    CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

  14. Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator.

    Science.gov (United States)

    Sasano, Yu; Kariya, Takahiro; Usugi, Shogo; Sugiyama, Minetaka; Harashima, Satoshi

    2017-12-01

    As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400-700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator.

  15. Structure and Molecular Dynamics Simulations of Protein Tyrosine Phosphatase Non-Receptor 12 Provide Insights into the Catalytic Mechanism of the Enzyme

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2017-12-01

    Full Text Available Protein tyrosine phosphatase non-receptor 12 (PTPN12 is an important protein tyrosine phosphatase involved in regulating cell adhesion and migration as well as tumorigenesis. Here, we solved a crystal structure of the native PTPN12 catalytic domain with the catalytic cysteine (residue 231 in dual conformation (phosphorylated and unphosphorylated. Combined with molecular dynamics simulation data, we concluded that those two conformations represent different states of the protein which are realized during the dephosphorylation reaction. Together with docking and mutagenesis data, our results provide a molecular basis for understanding the catalytic mechanism of PTPN12 and its role in tumorigenesis.

  16. Molecular Diversity through RNA Editing: A Balancing Act

    Science.gov (United States)

    Farajollahi, Sanaz; Maas, Stefan

    2010-01-01

    RNA editing by adenosine deamination fuels the generation of RNA and protein diversity in eukaryotes, particularly in higher organisms. This includes the recoding of translated exons, widespread editing of retrotransposon-derived repeat elements and sequence modification of miRNA transcripts. Such changes can bring about specific amino acid substitutions, alternative splicing and changes in gene expression levels. Although the overall prevalence of A-to-I editing and its specific functional impact on many of the affected genes are not yet known, the importance of balancing RNA modification levels across time and space is becoming increasingly evident. In particular, transcriptome instabilities in form of too much or too little RNA editing activity, or misguided editing manifest in several human disease phenotypes which disrupt that balance. PMID:20395010

  17. Molecular sieving polymer for DNA/RNA separation in capillary electrophoresis

    Science.gov (United States)

    Liu, Chenchen; Yamaguchi, Yoshinori; Dou, Xiaoming

    2017-07-01

    In capillary polymer electrophoresis, the property of polymer sieving matrix dominates the migration behavior of DNA/RNA. We investigated the capillary electrophoresis of RNA ranging from 100 nt to 10,000 nt in polyacrylamide (PA) solutions with different molecular weights (Mw) and different concentrations. We observed that the resolution length (RSL) of RNA fragments was improved and the migration time was prolonged, when polymer concentration was increased. The resolution for small RNA fragments (3000 nt) became inseparable. In addition, we estimated the smallest resolvable nucleotide length (Ls) by the plot of RSL against RNA size.

  18. Steady-state NTPase activity of Dengue virus NS3: number of catalytic sites, nucleotide specificity and activation by ssRNA.

    Directory of Open Access Journals (Sweden)

    J Jeremías Incicco

    Full Text Available Dengue virus nonstructural protein 3 (NS3 unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat /KM - was GTP[Formula: see text]ATP[Formula: see text]CTP [Formula: see text] UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A and poly(C. Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat and a 100 fold decrease in the apparent affinity (KM for ATP. When the ratio [RNA bases]/[NS3] was between 0 and [Formula: see text]20 the ATPase activity was inhibited by increasing both poly(A and poly(C. Using the theory of binding of large ligands (NS3 to a one-dimensional homogeneous lattice of infinite length (RNA we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.

  19. Characterization of molecular interactions between E. coli RNA polymerase and topoisomerase I by molecular simulations

    Science.gov (United States)

    Tiwari, Purushottam Babu; Chapagain, Prem P.; Banda, Srikanth; Darici, Yesim; Üren, Aykut; Tse-Dinh, Yuk-Ching

    2016-01-01

    Escherichia coli topoisomerase I (EctopoI), a type IA DNA topoisomerase, relaxes the negative DNA supercoiling generated by RNA polymerase (RNAP) during transcription elongation. Due to the lack of structural information on the complex, the exact nature of the RNAP-EctopoI interactions remains unresolved. Herein, we report for the first time, the structure-based modeling of the RNAP-EctopoI interactions using computational methods. Our results predict that the salt-bridge as well as hydrogen bond interactions are responsible for the formation and stabilization of the RNAP-EctopoI complex. Our investigations provide molecular insights for understanding how EctopoI interacts with RNAP, a critical step for preventing hypernegative DNA supercoiling during transcription. PMID:27448274

  20. Exploring the RNA-bound and RNA-free human Argonaute-2 by molecular dynamics simulation method.

    Science.gov (United States)

    Kong, Ren; Xu, Lei; Piao, Lianhua; Zhang, Dawei; Hou, Ting-Jun; Chang, Shan

    2017-11-01

    Argonaute 2 (Ago2) protein is the major vehicle of microRNAs (miRNAs)-guided gene repression and silencing processes. Although the crystal structure of human Ago2 (hAgo2) has recently been disclosed, the information of dynamically structural character of protein-RNA recognition is still lacking. Molecular dynamics simulations were used to systematically explore hAgo2 in the presence and absence of RNA duplex. Stable direct and water-mediated hydrogen bonds were observed between guide RNA backbone atoms and hAgo2, especially for nucleotides 2-7. In addition, water-mediated hydrogen bonds are indicated to be critical in the specific recognition between hAgo2 and the conserved adenine in position 1 of target RNA. The core domains (N, PAZ, MID, and PIWI) possess rigid body movements during the simulations. The motions of N-PAZ and PIWI-MID are negatively correlated with or without RNA binding and PAZ domain is identified as the most mobile domain in both systems. The reorientation of PAZ domain not only influences the binding of helix-7 and RNA duplex, the initial pairing process, but also the shape of N-PAZ cleft, where the supplemental base pairing occurs. It is speculated that PAZ domain could be a key regulator in hAgo2-mediated miRNA-induced gene regulation. © 2017 John Wiley & Sons A/S.

  1. RNA three-way junctions can act as flexible RNA structural elements in large RNA molecules: a molecular simulation analysis

    Czech Academy of Sciences Publication Activity Database

    Beššeová, Ivana; Réblová, Kamila; Leontis, N.B.; Šponer, Jiří

    2009-01-01

    Roč. 26, č. 6 (2009), s. 830-831 ISSN 0739-1102. [The 17th Conversation. 16.06.2009-20.06.2009, Albany] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA three-way junctions * RNA Subject RIV: BO - Biophysics

  2. Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity

    Czech Academy of Sciences Publication Activity Database

    Bjorkman, J.; Švec, David; Kubista, Mikael; Sjöback, R.

    2016-01-01

    Roč. 6, Jan 2016 (2016), s. 4-12 ISSN 2214-7535 R&D Projects: GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : RNA integrity * RNA quality * Endogenous RNase resistant marker Subject RIV: EB - Genetics ; Molecular Biology

  3. Low molecular weight chitosan conjugated with folate for siRNA delivery in vitro: optimization studies

    Science.gov (United States)

    Fernandes, Julio C; Qiu, Xingping; Winnik, Francoise M; Benderdour, Mohamed; Zhang, Xiaoling; Dai, Kerong; Shi, Qin

    2012-01-01

    The low transfection efficiency of chitosan is one of its drawbacks as a gene delivery carrier. Low molecular weight chitosan may help to form small-sized polymer-DNA or small interfering RNA (siRNA) complexes. Folate conjugation may improve gene transfection efficiency because of the promoted uptake of folate receptor-bearing cells. In the present study, chitosan was conjugated with folate and investigated for its efficacy as a delivery vector for siRNA in vitro. We demonstrate that the molecular weight of chitosan has a major influence on its biological and physicochemical properties, and very low molecular weight chitosan (below 10 kDa) has difficulty in forming stable complexes with siRNA. In this study, chitosan 25 kDa and 50 kDa completely absorbed siRNA and formed nanoparticles (≤220 nm) at a chitosan to siRNA weight ratio of 50:1. The introduction of a folate ligand onto chitosan decreased nanoparticle toxicity. Compared with chitosan-siRNA, folate-chitosan-siRNA nanoparticles improved gene silencing transfection efficiency. Therefore, folate-chitosan shows potential as a viable candidate vector for safe and efficient siRNA delivery. PMID:23209368

  4. Catalytic strategies of the hepatitis delta virus ribozyme as probed by molecular dynamics simulations

    Czech Academy of Sciences Publication Activity Database

    Krasovská, Maryna V.; Sefcikova, J.; Špačková, Naďa; Šponer, Jiří; Walter, N. G.

    2005-01-01

    Roč. 22, č. 6 (2005), s. 774 ISSN 0739-1102. [Albany 2005. Conversation /14./. 14.06.2005-18.06.2005, Albany] Institutional research plan: CEZ:AV0Z50040507 Keywords : catalytic strategies * hepatitis delta virus Subject RIV: BO - Biophysics

  5. Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

    Directory of Open Access Journals (Sweden)

    Jailson F. B. Querido

    2013-01-01

    Full Text Available Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV, a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae, one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.

  6. Kinetic proofreading at single molecular level: aminoacylation of tRNA(Ile and the role of water as an editor.

    Directory of Open Access Journals (Sweden)

    Mantu Santra

    Full Text Available Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA(Ile which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively, in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10(2.(4.33×10(2 = 7.8×10(4, (ii the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA(Ile. The present study shows a non-michaelis type dependence of rate of reaction on tRNA(Ile concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA(Ile whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.

  7. Cascade Amplification-Mediated In Situ Hot-Spot Assembly for MicroRNA Detection and Molecular Logic Gate Operations.

    Science.gov (United States)

    Yu, Sha; Wang, Yingying; Jiang, Li-Ping; Bi, Sai; Zhu, Jun-Jie

    2018-03-23

    MicroRNAs (miRNAs) play important roles in many biological processes and are associated with various diseases, especially cancers. Combination of technological developments such as nanomaterials, functional enzyme-mediated reactions, and DNA nanotechnology holds great potential for high-performance detection of miRNAs in molecular diagnostic systems. In this work, we have fabricated a cascade signal amplification platform through integrating duplex-specific nuclease (DSN)-assisted target recycling with catalytic hairpin assembly (CHA) reaction for the detection of microRNA-141 (miR-141). The target recycling process driven by DSN results in highly amplified translation of target miRNA to single-stranded connector DNA fragments. The CHA reaction is further initiated by connector DNAs using hairpin-modified gold nanoparticles (HP-AuNPs) as the sensing unit, leading to the formation of AuNP network architecture on the electrode for electrochemical and photoelectrochemical detection of miR-141 in signal-on and signal-off modes, respectively. The developed electrochemical biosensor exhibits a detection limit down to 25.1 aM miR-141 (60 copies in 4 μL sample) and excellent selectivity to discriminate a single base-mismatched sequence and other miRNAs. This assay is also applied to the determination of miR-141 in total RNAs extracted from human breast cancer cells (MDA-MB-231), confirming the applicability of this method for absolute quantification of specific miRNAs in real-world samples. Furthermore, two-input AND and INHIBIT (INH) logic gates are constructed to detect miRNAs. In particular, the AND gate achieves cell-specific gate activation based on expression profiles of miR-141 and microRNA-21 (miR-21). Therefore, our proposed cascade amplification platform has great potential applications in miRNA-related clinical diagnostics and biochemical research.

  8. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Directory of Open Access Journals (Sweden)

    Galiano V

    2016-10-01

    Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

  9. Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches

    KAUST Repository

    Jiang, Hanlun

    2016-12-06

    MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

  10. Exploration of the molecular mechanism of prostate cancer based on mRNA and miRNA expression profiles

    Directory of Open Access Journals (Sweden)

    Zhang X

    2017-06-01

    Full Text Available Xing Zhang,1 YuYan Sun,1 Peng Wang,1 Changfu Yang,1 Shengwei Li2 1Department of Urology, 2Surgery of Chinese Medicine, Yangzhou TCM Hospital, Nanjing University of Chinese Medicine, Yangzhou, People’s Republic of China Abstract: Prostate cancer, the second most common cancer in men, has been rarely explored by integrating mRNA and miRNA expression profiles. In this study, we combined two mRNA expression datasets and six documented miRNAs to uncover the comprehensive molecular mechanism of prostate cancer. Results showed that a total of 30 genes were significantly differentially expressed in 49 tumor samples by comparing with 22 normal samples. Importantly, all samples from the two datasets can be clearly classified into two groups, tumor group and normal group, based on the selected differentially expressed genes (DEGs. The miRNA–mRNA regulation network indicated that 4 out of 30 DEGs can be regulated by three miRNAs. In addition, prognostic performance validation using in silico databases showed that the DEGs can significantly differentiate between low-risk and high-risk prostate cancer. In summary, multiple biological processes are probably involved in the development and progression of prostate cancer. First, dysregulation of SV2 can regulate transporter and transmembrane transporter activity and then provide the necessary nutrients for tumor cell proliferation. Second, HOXD10 can induce cell proliferation via TCF-4. Finally, dysregulation of CACNA1D can further suppress tumor apoptosis in prostate cancer. The identification of critical genes and valuable biological processes can be useful for the understanding of the molecular mechanism of prostate cancer. Keywords: mRNA, DEGs, miRNA, prognostic

  11. Reduction of nitrogen oxides with catalytic acid resistant aluminosilicate molecular sieves and ammonia

    Science.gov (United States)

    Pence, Dallas T.; Thomas, Thomas R.

    1980-01-01

    Noxious nitrogen oxides in a waste gas stream such as the stack gas from a fossil-fuel-fired power generation plant or other industrial plant off-gas stream is catalytically reduced to elemental nitrogen and/or innocuous nitrogen oxides employing ammonia as reductant in the presence of a zeolite catalyst in the hydrogen or sodium form having pore openings of about 3 to 10 A.

  12. Active RNA polymerases: mobile or immobile molecular machines?

    Directory of Open Access Journals (Sweden)

    Argyris Papantonis

    2010-07-01

    Full Text Available It is widely assumed that active RNA polymerases track along their templates to produce a transcript. We test this using chromosome conformation capture and human genes switched on rapidly and synchronously by tumour necrosis factor alpha (TNFalpha; one is 221 kbp SAMD4A, which a polymerase takes more than 1 h to transcribe. Ten minutes after stimulation, the SAMD4A promoter comes together with other TNFalpha-responsive promoters. Subsequently, these contacts are lost as new downstream ones appear; contacts are invariably between sequences being transcribed. Super-resolution microscopy confirms that nascent transcripts (detected by RNA fluorescence in situ hybridization co-localize at relevant times. Results are consistent with an alternative view of transcription: polymerases fixed in factories reel in their respective templates, so different parts of the templates transiently lie together.

  13. Analysis of the complement and molecular evolution of tRNA genes in cow

    Directory of Open Access Journals (Sweden)

    Barris Wesley C

    2009-04-01

    set of cow tRNA genes that will facilitate further studies in understanding the molecular evolution of cow tRNA genes.

  14. Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition.

    Science.gov (United States)

    Schlundt, Andreas; Tants, Jan-Niklas; Sattler, Michael

    2017-04-15

    Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain

  15. Structural origins for the loss of catalytic activities of bifunctional human LTA4H revealed through molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Sundarapandian Thangapandian

    Full Text Available Human leukotriene A4 hydrolase (hLTA4H, which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents.

  16. Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity.

    Science.gov (United States)

    Björkman, J; Švec, D; Lott, E; Kubista, M; Sjöback, R

    2016-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

  17. Differential amplicons (ΔAmp—a new molecular method to assess RNA integrity

    Directory of Open Access Journals (Sweden)

    J. Björkman

    2016-01-01

    Full Text Available Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS, Quantitative real-time PCR (qPCR or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp of an Endogenous RNase Resistant (ERR marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

  18. Molecular modeling and molecular dynamics simulation study of archaeal leucyl-tRNA synthetase in complex with different mischarged tRNA in editing conformation.

    Science.gov (United States)

    Rayevsky, A V; Sharifi, M; Tukalo, M A

    2017-09-01

    Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS (PhLeuRS) with misacylated tRNA Leu was modeled wherever tRNA's acceptor stem was oriented directly into the editing site. To understand the distinctive features of organization we reconstructed a complex of PhLeuRS with tRNA and visualize post-transfer editing interactions mode by performing molecular dynamics (MD) simulation studies. To study molecular basis for substrate selectivity by PhLeuRS's editing site we utilized MD simulation of the entire LeuRS complexes using a diverse charged form of tRNAs, namely norvalyl-tRNA Leu and isoleucyl-tRNA Leu . In general, the editing site organization of LeuRS from P.horikoshii has much in common with bacterial LeuRS. The MD simulation results revealed that the post-transfer editing substrate norvalyl-A76, binds more strongly than isoleucyl-A76. Moreover, the branched side chain of isoleucine prevents water molecules from being closer and hence the hydrolysis reaction slows significantly. To investigate a possible mechanism of the post-transfer editing reaction, by PhLeuRS we have determined that two water molecules (the attacking and assisting water molecules) are localized near the carbonyl group of the amino acid to be cleaved off. These water molecules approach the substrate from the opposite side to that observed for Thermus thermophilus LeuRS (TtLeuRS). Based on the results obtained, it was suggested that the post-transfer editing mechanism of PhLeuRS differs from that of prokaryotic TtLeuRS. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study

    Directory of Open Access Journals (Sweden)

    P. V. G. M. Rathnayake

    2017-01-01

    Full Text Available The use of gene therapeutics, including short interfering RNA (siRNA, is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules.

  20. MicroRNA-triggered, cascaded and catalytic self-assembly of functional ``DNAzyme ferris wheel'' nanostructures for highly sensitive colorimetric detection of cancer cells

    Science.gov (United States)

    Zhou, Wenjiao; Liang, Wenbin; Li, Xin; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2015-05-01

    The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional ``DNAzyme ferris wheel'' nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into ``DNAzyme ferris wheel''-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

  1. MicroRNA-triggered, cascaded and catalytic self-assembly of functional "DNAzyme ferris wheel" nanostructures for highly sensitive colorimetric detection of cancer cells.

    Science.gov (United States)

    Zhou, Wenjiao; Liang, Wenbin; Li, Xin; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2015-05-21

    The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional "DNAzyme ferris wheel" nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into "DNAzyme ferris wheel"-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

  2. Decoupled catalytic hydrogen evolution from a molecular metal oxide redox mediator in water splitting.

    Science.gov (United States)

    Rausch, Benjamin; Symes, Mark D; Chisholm, Greig; Cronin, Leroy

    2014-09-12

    The electrolysis of water using renewable energy inputs is being actively pursued as a route to sustainable hydrogen production. Here we introduce a recyclable redox mediator (silicotungstic acid) that enables the coupling of low-pressure production of oxygen via water oxidation to a separate, catalytic hydrogen production step outside the electrolyzer that requires no post-electrolysis energy input. This approach sidesteps the production of high-pressure gases inside the electrolytic cell (a major cause of membrane degradation) and essentially eliminates the hazardous issue of product gas crossover at the low current densities that characterize renewables-driven water-splitting devices. We demonstrated that a platinum-catalyzed system can produce pure hydrogen over 30 times faster than state-of-the-art proton exchange membrane electrolyzers at equivalent platinum loading. Copyright © 2014, American Association for the Advancement of Science.

  3. Structural and optical properties of self-catalytic GaAs:Mn nanowires grown by molecular beam epitaxy on silicon substrates

    DEFF Research Database (Denmark)

    Gas, Katarzyna; Sadowski, Janusz; Kasama, Takeshi

    2013-01-01

    Mn-doped GaAs nanowires were grown in the self-catalytic growth mode on the oxidized Si(100) surface by molecular beam epitaxy and characterized by scanning and transmission electron microscopy, Raman scattering, photoluminescence, cathodoluminescence, and electron transport measurements. The tra...

  4. Atomistic details of the molecular recognition of DNA-RNA hybrid ...

    Indian Academy of Sciences (India)

    127, No. 10, October 2015, pp. 1701–1713. c Indian Academy of Sciences. DOI 10.1007/s12039-015-0942-7. Atomistic details of the molecular recognition of DNA-RNA hybrid duplex by ribonuclease H enzyme. GORLE SURESH and U DEVA PRIYAKUMAR∗. Center for Computational Natural Sciences and Bioinformatics, ...

  5. Molecular dynamics simulations and quantum chemistry as useful complements to RNA structural bioinformatics

    Czech Academy of Sciences Publication Activity Database

    Šponer, Jiří

    2009-01-01

    Roč. 26, č. 6 (2009), s. 824-825 ISSN 0739-1102. [The 17th Conversation. 16.06.2009-20.06.2009, Albany] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : molecular dynamics * RNA Subject RIV: BO - Biophysics

  6. Development of molecular cytogenetics and physical mapping of ribosomal RNA genes in Lupinus

    Czech Academy of Sciences Publication Activity Database

    Naganowska, B.; Doležel, Jaroslav; Swiecicki, W. K.

    2003-01-01

    Roč. 46, - (2003), s. 211-215 ISSN 0006-3134 R&D Projects: GA ČR GA521/03/0595 Institutional research plan: CEZ:AV0Z5038910 Keywords : fluorescent in situ hybridization * Lupinus sp. * rRNA genes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.919, year: 2003

  7. Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

    Science.gov (United States)

    Grierson, D; Hemleben, V

    1977-04-04

    The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise

  8. [Molecular relationship of Eurytrema coelmaticum inferred from 18S rRNA sequence].

    Science.gov (United States)

    Zheng, Ya-dong; Luo, Xue-nong; Shi, Cheng-hong; Zong, Rui-qian; Jing, Zhi-zhong; Cai, Xue-peng

    2006-10-01

    To elucidate the taxonomic position of Eurytrema coelmaticum by using molecular technology. 18S rRNA fragment was amplified from E. coelmaticum genomic DNA by specific conservative primers and sequenced. Homology and phylogenic tree of 18S rRNA sequences between E. coelmaticum and other Dicrocoeliidae trematodes were analyzed and constructed by DNAStar and MEGA3 respectively, and their evolutionary relationship was determined. E. coelmaticum 18S rRNA sequence was with high homology to those from Dicrocoelium dendriticum, Lyperosomum collurionis and Brachylecithum lobatum. Among them, the diversity of E. coelmaticum from D. dendriticum was 2.42%, and that from L. collurionis was 1.75%; D. dendriticum and B. lobatum were closer in evolution only with 1.09% diversity. For Dicrocoeliidae trematodes, classification based on 18S rRNA target is valid and the sequences are highly conservative. E. coelmaticum is evolutionarily closer to L. collurionis than to D. dendriticum and B. lobatum.

  9. Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions

    Science.gov (United States)

    Nakamura, Hideki; Lee, Albert A.; Afshar, Ali Sobhi; Watanabe, Shigeki; Rho, Elmer; Razavi, Shiva; Suarez, Allister; Lin, Yu-Chun; Tanigawa, Makoto; Huang, Brian; Derose, Robert; Bobb, Diana; Hong, William; Gabelli, Sandra B.; Goutsias, John; Inoue, Takanari

    2018-01-01

    Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

  10. Molecular characterisation of the thermostability and catalytic properties of enzymes from hyperthermophiles

    NARCIS (Netherlands)

    Lebbink, J.H.G.

    1999-01-01

    Hyperthermophilic organisms are able to survive and reproduce optimally between 80°C and 113°C. Most of them belong to the domain of the Archaea, although several hyperthermophilic Bacteria have been described. One of the major questions regarding hyperthermophiles concerns the molecular

  11. A new insight into electrochemical microRNA detection: a molecular caliper, p19 protein.

    Science.gov (United States)

    Kilic, Tugba; Nur Topkaya, Seda; Ozsoz, Mehmet

    2013-10-15

    microRNA (miRNA) has drawn a great attention in biomedical research due to its functions on biological processes. Detection of miRNAs is a big challenge since the amount present in real samples is very low and the length of them is short. In this study, for the first time an electrochemical biosensor for detection of mir21 using the oxidation signal of protein 19 (p19) as a molecular caliper was designed. The proposed method enables detection of mir21 in direct, rapid, sensitive, inexpensive and label-free way. Binding specificity of the p19 to 20-23 base pair length double stranded RNA (dsRNA) and direct/water-mediated intermolecular contacts between the fusion protein and miRNA allows detection of miRNA-antimiRNA hybrid structure. The detection of mir21 was achieved in picomole sensitivity through the changes of intrinsic p19 oxidation signals observed at +0.80 V with Differential Pulse Voltammetry (DPV) and the specifity of the designed sensor was proved by control studies. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Catalytic Synthesis of n-Butyl Oleate by Cerium Complex Doped Y/SBA-15 Composite Molecular Sieve

    Science.gov (United States)

    Shi, Chunwei; Bian, Xue; Wu, Yongfu; Cong, Yufeng; Pei, Mingyuan

    2018-01-01

    Cerium ion was successfully incorporated into Y/SBA-15 micro-mesoporous molecular sieves via the hydrothermal synthesis method to give a series of composite materials. The prepared materials were thoroughly characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray fluorescence spectroscopy (XRF), scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and differential thermo gravimetric analysis (TG-DTG). The results showed that the prepared composite materials retained the highly ordered mesoporous two-dimensional hexagonal structure of SBA-15 and the octagonal structure of Y. The catalyst Ce-Y/SBA-15 was prepared and characterized, then the esterification of n-butanol and oleic acid was studied with bismuth phosphotungstate as a catalyst. Using this model reaction, the effects of Ce-HY/SBA-15, molar ratio of alcohol to oleic acid, amount of catalysts, reaction time and reaction temperature were investigated. The experimental results show that the optimal reaction conditions were: 1.8:1 molar ratio of alcohol to acid, 5 % catalyst amount (based on weight of oleic acid), 4 h reaction time and reflux conditions. Under these conditions, the yield of esterification was 90.6 %. The results suggest that the addition of Ce can effectively improve the catalytic properties of composite molecular sieves.

  13. Catalytic and molecular properties of rabbit liver carboxylesterase acting on 1,8-cineole derivatives.

    Science.gov (United States)

    del Loandos, María H; Muro, Ana C; Villecco, Margarita B; Masman, Marcelo F; Luiten, Paul G M; Andujar, Sebastian A; Suvirec, Fernando D; Enriz, Ricardo D

    2012-09-01

    Rabbit liver carboxylesterase (rCE) was evaluated as the catalyst for the enantioselective hydrolysis of (+/-)-3-endo-acetyloxy-1 ,8-cineole [(+/-)-4], which yields (1S,3S,4R)-(+)-3-acetyloxy-1,8-cineole [(+)-4] and (1R,3R,4S)-(-)-3-hydroxy-1,8-cineole [(-)-3]. Enantioselective asymmetrization of meso-3,5-diacetoxy-1,8-cineol (5) gives (1S,3S,4R,5R)-(-)-3-acetyloxy-5-hydroxy-1,8-cineole (6), with high enantioselectivity. rCE has been chosen to perform both experiments and molecular modeling simulations. Docking simulations combined with molecular dynamics calculations were used to study rCE-catalyzed enantioselective hydrolysis of cineol derivatives. Both compounds were found to bind with their acetyl groups stabilized by hydrogen bond interactions between their oxygen atoms and Ser221.

  14. Capillary electrophoresis of RNA in hydroxyethylcellulose polymer with various molecular weights.

    Science.gov (United States)

    Li, Zhenqing; Liu, Chenchen; Zhang, Dawei; Luo, Shaopeng; Yamaguchi, Yoshinori

    2016-02-01

    Recent research demonstrates that large numbers of long noncoding RNAs (lncRNAs) in mammals exhibit indices of functionality, and thus analysis of longer RNAs is of great significance. In the present work, we investigated the effect of molecular weight on the separation performance of long RNA by capillary electrophoresis (CE). Results demonstrate that (1) low molecular weight of hydroxyethylcellulose (HEC) (90k) favors the separation of short RNA (RNA was improved and the migration time was linearly extended with the increase of polymer concentration. (2) In the longer chain HEC (250k, 720k and 1300k), the resolution for the small RNA fragment (3000 nt) deteriorated. (3) Based on logarithmic plot, there exist two migration regimes for RNA in short chain HEC (90k), three regimes in moderate chain HEC (250k and 720k), and four regimes in the long chain HEC (1300k). Such a systematic investigation of long RNAs may be useful for research on lncRNAs in the length range of 100-10,000 nt. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

    Energy Technology Data Exchange (ETDEWEB)

    Makino, Debora Lika, E-mail: dmakino@biochem.mpg.de; Conti, Elena [Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany)

    2013-11-01

    The crystallographic steps towards the structure determination of a complete eukaryotic exosome complex bound to RNA are presented. Phasing of this 11-protein subunit complex was carried out via molecular replacement. The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.

  16. Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets.

    Science.gov (United States)

    Fernando, Deepani D; Marr, Edward J; Zakrzewski, Martha; Reynolds, Simone L; Burgess, Stewart T G; Fischer, Katja

    2017-06-10

    Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools. We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi). We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls. A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

  17. A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

    Science.gov (United States)

    Albert, Benjamin; Knight, Britta; Merwin, Jason; Martin, Victoria; Ottoz, Diana; Gloor, Yvonne; Bruzzone, Maria Jessica; Rudner, Adam; Shore, David

    2016-11-17

    Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  19. Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M., E-mail: sean.burrows@oregonstate.edu

    2016-02-25

    Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

  20. Catalytic degradation of Acid Orange 7 by manganese oxide octahedral molecular sieves with peroxymonosulfate under visible light irradiation.

    Science.gov (United States)

    Duan, Lian; Sun, Binzhe; Wei, Mingyu; Luo, Shilu; Pan, Fei; Xu, Aihua; Li, Xiaoxia

    2015-03-21

    In this paper, the photodegradation of Acid Orange 7 (AO7) in aqueous solutions with peroxymonosulfate (PMS) was studied with manganese oxide octahedral molecular sieves (OMS-2) as the catalyst. The activities of different systems including OMS-2 under visible light irradiation (OMS-2/Vis), OMS-2/PMS and OMS-2/PMS/Vis were evaluated. It was found that the efficiency of OMS-2/PMS was much higher than that of OMS-2/Vis and could be further enhanced by visible light irradiation. The catalyst also exhibited stable performance for multiple runs. Results from ESR and XPS analyses suggested that the highly catalytic activity of the OMS-2/PMS/Vis system possible involved the activation of PMS to sulfate radicals meditated by the redox pair of Mn(IV)/Mn(III) and Mn(III)/Mn(II), while in the OMS-2/PMS system, only the redox reaction between Mn(IV)/Mn(III) occurred. Several operational parameters, such as dye concentration, catalyst load, PMS concentration and solution pH, affected the degradation of AO7. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Molecular hydrogen and catalytic combustion in the production of hyperpolarized 83Kr and 129Xe MRI contrast agents.

    Science.gov (United States)

    Rogers, Nicola J; Hill-Casey, Fraser; Stupic, Karl F; Six, Joseph S; Lesbats, Clémentine; Rigby, Sean P; Fraissard, Jacques; Pavlovskaya, Galina E; Meersmann, Thomas

    2016-03-22

    Hyperpolarized (hp) (83)Kr is a promising MRI contrast agent for the diagnosis of pulmonary diseases affecting the surface of the respiratory zone. However, the distinct physical properties of (83)Kr that enable unique MRI contrast also complicate the production of hp (83)Kr. This work presents a previously unexplored approach in the generation of hp (83)Kr that can likewise be used for the production of hp (129)Xe. Molecular nitrogen, typically used as buffer gas in spin-exchange optical pumping (SEOP), was replaced by molecular hydrogen without penalty for the achievable hyperpolarization. In this particular study, the highest obtained nuclear spin polarizations were P =29% for(83)Kr and P= 63% for (129)Xe. The results were reproduced over many SEOP cycles despite the laser-induced on-resonance formation of rubidium hydride (RbH). Following SEOP, the H2 was reactively removed via catalytic combustion without measurable losses in hyperpolarized spin state of either (83)Kr or (129)Xe. Highly spin-polarized (83)Kr can now be purified for the first time, to our knowledge, to provide high signal intensity for the advancement of in vivo hp (83)Kr MRI. More generally, a chemical reaction appears as a viable alternative to the cryogenic separation process, the primary purification method of hp(129)Xe for the past 2 1/2 decades. The inherent simplicity of the combustion process will facilitate hp (129)Xe production and should allow for on-demand continuous flow of purified and highly spin-polarized (129)Xe.

  2. Textural mesoporosity and the catalytic activity of mesoporous molecular sieves with wormhole framework structures

    International Nuclear Information System (INIS)

    Pauly, T.R.; Liu, Y.; Pinnavaia, T.J.; Billinge, S.J.L.; Rieker, T.P.

    1999-01-01

    Three different water-alcohol cosolvent systems were used to assemble mesoporous molecular sieve silicas with wormhole framework structures (previously denoted HMS silicas) from an electrically neutral amine surfactant (Sdegree) and a silicon alkoxide precursor (Idegree). The fundamental particle size and associated textural (interparticle) porosity of the disordered structures were correlated with the solubility of the surfactant in the water-alcohol cosolvents used for the SdegreeIdegree assembly process. Polar cosolvents containing relatively low volume fractions of C n H 2n+1 OH alcohols (n = 1--3) gave heterogeneous surfactant emulsions that assembled intergrown aggregates of small primary particles with high textural pore volumes (designated HMS-HTx). Conversely, three-dimensional, monolithic particles with little or no textural porosity (designated HMS-LTx) were formed from homogeneous surfactant solutions in lower polarity cosolvents. Aluminum substituted AL-HMS-HTx analogues with high textural porosity and improved framework accessibility also were shown to be much more efficient catalysts than AL-HMS-LTx or monolithic forms of hexagonal AL-MCM-41 for the sterically demanding condensed phase alkylation of 2,4-di-tert-butylphenol with cinnamyl alcohol. Transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) studies verified the textural differences between wormhole HMS and electrostatically assembled hexagonal MCM-41 and SBA-3 molecular sieves. Power law fits to the scattering data indicated a surface fractal (D s = 2.76) for HMS-HTx, consistent with rough surfaces. A second power law at lower-q indicated the formation of a mass fractal (D m = 1.83) consistent with branching of small fundamental particles. Hexagonal MCM-41 and SBA-3 silicas, on the other hand, exhibited scattering properties consistent with moderately rough surfaces (D s = 2.35 and 2.22, respectively) and large particle diameters (much g t1 micro m). HMS-LTx silicas

  3. Molecular characterization and expression of maternally expressed gene 3 (Meg3/Gtl2) RNA in the mouse inner ear

    DEFF Research Database (Denmark)

    Manji, S.S.; Sørensen, Brita Singers; Klockars, T.

    2006-01-01

    noncoding RNA. Real-time PCR analysis demonstrated that Meg3/Gtl2 was highly expressed in the cochlea, brain, and eye. Molecular studies revealed the presence of several Meg3/Gtl2 RNA splice variants in the mouse cochlea, brain, and eye. In situ hybridizations showed intense Meg3/Gtl2 RNA staining...... in the nuclei of type I spiral ganglion cells and in cerebellum near the dorsal vestibular region of the cochlea. In embryonic mouse head sections, Meg3/Gtl2 RNA expression was observed in the otocyst, brain, eye, cartilage, connective tissue, and muscle. Meg3/Gtl2 RNA expression increased in the developing...

  4. Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Moheshwarnath Issur

    Full Text Available RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5' ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

  5. Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics

    Science.gov (United States)

    Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

    2014-01-01

    The effects of “molecular crowding” on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop–receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high–molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ −2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop–receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple “hard-sphere” treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ −5 kcal/mol free-energy stabilization at excluded volume fractions of 30%. PMID:24850865

  6. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

    Directory of Open Access Journals (Sweden)

    Mykuliak V. V.

    2014-03-01

    Full Text Available Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS. Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

  7. Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia).

    Science.gov (United States)

    Douzery, E; Catzeflis, F M

    1995-11-01

    The complete 12S rRNA gene has been sequenced in 4 Ungulata (hoofed eutherians) and 1 marsupial and compared to 38 available mammalian sequences in order to investigate the molecular evolution of the mitochondrial small-subunit ribosomal RNA molecule. Ungulata were represented by one artiodactyl (the collared peccary, Tayassu tajacu, suborder Suiformes), two perissodactyls (the Grevy's zebra, Equus grevyi, suborder Hippomorpha; the white rhinoceros, Ceratotherium simum, suborder Ceratomorpha), and one hyracoid (the tree hyrax, Dendrohyrax dorsalis). The fifth species was a marsupial, the eastern gray kangaroo (Macropus giganteus). Several transition/transversion biases characterized the pattern of changes between mammalian 12S rRNA molecules. A bias toward transitions was found among 12S rRNA sequences of Ungulata, illustrating the general bias exhibited by ribosomal and protein-encoding genes of the mitochondrial genome. The derivation of a mammalian 12S rRNA secondary structure model from the comparison of 43 eutherian and marsupial sequences evidenced a pronounced bias against transversions in stems. Moreover, transversional compensatory changes were rare events within double-stranded regions of the ribosomal RNA. Evolutionary characteristics of the 12S rRNA were compared with those of the nuclear 18S and 28S rRNAs. From a phylogenetic point of view, transitions, transversions and indels in stems as well as transversional and indels events in loops gave congruent results for comparisons within orders. Some compensatory changes in double-stranded regions and some indels in single-stranded regions also constituted diagnostic events. The 12S rRNA molecule confirmed the monophyly of infraorder Pecora and order Cetacea and demonstrated the monophyly of the suborder Ruminantia was not supported and the branching pattern between Cetacea and the artiodacytyl suborders Ruminantia and Suiformes was not established. The monophyly of the order Perissodactyla was evidenced

  8. RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

    Directory of Open Access Journals (Sweden)

    Mamidala Praveen

    2012-01-01

    Full Text Available Abstract Background Bed bugs (Cimex lectularius are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq experiment to find differentially expressed genes between pesticide-resistant (PR and pesticide-susceptible (PS strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs and our previous 454 pyrosequenced database (21,088 ESTs. The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2 revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide

  9. Quantum Mechanics and Molecular Mechanics Study of the Catalytic Mechanism of Human AMSH-LP Domain Deubiquitinating Enzymes.

    Science.gov (United States)

    Zhu, Wenyou; Liu, Yongjun; Ling, Baoping

    2015-08-25

    Deubiquitinating enzymes (DUBs) catalyze the cleavage of the isopeptide bond in polyubiquitin chains to control and regulate the deubiquitination process in all known eukaryotic cells. The human AMSH-LP DUB domain specifically cleaves the isopeptide bonds in the Lys63-linked polyubiquitin chains. In this article, the catalytic mechanism of AMSH-LP has been studied using a combined quantum mechanics and molecular mechanics method. Two possible hydrolysis processes (Path 1 and Path 2) have been considered. Our calculation results reveal that the activation of Zn(2+)-coordinated water molecule is the essential step for the hydrolysis of isopeptide bond. In Path 1, the generated hydroxyl first attacks the carbonyl group of Gly76, and then the amino group of Lys63 is protonated, which is calculated to be the rate limiting step with an energy barrier of 13.1 kcal/mol. The energy barrier of the rate limiting step and the structures of intermediate and product are in agreement with the experimental results. In Path 2, the protonation of amino group of Lys63 is prior to the nucleophilic attack of activated hydroxyl. The two proton transfer processes in Path 2 correspond to comparable overall barriers (33.4 and 36.1 kcal/mol), which are very high for an enzymatic reaction. Thus, Path 2 can be ruled out. During the reaction, Glu292 acts as a proton transfer mediator, and Ser357 mainly plays a role in stabilizing the negative charge of Gly76. Besides acting as a Lewis acid, Zn(2+) also influences the reaction by coordinating to the reaction substrates (W1 and Gly76).

  10. Novel hypovirulence-associated RNA mycovirus in the plant pathogenic fungus botrytis cinerea: molecular and biological characterization

    Science.gov (United States)

    Botrytis cinerea is a pathogenic fungus causing gray mold disease on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain ...

  11. Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

    International Nuclear Information System (INIS)

    Brock, K.V.

    1987-01-01

    Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

  12. Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

    Directory of Open Access Journals (Sweden)

    Gea Guerriero

    2016-07-01

    Full Text Available In plants there is no universal protocol for RNA extraction, since optimizations are required depending on the species, tissues and developmental stages. Some plants/tissues are rich in secondary metabolites or synthesize thick cell walls, which hinder an efficient RNA extraction. One such example is bast fibres, long extraxylary cells characterized by a thick cellulosic cell wall. Given the economic importance of bast fibres, which are used in the textile sector, as well as in biocomposites as green substitutes of glass fibres, it is desirable to better understand their development from a molecular point of view. This knowledge favours the development of biotechnological strategies aimed at improving specific properties of bast fibres. To be able to perform high-throughput analyses, such as, for instance, transcriptomics of bast fibres, RNA extraction is a crucial and limiting step. We here detail a protocol enabling the rapid extraction of high quality RNA from the bast fibres of textile hemp, Cannabis sativa L., a multi-purpose fibre crop standing in the spotlight of research.

  13. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

    Science.gov (United States)

    Dahlman, James E.; Barnes, Carmen; Khan, Omar F.; Thiriot, Aude; Jhunjunwala, Siddharth; Shaw, Taylor E.; Xing, Yiping; Sager, Hendrik B.; Sahay, Gaurav; Speciner, Lauren; Bader, Andrew; Bogorad, Roman L.; Yin, Hao; Racie, Tim; Dong, Yizhou; Jiang, Shan; Seedorf, Danielle; Dave, Apeksha; Singh Sandhu, Kamaljeet; Webber, Matthew J.; Novobrantseva, Tatiana; Ruda, Vera M.; Lytton-Jean, Abigail K. R.; Levins, Christopher G.; Kalish, Brian; Mudge, Dayna K.; Perez, Mario; Abezgauz, Ludmila; Dutta, Partha; Smith, Lynelle; Charisse, Klaus; Kieran, Mark W.; Fitzgerald, Kevin; Nahrendorf, Matthias; Danino, Dganit; Tuder, Rubin M.; von Andrian, Ulrich H.; Akinc, Akin; Panigrahy, Dipak; Schroeder, Avi; Koteliansky, Victor; Langer, Robert; Anderson, Daniel G.

    2014-08-01

    Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

  14. Performance of molecular mechanics force fields for RNA simulations: Stability of UUCG and GNRA hairpins

    Czech Academy of Sciences Publication Activity Database

    Banáš, P.; Hollas, D.; Zgarbová, M.; Jurečka, P.; Orozco, M.; Cheatham III, T.E.; Šponer, Jiří; Otyepka, M.

    2010-01-01

    Roč. 6, č. 12 (2010), s. 3836-3849 ISSN 1549-9618 R&D Projects: GA MŠk(CZ) LC06030; GA ČR(CZ) GA203/09/1476; GA ČR(CZ) GD203/09/H046; GA AV ČR(CZ) IAA400040802 Grant - others:GA MŠk(CZ) LC512 Program:LC Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : molecular dynamics * force fields * RNA * tetraloops Subject RIV: BO - Biophysics Impact factor: 5.138, year: 2010

  15. Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

    OpenAIRE

    Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2012-01-01

    A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer e...

  16. Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by molecular dynamics

    Czech Academy of Sciences Publication Activity Database

    Réblová, Kamila; Fadrná, E.; Sarzynska, J.; Kulinski, T.; Kulhánek, P.; Ennifar, E.; Koča, J.; Šponer, Jiří

    2007-01-01

    Roč. 93, č. 11 (2007), s. 3932-3949 ISSN 0006-3495 R&D Projects: GA MŠk(CZ) LC06030; GA ČR(CZ) GA203/05/0009; GA ČR(CZ) GA203/05/0388; GA AV ČR(CZ) 1QS500040581 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : molecular dynamics * RNA * virus Subject RIV: BO - Biophysics Impact factor: 4.627, year: 2007

  17. Molecular dynamics simulations of RNA: An in silico single molecule approach

    Czech Academy of Sciences Publication Activity Database

    McDowell, S.E.; Špačková, Naďa; Šponer, Jiří; Walter, N.G.

    2006-01-01

    Roč. 85, č. 2 (2006), s. 169-184 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA203/05/0009; GA ČR(CZ) GA203/05/0388; GA AV ČR(CZ) 1QS500040581; GA MŠk(CZ) LC06030; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z40550506 Keywords : hydration and cation binding * molecular dynamics * RNA Subject RIV: BO - Biophysics Impact factor: 2.480, year: 2006

  18. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers

    Directory of Open Access Journals (Sweden)

    Jinwei Zhang

    2016-04-01

    Full Text Available Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers.

  19. Molecular Properties, Functional Mechanisms, and Applications of Sliced siRNA

    Directory of Open Access Journals (Sweden)

    Guihua Sun

    2015-01-01

    Full Text Available Using pre-miR-451 as a model molecule, we have characterized the general molecular properties of small hairpin RNAs that are processed into potent small interfering RNAs (siRNA by Argonaute2 (Ago2. The Ago2-sliced siRNAs (sli-siRNAs have the same silencing potency as the classical Dicer diced siRNAs (di-siRNAs but have dramatically reduced unwanted sense strand activities. We have built vectors with the constitutive or inducible U6 promoter that can express sli-siRNAs in mammalian cells, in which the sli-siRNAs can be correctly processed to repress target genes. As a proof of principle for potential applications of sli-siRNAs in vivo, we show that the expression of one Ago2 shRNA-1148 in HCT-116 colon cancer cells knocked down RRM2 expression and reduced the proliferation and invasiveness of the cells. The defined sli-siRNA model molecules and the expression systems established in this study will facilitate the design and application of sli-siRNAs as novel potent RNAi triggers with reduced off-target effects.

  20. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations.

    Science.gov (United States)

    Fu, Glenn K; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N; Davis, Ronald W; Xiao, Wenzhong; Fodor, Stephen P A

    2014-02-04

    We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

  1. Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA.

    Science.gov (United States)

    Wang, Xueyin; Paucek, Richard D; Gooding, Anne R; Brown, Zachary Z; Ge, Eva J; Muir, Tom W; Cech, Thomas R

    2017-12-01

    Many studies have revealed pathways of epigenetic gene silencing by Polycomb repressive complex 2 (PRC2) in vivo, but understanding the underlying molecular mechanisms requires biochemistry. Here we analyze interactions of reconstituted human PRC2 with nucleosome complexes. Histone modifications, the H3K27M cancer mutation, and inclusion of JARID2 or EZH1 in the PRC2 complex have unexpectedly minor effects on PRC2-nucleosome binding. Instead, protein-free linker DNA dominates the PRC2-nucleosome interaction. Specificity for CG-rich sequences is consistent with PRC2 occupying CG-rich DNA in vivo. PRC2 preferentially binds methylated DNA regulated by its AEBP2 subunit, suggesting how DNA and histone methylation collaborate to repress chromatin. We find that RNA, known to inhibit PRC2 activity, is not a methyltransferase inhibitor per se. Instead, RNA sequesters PRC2 from nucleosome substrates, because PRC2 binding requires linker DNA, and RNA and DNA binding are mutually exclusive. Together, we provide a model for PRC2 recruitment and an explanation for how actively transcribed genomic regions bind PRC2 but escape silencing.

  2. Spray-dried powders enhance vaginal siRNA delivery by potentially modulating the mucus molecular sieve structure.

    Science.gov (United States)

    Wu, Na; Zhang, Xinxin; Li, Feifei; Zhang, Tao; Gan, Yong; Li, Juan

    2015-01-01

    Vaginal small interfering RNA (siRNA) delivery provides a promising strategy for the prevention and treatment of vaginal diseases. However, the densely cross-linked mucus layer on the vaginal wall severely restricts nanoparticle-mediated siRNA delivery to the vaginal epithelium. In order to overcome this barrier and enhance vaginal mucus penetration, we prepared spray-dried powders containing siRNA-loaded nanoparticles. Powders with Pluronic F127 (F127), hydroxypropyl methyl cellulose (HPMC), and mannitol as carriers were obtained using an ultrasound-assisted spray-drying technique. Highly dispersed dry powders with diameters of 5-15 μm were produced. These powders showed effective siRNA protection and sustained release. The mucus-penetrating properties of the powders differed depending on their compositions. They exhibited different potential of opening mesh size of molecular sieve in simulated vaginal mucus system. A powder formulation with 0.6% F127 and 0.1% HPMC produced the maximum increase in the pore size of the model gel used to simulate vaginal mucus by rapidly extracting water from the gel and interacting with the gel; the resulting modulation of the molecular sieve effect achieved a 17.8-fold improvement of siRNA delivery in vaginal tract and effective siRNA delivery to the epithelium. This study suggests that powder formulations with optimized compositions have the potential to alter the steric barrier posed by mucus and hold promise for effective vaginal siRNA delivery.

  3. RNA

    African Journals Online (AJOL)

    SARAH

    30 nov. 2013 ... RÉSUMÉ. Objectif : La présente étude est conduite dans les régions de Maradi et Zinder situées dans le Centre-Sud du. Niger où la pratique de la régénération naturelle assistée des ligneux dans les champs (RNA) a permis de reverdir plus de 5 millions d'hectares. Le but de ce travail est d'évaluer ...

  4. Comparative mRNA and microRNA expression profiling of three genitourinary cancers reveals common hallmarks and cancer-specific molecular events.

    Directory of Open Access Journals (Sweden)

    Xianxin Li

    Full Text Available Genome-wide gene expression profile using deep sequencing technologies can drive the discovery of cancer biomarkers and therapeutic targets. Such efforts are often limited to profiling the expression signature of either mRNA or microRNA (miRNA in a single type of cancer.Here we provided an integrated analysis of the genome-wide mRNA and miRNA expression profiles of three different genitourinary cancers: carcinomas of the bladder, kidney and testis.Our results highlight the general or cancer-specific roles of several genes and miRNAs that may serve as candidate oncogenes or suppressors of tumor development. Further comparative analyses at the systems level revealed that significant aberrations of the cell adhesion process, p53 signaling, calcium signaling, the ECM-receptor and cell cycle pathways, the DNA repair and replication processes and the immune and inflammatory response processes were the common hallmarks of human cancers. Gene sets showing testicular cancer-specific deregulation patterns were mainly implicated in processes related to male reproductive function, and general disruptions of multiple metabolic pathways and processes related to cell migration were the characteristic molecular events for renal and bladder cancer, respectively. Furthermore, we also demonstrated that tumors with the same histological origins and genes with similar functions tended to group together in a clustering analysis. By assessing the correlation between the expression of each miRNA and its targets, we determined that deregulation of 'key' miRNAs may result in the global aberration of one or more pathways or processes as a whole.This systematic analysis deciphered the molecular phenotypes of three genitourinary cancers and investigated their variations at the miRNA level simultaneously. Our results provided a valuable source for future studies and highlighted some promising genes, miRNAs, pathways and processes that may be useful for diagnostic or

  5. Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II.

    Science.gov (United States)

    Akhrymuk, Ivan; Kulemzin, Sergey V; Frolova, Elena I

    2012-07-01

    The Old World alphaviruses are emerging human pathogens with an ability to cause widespread epidemics. The latest epidemic of Chikungunya virus, from 2005 to 2007, affected over 40 countries in Africa, Asia, and Europe. The Old World alphaviruses are highly cytopathic and known to evade the cellular antiviral response by inducing global inhibition of transcription in vertebrate cells. This function was shown to be mediated by their nonstructural nsP2 protein; however, the detailed mechanism of this phenomenon has remained unknown. Here, we report that nsP2 proteins of Sindbis, Semliki Forest, and Chikungunya viruses inhibit cellular transcription by inducing rapid degradation of Rpb1, a catalytic subunit of the RNAPII complex. This degradation of Rpb1 is independent of the nsP2-associated protease activity, but, instead, it proceeds through nsP2-mediated Rpb1 ubiquitination. This function of nsP2 depends on the integrity of the helicase and S-adenosylmethionine (SAM)-dependent methyltransferase-like domains, and point mutations in either of these domains abolish Rpb1 degradation. We go on to show that complete degradation of Rpb1 in alphavirus-infected cells occurs within 6 h postinfection, before other previously described virus-induced changes in cell physiology, such as apoptosis, autophagy, and inhibition of STAT1 phosphorylation, are detected. Since Rpb1 is a subunit that catalyzes the polymerase reaction during RNA transcription, degradation of Rpb1 plays an indispensable role in blocking the activation of cellular genes and downregulating cellular antiviral response. This indicates that the nsP2-induced degradation of Rpb1 is a critical mechanism utilized by the Old World alphaviruses to subvert the cellular antiviral response.

  6. Mimicry of molecular pretenders: the terminal structures of satellites associated with plant RNA viruses.

    Science.gov (United States)

    Huang, Ying-Wen; Hu, Chung-Chi; Lin, Na-Sheng; Hsu, Yau-Heiu

    2010-01-01

    Satellite RNAs (satRNAs) and satellite viruses depend on the replicase complexes provided by their cognate helper viruses and host plants for replication, pretending that they are part of the viral genomes. Although satRNAs and satellite viruses do not share significant nucleotide sequence similarity with the helper viruses, the essential cis-acting elements recognized by the replicase complexes must reside on their genomes, acting as the mimicry for the molecular pretenders. By understanding how this molecular mimicry deceives the helper viruses into supporting the satellites, a significant amount of knowledge of the basic requirements and mechanisms for replication of viruses and satellites has been obtained. Here we review the recent advances in understanding the effects of the cis elements at the termini of satRNAs and satellite viruses on their accumulation. Several well-characterized satellite/helper virus systems, representing the non-coding short satRNAs, mRNA-type long satRNAs, circular satRNAs and satellite viruses, are compared and contrasted. It is concluded that different satellites may adopt different strategies to exploit the replication/transcription/translation machineries of their helper viruses, and different mimicries may be implemented by the same molecular pretender for different biological functions.

  7. Molecular mechanisms of isocitrate dehydrogenase 1 (IDH1) mutations identified in tumors: The role of size and hydrophobicity at residue 132 on catalytic efficiency.

    Science.gov (United States)

    Avellaneda Matteo, Diego; Grunseth, Adam J; Gonzalez, Eric R; Anselmo, Stacy L; Kennedy, Madison A; Moman, Precious; Scott, David A; Hoang, An; Sohl, Christal D

    2017-05-12

    Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP + -dependent conversion of isocitrate (ICT) to α-ketoglutarate (αKG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of αKG to d-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit αKG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ∼640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Role of Carboxylate ligands in the Synthesis of AuNPs: Size Control, Molecular Interaction and Catalytic Activity

    KAUST Repository

    Aljohani, Hind Abdullah

    2016-05-22

    then describe the effect of the concentrations and of various type of the stabilizer, and the post-synthesis treatment on gold nanoparticles size. In Chapter 4, we focus on determining the nature of the interactions at molecular level between citrate (and other carboxylate-containing ligands) and AuNP in terms of the mode of coordination at the surface, and the formal oxidation state of Au when interacting with these negatively charged carboxylate ligands (i.e., LX- in the Green formalism). We achieve this by combining very advanced 13C CP/MAS, 23Na MAS and low-temperature SSNMR, high-resolution transmission electron microscopy (HRTEM) and density functional theory (DFT) calculations. A particular emphasis will be based on SS-NMR. In Chapter 5, we study the influence of pretreatment of 1% Au/TiO2 catalysts on the resulting activity in the oxidation of carbon monoxide, the effect of the concentration and the type of the ligands on the catalytic activity. The catalysts were characterized by TPO, XRD, and TEM spectroscopy.

  9. Functional mimicry of carboxypeptidase A by a combination of transition state stabilization and a defined orientation of catalytic moieties in molecularly imprinted polymers.

    Science.gov (United States)

    Liu, Jun-qiu; Wulff, Günter

    2008-06-25

    An artificial model for the natural enzyme carboxypeptidase A has been constructed by molecular imprinting in synthetic polymers. The tetrahedral transition state analogues (TSAs 4 and 5) for the carbonate hydrolysis have been designed as templates to allow incorporation of the main catalytic elements, an amidinium group and a Zn(2+) or Cu(2+) center, in a defined orientation in the transition state imprinted active site. The complexation of the functional monomer and the template in presence of Cu(2+) through stoichiometric noncovalent interaction was established on the basis of (1)H NMR studies and potentiometric titration. The Cu(2+) center was introduced into the imprinted cavity during polymerization or by substitution of Zn(2+) in Zn(2+) imprinted polymers. The direct introduction displayed obvious advantages in promoting catalytic efficiency. With substrates exhibiting a very similar structure to the template, an extraordinarily high enhancement of the rate of catalyzed to uncatalyzed reaction (k(cat)/k(uncat)) of 10(5)-fold was observed. If two amidinium moieties are introduced in proximity to one Cu(2+) center in the imprinted cavity by complexation of the functional monomer 3 with the template 5, the imprinted catalysts exhibited even higher activities and efficiencies for the carbonate hydrolysis with k(cat)/k(uncat) as high as 410,000. These are by far the highest values obtained for molecularly imprinted catalysts, and they are also considerably higher compared to catalytic antibodies. Our kinetic studies and competitive inhibition experiments with the TSA template showed a clear indication of a very efficient imprinting procedure. In addition, this demonstrates the important role of the transition state stabilization during the catalysis of this reaction.

  10. Molecular Mechanisms of Mild and Severe Pneumonia: Insights from RNA Sequencing.

    Science.gov (United States)

    Huang, Sai; Feng, Cong; Chen, Li; Huang, Zhi; Zhou, Xuan; Li, Bei; Wang, Li-Li; Chen, Wei; Lv, Fa-Qin; Li, Tan-Shi

    2017-04-06

    BACKGROUND This study aimed to uncover the molecular mechanisms underlying mild and severe pneumonia by use of mRNA sequencing (RNA-seq). MATERIAL AND METHODS RNA was extracted from the peripheral blood of patients with mild pneumonia, severe pneumonia, and healthy controls. Sequencing was performed on the HiSeq4000 platform. After filtering, clean reads were mapped to the human reference genome hg19. Differentially expressed genes (DEGs) were identified between the control group and the mild or severe group. A transcription factor-gene network was constructed for each group. Biological process (BP) terms enriched by DEGs in the network were analyzed and these genes were also mapped to the Connectivity map to search for small-molecule drugs. RESULTS A total of 199 and 560 DEGs were identified from the mild group and severe group, respectively. A transcription factor-gene network consisting of 215 nodes and another network consisting of 451 nodes were constructed in the mild group and severe group, respectively, and 54 DEGs (e.g., S100A9 and S100A12) were found to be common, with consistent differential expression changes in the 2 groups. Genes in the transcription factor-gene network for the mild group were mainly enriched in 13 BP terms, especially defense and inflammatory response (e.g., S100A8) and spermatogenesis, while the top BP terms enriched by genes in the severe group include response to oxidative stress (CCL5), wound healing, and regulation of cell differentiation (CCL5), and of the cellular protein metabolic process. CONCLUSIONS S100A9 and S100A12 may have a role in the pathogenesis of pneumonia: S100A9 and CXCL1 may contribute solely in mild pneumonia, and CCL5 and CXCL11 may contribute in severe pneumonia.

  11. Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery

    Science.gov (United States)

    Zhou, Jihan; Li, Dong; Wen, Hao; Zheng, Shuquan; Su, Cuicui; Yi, Fan; Wang, Jue; Liang, Zicai; Tang, Tao; Zhou, Demin; Zhang, Li-He; Liang, Dehai; Du, Quan

    2016-03-01

    Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications.

  12. The emerging molecular biology toolbox for the study of long noncoding RNA biology.

    Science.gov (United States)

    Fok, Ezio T; Scholefield, Janine; Fanucchi, Stephanie; Mhlanga, Musa M

    2017-10-01

    Long noncoding RNAs (lncRNAs) have been implicated in many biological processes. However, due to the unique nature of lncRNAs and the consequential difficulties associated with their characterization, there is a growing disparity between the rate at which lncRNAs are being discovered and the assignment of biological function to these transcripts. Here we present a molecular biology toolbox equipped to help dissect aspects of lncRNA biology and reveal functionality. We outline an approach that begins with a broad survey of genome-wide, high-throughput datasets to identify potential lncRNA candidates and then narrow the focus on specific methods that are well suited to interrogate the transcripts of interest more closely. This involves the use of imaging-based strategies to validate these candidates and observe the behaviors of these transcripts at single molecule resolution in individual cells. We also describe the use of gene editing tools and interactome capture techniques to interrogate functionality and infer mechanism, respectively. With the emergence of lncRNAs as important molecules in healthy and diseased cellular function, it remains crucial to deepen our understanding of their biology.

  13. Synthesis of Hβ (core)/SAPO-11 (shell) Composite Molecular Sieve and its Catalytic Performances in the Methylation of Naphthalene with Methanol

    International Nuclear Information System (INIS)

    Wang, Xiaoxiao; Zhao, Liangfu; Guo, Shaoqing

    2013-01-01

    Hβ (core)/SAPO-11 (shell) composite molecular sieve was synthesized by the hydrothermal method in order to combine the advantages of Hβ and SAPO-11 for the methylation of naphthalene with methanol. For comparison, the mechanical mixture was prepared through the blending of Hβ and SAPO-11. The physicochemical properties of Hβ, SAPO-11, the composite and the mechanical mixture were characterized by various characterization methods. The characterization results indicated that Hβ/SAPO-11 composite molecular sieve exhibited a core-shell structure, with the Hβ phase as the core and the SAPO-11 phase as the shell. The pore diameter of the composite was between that of Hβ and SAPO-11. The composite had fewer acid sites than Hβ and mechanical mixture while more acid sites than SAPO-11. The experimental results indicated that the composite exhibited high catalytic performances for the methylation of naphthalene with methanol

  14. Molecular insights into the specific recognition between the RNA binding domain qRRM2 of hnRNP F and G-tract RNA: A molecular dynamics study.

    Science.gov (United States)

    Wang, Lingyun; Yan, Feng

    2017-12-09

    Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Electrochemical characterization of praseodymia doped zircon. Catalytic effect on the electrochemical reduction of molecular oxygen in polar organic solvents

    Energy Technology Data Exchange (ETDEWEB)

    Domenech, Antonio, E-mail: antonio.domenech@uv.es [Departament de Quimica Analitica, Universitat de Valencia, Dr. Moliner, 50, 46100 Burjassot, Valencia (Spain); Montoya, Noemi; Alarcon, Javier [Departament de Quimica Inorganica, Universitat de Valencia, Dr. Moliner, 50, 46100 Burjassot, Valencia (Spain)

    2011-08-01

    Highlights: > Electrochemical characterization of Pr centers in praseodymia-doped zircon. > Study of the catalytic effect on the reduction of peroxide radical anion in nonaqueous solvents. > Assessment of non-uniform distribution of Pr centers in the zircon grains. - Abstract: The voltammetry of microparticles and scanning electrochemical microscopy methodologies are applied to characterize praseodymium centers in praseodymia-doped zircon (Pr{sub x}Zr{sub (1-y)}Si{sub (1-z)}O{sub 4}; y + z = x; 0.02 < x < 0.10) specimens prepared via sol-gel synthetic routes. In contact with aqueous electrolytes, two overlapping Pr-centered cathodic processes, attributable to the Pr (IV) to Pr (III) reduction of Pr centers in different sites are obtained. In water-containing, air-saturated acetone and DMSO solutions as solvent, Pr{sub x}Zr{sub (1-y)}Si{sub (1-z)}O{sub 4} materials produce a significant catalytic effect on the electrochemical reduction of peroxide radical anion electrochemically generated. These electrochemical features denote that most of the Pr centers are originally in its 4+ oxidation state in the parent Pr{sub x}Zr{sub (1-y)}Si{sub (1-z)}O{sub 4} specimens. The variation of the catalytic performance of such specimens with potential scan rate, water concentration and Pr loading suggests that Pr is not uniformly distributed within the zircon grains, being concentrated in the outer region of such grains.

  16. Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, Srirupa; Basler, Christopher F.; Amarasinghe, Gaya K.; Leung, Daisy W.

    2016-08-01

    The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most open reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. A greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures.

  17. Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses.

    Science.gov (United States)

    Chatterjee, Srirupa; Basler, Christopher F; Amarasinghe, Gaya K; Leung, Daisy W

    2016-08-28

    The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most open reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. A greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

  19. Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis)

    Science.gov (United States)

    Zheng, Chao; Zhao, Lei; Wang, Yu; Shen, Jiazhi; Zhang, Yinfei; Jia, Sisi; Li, Yusheng; Ding, Zhaotang

    2015-01-01

    Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants’ growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4℃) and freezing (-5℃) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., ‘Photosynthesis’), GO terms (e.g., ‘response to karrikin’) and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and β-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology. PMID:25901577

  20. Molecular Basis of Transcription-Coupled Pre-mRNA Capping

    NARCIS (Netherlands)

    Martinez-Rucobo, Fuensanta W.; Kohler, Rebecca; van de Waterbeemd, Michiel|info:eu-repo/dai/nl/412537761; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Hemann, Matthias; Herzog, Franz; Stark, Holger; Cramer, Patrick

    2015-01-01

    Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of

  1. Origins of biological function in DNA and RNA hairpin loop motifs from replica exchange molecular dynamics simulation.

    Science.gov (United States)

    Swadling, Jacob B; Ishii, Kunihiko; Tahara, Tahei; Kitao, Akio

    2018-01-31

    Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have remarkably similar chemical structures, but despite this, they play significantly different roles in modern biology. In this article, we explore the possible conformations of DNA and RNA hairpins to better understand the fundamental differences in structure formation and stability. We use large parallel temperature replica exchange molecular dynamics ensembles to sample the full conformational landscape of these hairpin molecules so that we can identify the stable structures formed by the hairpin sequence. Our simulations show RNA adopts a narrower distribution of folded structures compared to DNA at room temperature, which forms both hairpins and many unfolded conformations. RNA is capable of forming twice as many hydrogen bonds than DNA which results in a higher melting temperature. We see that local chemical differences lead to emergent molecular properties such as increased persistence length in RNA that is weakly temperature dependant. These discoveries provide fundamental insight into how RNA forms complex folded tertiary structures which confer enzymatic-like function in ribozymes, whereas DNA retains structural motifs in order to facilitate function such as translation of sequence.

  2. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.

    Directory of Open Access Journals (Sweden)

    Cornelius Schneider

    Full Text Available Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

  3. Molecular pathogenesis of interstitial cystitis based on microRNA expression signature: miR-320 family-regulated molecular pathways and targets.

    Science.gov (United States)

    Arai, Takayuki; Fuse, Miki; Goto, Yusuke; Kaga, Kanya; Kurozumi, Akira; Yamada, Yasutaka; Sugawara, Sho; Okato, Atsushi; Ichikawa, Tomohiko; Yamanishi, Tomonori; Seki, Naohiko

    2018-03-12

    Interstitial cystitis (IC), also known as bladder pain syndrome, is a chronic inflammatory disease that affects the bladder. The symptoms of IC vary, including feeling an urgent need for immediate urination and of needing to urinate often, as well as bladder or pelvic pain. Despite its high incidence, no molecular diagnostic methods are available for IC, and the molecular pathogenesis is unknown. microRNAs (miRNA) can regulate expression of RNA transcripts in cells and aberrant expression of miRNAs is associated with several human diseases. Here, we investigated the molecular pathogenesis of IC based on miRNA expression signatures. RNA sequencing of miRNA levels in IC tissues and comparison with levels in normal bladder tissue and bladder cancer revealed dysregulated expression of 366 miRNAs (203 and 163 down- and upregulated miRNAs, respectively). In particular, miR-320 family miRNAs(miR-320a, miR-320b, miR-320c, miR-320d and miR-320e) had downregulated expression in IC tissues. Genome-wide gene expression analyses and in silico database analyses showed that three transcription factors, E2F-1, E2F-2 and TUB, are regulated by miR-320 family miRNAs. Immunostaining of IC tissues confirmed that these transcription factors are overexpressed in IC tissues. Novel approaches that identify aberrantly expressed miRNA regulatory networks in IC could provide new prognostic markers and therapeutic targets for this disease.

  4. Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNA-seq, RNA interference and irradiation approach

    Science.gov (United States)

    2012-01-01

    Background Planarian stem cells, or neoblasts, drive the almost unlimited regeneration capacities of freshwater planarians. Neoblasts are traditionally described by their morphological features and by the fact that they are the only proliferative cell type in asexual planarians. Therefore, they can be specifically eliminated by irradiation. Irradiation, however, is likely to induce transcriptome-wide changes in gene expression that are not associated with neoblast ablation. This has affected the accurate description of their specific transcriptomic profile. Results We introduce the use of Smed-histone-2B RNA interference (RNAi) for genetic ablation of neoblast cells in Schmidtea mediterranea as an alternative to irradiation. We characterize the rapid, neoblast-specific phenotype induced by Smed-histone-2B RNAi, resulting in neoblast ablation. We compare and triangulate RNA-seq data after using both irradiation and Smed-histone-2B RNAi over a time course as means of neoblast ablation. Our analyses show that Smed-histone-2B RNAi eliminates neoblast gene expression with high specificity and discrimination from gene expression in other cellular compartments. We compile a high confidence list of genes downregulated by both irradiation and Smed-histone-2B RNAi and validate their expression in neoblast cells. Lastly, we analyze the overall expression profile of neoblast cells. Conclusions Our list of neoblast genes parallels their morphological features and is highly enriched for nuclear components, chromatin remodeling factors, RNA splicing factors, RNA granule components and the machinery of cell division. Our data reveal that the regulation of planarian stem cells relies on posttranscriptional regulatory mechanisms and suggest that planarians are an ideal model for this understudied aspect of stem cell biology. PMID:22439894

  5. Non-Watson-Crick basepairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E

    Czech Academy of Sciences Publication Activity Database

    Réblová, K.; Špačková, Naďa; Štefl, R.; Csaszar, K.; Koča, J.; Leontis, N. B.; Šponer, Jiří

    2003-01-01

    Roč. 84, č. 6 (2003), s. 3564-3582 ISSN 0006-3495 R&D Projects: GA MŠk LN00A016 Grant - others:National Institutes of Health(US) 2R15 GM55898; National Science Foundation(US) CHE-9732563 Institutional research plan: CEZ:AV0Z5004920 Keywords : non- Watson -Crick base pairs * ribosomal RNA * Loop E Subject RIV: BO - Biophysics Impact factor: 4.463, year: 2003

  6. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast.

    Science.gov (United States)

    Belew, Ashton T; Advani, Vivek M; Dinman, Jonathan D

    2011-04-01

    Although first discovered in viruses, previous studies have identified operational -1 ribosomal frameshifting (-1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast -1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five -1 RF signals. Ablation of the -1 RF signals or of NMD stabilizes this mRNA, and changes in -1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous -1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that -1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence -1 RF signals in ∼10% of genes in all eukaryotic genomes surveyed suggests that -1 RF is a broadly used post-transcriptional regulator of gene expression.

  7. Valyl-tRNA synthetase gene of Escherichia coli K12: Molecular genetic characterization and homology within a family of aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Heck, J.D. III.

    1988-01-01

    This work reports the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene encoding valyl-tRNA synthetase. The valS gene was subcloned from plasmid pLC26-22 by genetic complementation of a valS ts strain. The DNA region encoding the valS structural gene was determined by in vitro coupled transcription-translation assays. Cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase specific activity twelve-fold. DNA sequences flanking the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both [α- 32 P]labeled and [γ- 32 P]end-labeled in vitro transcription assays. The DNA sequence of the valS gene of Escherichia coli has been determined. Significant similarity at the primary sequence level was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. An extended open reading frame (ORF) encoded on the DNA strand opposite the valS structural gene is described

  8. Molecular markers and mechanisms of stroke: RNA studies of blood in animals and humans

    Science.gov (United States)

    Sharp, Frank R; Jickling, Glen C; Stamova, Boryana; Tian, Yingfang; Zhan, Xinhua; Liu, DaZhi; Kuczynski, Beth; Cox, Christopher D; Ander, Bradley P

    2011-01-01

    Whole genome expression microarrays can be used to study gene expression in blood, which comes in part from leukocytes, immature platelets, and red blood cells. Since these cells are important in the pathogenesis of stroke, RNA provides an index of these cellular responses to stroke. Our studies in rats have shown specific gene expression changes 24 hours after ischemic stroke, hemorrhage, status epilepticus, hypoxia, hypoglycemia, global ischemia, and following brief focal ischemia that simulated transient ischemic attacks in humans. Human studies show gene expression changes following ischemic stroke. These gene profiles predict a second cohort with >90% sensitivity and specificity. Gene profiles for ischemic stroke caused by large-vessel atherosclerosis and cardioembolism have been described that predict a second cohort with >85% sensitivity and specificity. Atherosclerotic genes were associated with clotting, platelets, and monocytes, and cardioembolic genes were associated with inflammation, infection, and neutrophils. These gene profiles predicted the cause of stroke in 58% of cryptogenic patients. These studies will provide diagnostic, prognostic, and therapeutic markers, and will advance our understanding of stroke in humans. New techniques to measure all coding and noncoding RNAs along with alternatively spliced transcripts will markedly advance molecular studies of human stroke. PMID:21505474

  9. 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.

  10. A-minor tertiary interactions in RNA kink-turns. Molecular dynamics and quantum chemical analysis.

    Science.gov (United States)

    Réblová, Kamila; Šponer, Judit E; Špačková, Naďa; Beššeová, Ivana; Šponer, Jiří

    2011-12-01

    The RNA kink-turn is an important recurrent RNA motif, an internal loop with characteristic consensus sequence forming highly conserved three-dimensional structure. Functional arrangement of RNA kink-turns shows a sharp bend in the phosphodiester backbone. Among other signature interactions, kink-turns form A-minor interaction between their two stems. Most kink-turns possess extended A-minor I (A-I) interaction where adenine of the second A•G base pair of the NC-stem interacts with the first canonical pair of the C-stem (i.e., the receptor pair) via trans-sugar-edge/sugar-edge (tSS) and cis-sugar-edge/sugar-edge (cSS) interactions. The remaining kink-turns have less compact A-minor 0 (A-0) interaction with just one tSS contact. We show that kink-turns with A-I in ribosomal X-ray structures keep G═C receptor base pair during evolution while the inverted pair (C═G) is not realized. In contrast, kink-turns with A-0 in the observed structures alternate G═C and C═G base pairs in sequences. We carried out an extended set (~5 μs) of explicit-solvent molecular dynamics simulations of kink-turns to rationalize this structural/evolutionary pattern. The simulations were done using a net-neutral Na(+) cation atmosphere (with ~0.25 M cation concentration) supplemented by simulations with either excess salt KCl atmosphere or inclusion of Mg(2+). The results do not seem to depend on the treatment of ions. The simulations started with X-ray structures of several kink-turns while we tested the response of the simulated system to base substitutions, modest structural perturbations and constraints. The trends seen in the simulations reveal that the A-I/G═C arrangement is preferred over all three other structures. The A-I/C═G triple appears structurally entirely unstable, consistent with the covariation patterns seen during the evolution. The A-0 arrangements tend to shift toward the A-I pattern in simulations, which suggests that formation of the A-0 interaction is

  11. Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

    Science.gov (United States)

    Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

    1982-01-01

    The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

  12. Molecular approaches for forensic cell type identification: On mRNA, miRNA, DNA methylation and microbial markers.

    Science.gov (United States)

    Sijen, Titia

    2015-09-01

    Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Polymerisation of activated RNA in eutectic ice phases

    DEFF Research Database (Denmark)

    Dörr, Mark; Maurer, Sarah Elisabeth; Monnard, Pierre-Alain

    The non enzymatic elongation of RNA oligomers by activated RNA dimers and other oligomers in eutectic ice phases is investigated. Eutectic salt-ice mixtures contain channels of liquid aqueous solutions with a high molecule concentration or brines, which form an environment conductive to spontaneous...... (“cooperative sequences”) or degrading (“parasitic sequences”) the RNA population. These eutectic phases in water-ice are plausible prebiotic micro-environments that should help to overcome the dilution problem in origin of life scenarios. They might have supported the production of libraries....../populations of longer RNA chains rising the potential to produce (auto-)catalytic active molecular species (e.g. ribozymes)....

  14. Atomistic insight into the catalytic mechanism of glycosyltransferases by combined quantum mechanics/molecular mechanics (QM/MM) methods.

    Science.gov (United States)

    Tvaroška, Igor

    2015-02-11

    Glycosyltransferases catalyze the formation of glycosidic bonds by assisting the transfer of a sugar residue from donors to specific acceptor molecules. Although structural and kinetic data have provided insight into mechanistic strategies employed by these enzymes, molecular modeling studies are essential for the understanding of glycosyltransferase catalyzed reactions at the atomistic level. For such modeling, combined quantum mechanics/molecular mechanics (QM/MM) methods have emerged as crucial. These methods allow the modeling of enzymatic reactions by using quantum mechanical methods for the calculation of the electronic structure of the active site models and treating the remaining enzyme environment by faster molecular mechanics methods. Herein, the application of QM/MM methods to glycosyltransferase catalyzed reactions is reviewed, and the insight from modeling of glycosyl transfer into the mechanisms and transition states structures of both inverting and retaining glycosyltransferases are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus.

    Science.gov (United States)

    Villar, Emilie; Klopp, Christophe; Noirot, Céline; Novaes, Evandro; Kirst, Matias; Plomion, Christophe; Gion, Jean-Marc

    2011-11-02

    In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit. Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed. For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance of biomass growth during the

  16. In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis.

    Science.gov (United States)

    Weiss, Victor U; Bliem, Christina; Gösler, Irene; Fedosyuk, Sofiya; Kratzmeier, Martin; Blaas, Dieter; Allmaier, Günter

    2016-06-01

    Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.

  17. Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels

    OpenAIRE

    Sugimoto, Shinya; Arita-Morioka, Ken-ichi; Mizunoe, Yoshimitsu; Yamanaka, Kunitoshi; Ogura, Teru

    2015-01-01

    The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides pr...

  18. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    Science.gov (United States)

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Catalytic NH3 Synthesis using N2 /H2 at Molecular Transition Metal Complexes: Concepts for Lead Structure Determination using Computational Chemistry.

    Science.gov (United States)

    Hölscher, Markus; Leitner, Walter

    2017-09-07

    While industrial NH 3 synthesis based on the Haber-Bosch-process was invented more than a century ago, there is still no molecular catalyst available which reduces N 2 in the reaction system N 2 /H 2 to NH 3 . As the many efforts of experimentally working research groups to develop a molecular catalyst for NH 3 synthesis from N 2 /H 2 have led to a variety of stoichiometric reductions it seems justified to undertake the attempt of systematizing the various approaches of how the N 2 molecule might be reduced to NH 3 with H 2 at a transition metal complex. In this contribution therefore a variety of intuition-based concepts are presented with the intention to show how the problem can be approached. While no claim for completeness is made, these concepts intend to generate a working plan for future research. Beyond this, it is suggested that these concepts should be evaluated with regard to experimental feasibility by checking barrier heights of single reaction steps and also by computation of whole catalytic cycles employing density functional theory (DFT) calculations. This serves as a tool which extends the empirically driven search process and expands it by computed insights which can be used to rationalize the various challenges which must be met. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

    Science.gov (United States)

    Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

    2014-04-01

    The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

  1. Entrapping ribosomes for viral translation: tRNA mimicry as a molecular Trojan horse.

    Science.gov (United States)

    Barends, Sharief; Bink, Hugo H J; van den Worm, Sjoerd H E; Pleij, Cornelis W A; Kraal, Barend

    2003-01-10

    Turnip yellow mosaic virus (TYMV) has a genomic plus-strand RNA with a 5' cap followed by overlapping and different reading frames for the movement protein and polyprotein, while the distal coat protein cistron is translated from a subgenomic RNA. The 3'-untranslated region harbors a tRNA-like structure (TLS) to which a valine moiety can be added and it is indispensable for virus viability. Here, we report about a surprising interaction between TYMV-RNA-programmed ribosomes and 3'-valylated TLS that yields polyprotein with the valine N terminally incorporated by a translation mechanism resistant to regular initiation inhibitors. Disruption of the TLS exclusively abolishes polyprotein synthesis, which can be restored by adding excess TLS in trans. Our observations imply a novel eukaryotic mechanism for internal initiation of mRNA translation.

  2. Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

    Science.gov (United States)

    Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

    2008-11-01

    The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

  3. Zeolites - the relationship between morphostructural parameters and adsorptive/molecular sieving/catalytic properties of porous solids (models and practices)

    International Nuclear Information System (INIS)

    Hartomo, A.J.; Soendoror, N.P.C.

    1989-01-01

    Zeolites receive great attention among researchers today primarily because of their numerous industrial uses and applications, ranging from water treatment to petroleum industries, concerning adsorption, molecular sieving and/or catalysis. Some important progress taking place during the last few years - theoretical and experimental are discussed. In Indonesia, zeolites of natural origin are found in many places including the western and eastern part of Java Island. Works preparing, modifying and characterizing their performance whether by spectroscopic, thermochemical or volumetric methods are presented. Cooperation in R and D is still to be seriously and constantly increased/intensified. (Auth.). 22 refs.; 10 figs.; 2 tabs

  4. Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

    Science.gov (United States)

    Sakajo, S; Nakamura, K; Asahi, T

    1987-06-01

    A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

  5. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  6. Altered MicroRNA Expression Is Associated with Tumor Grade, Molecular Background and Outcome in Childhood Infratentorial Ependymoma.

    Directory of Open Access Journals (Sweden)

    Magdalena Zakrzewska

    Full Text Available Ependymal tumors are the third most common group of brain tumors in children, accounting for about 10% of all primary brain neoplasms. According to the current WHO classification, they comprise four entities with the most frequent ependymoma and anaplastic ependymoma. The most of pediatric tumors are located within the posterior fossa, with a tendency to infiltrate the vital brain structures. This limits surgical resection and poses a considerable clinical problem. Moreover, there are no appropriate outcome prognostic factors besides the extent of surgical resection. Despite definition of molecular subgroups, the majority of childhood ependymomas present a balanced genome, which makes it difficult to establish molecular prognostic factors.The purpose of our study was to explore whether miRNA expression could be used as prognostic markers in pediatric infratentorial ependymomas. We also performed a mRNA expression pattern analysis of NELL2 and LAMA2 genes, with immunohistochemical illustrations of representative cases. The miRNA and mRNA expression was measured in 53 pediatric infratentorial ependymomas using a real-time quantitative PCR.Three miRNAs were shown to efficiently differentiate between grade II and III ependymomas: miR-17-5p, miR-19a-3p, and miR-106b-5p. Survival analysis showed that the probabilities of overall (p = 0.036 and event-free survival (p = 0.002 were reduced with higher than median miRNA expression levels of miR-17-5p. Using multivariate analysis adjusted for patient's age, sex, tumor grade and localization, we showed statistically significant associations with event-free survival (p = 0004 and borderline statistical significance with overall survival (p = 0.057 for miR-17-5p. Correlation analysis of miR-19a, miR-17-5p, miR-106b revealed that their expression levels were significantly correlated with EZH2 expression, suggested marker of PFA ependymomas. Furthermore, lower expression level of LAMA2 mRNA was shown to be

  7. Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

    Science.gov (United States)

    Yu, Lin; Sang, Wen; Wu, Ming-De; Zhang, Jing; Yang, Long; Zhou, Ying-Jun; Chen, Wei-Dong

    2015-01-01

    Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1. PMID:25595766

  8. An RNA Phage Lab: MS2 in Walter Fiers' laboratory of molecular biology in Ghent, from genetic code to gene and genome, 1963-1976.

    Science.gov (United States)

    Pierrel, Jérôme

    2012-01-01

    The importance of viruses as model organisms is well-established in molecular biology and Max Delbrück's phage group set standards in the DNA phage field. In this paper, I argue that RNA phages, discovered in the 1960s, were also instrumental in the making of molecular biology. As part of experimental systems, RNA phages stood for messenger RNA (mRNA), genes and genome. RNA was thought to mediate information transfers between DNA and proteins. Furthermore, RNA was more manageable at the bench than DNA due to the availability of specific RNases, enzymes used as chemical tools to analyse RNA. Finally, RNA phages provided scientists with a pure source of mRNA to investigate the genetic code, genes and even a genome sequence. This paper focuses on Walter Fiers' laboratory at Ghent University (Belgium) and their work on the RNA phage MS2. When setting up his Laboratory of Molecular Biology, Fiers planned a comprehensive study of the virus with a strong emphasis on the issue of structure. In his lab, RNA sequencing, now a little-known technique, evolved gradually from a means to solve the genetic code, to a tool for completing the first genome sequence. Thus, I follow the research pathway of Fiers and his 'RNA phage lab' with their evolving experimental system from 1960 to the late 1970s. This study illuminates two decisive shifts in post-war biology: the emergence of molecular biology as a discipline in the 1960s in Europe and of genomics in the 1990s.

  9. The evolution of catalytic function

    Science.gov (United States)

    Maurel, Marie-Christine; Ricard, Jacques

    2006-03-01

    It is very likely that the main driving force of enzyme evolution is the requirement to improve catalytic and regulatory efficiency which results from the intrinsic performance as well as from the spatial and functional organization of enzymes in living cells. Kinetic co-operativity may occur in simple monomeric proteins if they display “slow” conformational transitions, at the cost of catalytic efficiency. Oligomeric enzymes on the other hand can be both efficient and co-operative. We speculate that the main reason for the emergence of co-operative oligomeric enzymes is the need for catalysts that are both cooperative and efficient. As it is not useful for an enzyme to respond to a change of substrate concentration in a complex kinetic way, the emergence of symmetry has its probable origin in a requirement for “functional simplicity”. In a living cell, enzyme are associated with other macromolecules and membranes. The fine tuning of their activity may also be reached through mutations of the microenvironment. Our hypothesis is that these mutations are related to the vectorial transport of molecules, to achieve the hysteresis loops of enzyme reactions generated by the coupling of reaction and diffusion, through the co-operativity brought about by electric interactions between a charged substrate and a membrane, and last but not least, through oscillations. As the physical origins of these effects are very simple and do not require complex molecular devices, it is very likely that the functional advantage generated by the spatial and functional organization of enzyme molecules within the cell have appeared in prebiotic catalysis or very early during the primeval stages of biological evolution. We shall began this paper by presenting the nature of the probable earliest catalysts in the RNA world.

  10. Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

    Science.gov (United States)

    Maughan, P J; Kolano, B A; Maluszynska, J; Coles, N D; Bonifacio, A; Rojas, J; Coleman, C E; Stevens, M R; Fairbanks, D J; Parkinson, S E; Jellen, E N

    2006-07-01

    The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.

  11. Catalytic cracking with deasphalted oil

    Energy Technology Data Exchange (ETDEWEB)

    Beaton, W.I.; Taylor, J.L.; Peck, L.B.; Mosby, J.F.

    1990-07-10

    This patent describes a catalytic cracking process. It comprises: hydrotreating resid; thereafter deasphalting the hydrotreated resid to produce substantially deasphalted oil; catalytically cracking the hydrotreated oil in a catalytic cracking unit in the presence of a cracking catalyst to produce upgraded oil leaving coked catalyst; and regenerating the coked catalyst in the presence of a combustion-supporting gas comprising excess molecular oxygen in an amount greater than the stoichiometric amount required for substantially completely combusting the coke on the catalyst to carbon dioxide.

  12. Identification and molecular characterization of a naturally occurring RNA virus mutant defective in the initiation of host recovery

    International Nuclear Information System (INIS)

    Xin Hongwu; Ding Shouwei

    2003-01-01

    The host recovery response is characterized by the disappearance of disease symptoms and activation of the RNA silencing virus resistance in the new growth following an initial symptomatic infection. However, it is not clear what triggers the initiation of recovery, which occurs naturally only in some virus-host interactions. Here we report the identification and characterization of a spontaneous mutant of Tobacco streak virus (TSV) that became defective in triggering recovery in tobacco plants. Infectious full-length cDNA clones corresponding to the tripartite RNA genome were constructed from both the wild-type and the nonrecovery mutant of TSV (TSVnr), the first sets of infectious cDNA clones from an Ilarvirus. Genetic and molecular analyses identified an A → G mutation in the TSVnr genome that was sufficient to confer nonrecovery when introduced into TSV. The mutation was located in the intergenic region of RNA 3 upstream of the mapped transcriptional start site of the coat protein mRNA. Intriguingly, induction of recovery by TSV was not accompanied by virus clearance and TSV consistently accumulated to significantly higher levels than TSVnr did even though TSVnr-infected plants displayed severe symptoms throughout the course of infection. Thus, our findings indicate that recovery of host can be initiated by minimal genetic changes in a viral genome and may occur in the absence of virus clearance. Mechanisms possibly involved in the initiation of host recovery are discussed

  13. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    KAUST Repository

    Liu, Chenggang

    2012-04-03

    Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher Kcat and lower Km values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates. © 2012 American Society of Plant Biologists.

  14. Titanium(III, IV-Containing Catalytic Systems for Production of Ultrahigh Molecular Weight Polyethylene Nascent Reactor Powders, Suitable for Solventless Processing—Impact of Oxidation States of Transition Metal

    Directory of Open Access Journals (Sweden)

    Vladislav A. Tuskaev

    2017-12-01

    Full Text Available Catalytic systems containing TiCl4 or TiCl3, THF, organomagnesium (n-Bu2Mg and organoaluminum compounds capable of producing ultrahigh molecular weight polyethylene (UHMWPE were developed. The resulting polymers were characterized by a molecular weight in the range of (1.8–7.8 × 106 Da and desirable morphology, suitable for modern methods of polymer processing—the solvent-free solid-state processing of superhigh-strength (tensile strength up to 2.1 GPa and high-modulus (elastic modulus up to 125 GPa oriented films and film tapes. The impacts of a THF additive, the oxidation state of the titanium atom, and the composition and nature of the nontransition organometallic compounds on the formation of catalytic systems for UHMWPE production were evaluated. The results indicate the suitability of individual titanium chloride tetrahydrofuran complex application for the formation of THF-containing catalytic systems. This approach also results in a significant increase in the system catalytic activity and mechanical properties of UHMWPE. The catalysts based on Ti(III were inferior to systems containing Ti(IV in productivity but were markedly superior in the mechanical properties of UHMWPE.

  15. Molecular cloning and characterization of a novel mRNA present in the squid giant axon.

    Science.gov (United States)

    Chun, J T; Gioio, A E; Crispino, M; Eyman, M; Giuditta, A; Kaplan, B B

    1997-07-15

    Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.

  16. Molecular Modeling of the Catalytic Domain of CyaA Deepened the Knowledge of Its Functional Dynamics

    Directory of Open Access Journals (Sweden)

    Thérèse E Malliavin

    2017-06-01

    Full Text Available Although CyaA has been studied for over three decades and revealed itself to be a very good prototype for developing various biotechnological applications, only a little is known about its functional dynamics and about the conformational landscape of this protein. Molecular dynamics simulations helped to clarify the view on these points in the following way. First, the model of interaction between AC and calmodulin (CaM has evolved from an interaction centered on the surface between C-CaM hydrophobic patch and the α helix H of AC, to a more balanced view, in which the C-terminal tail of AC along with the C-CaM Calcium loops play an important role. This role has been confirmed by the reduction of the affinity of AC for calmodulin in the presence of R338, D360 and N347 mutations. In addition, enhanced sampling studies have permitted to propose a representation of the conformational space for the isolated AC. It remains to refine this representation using structural low resolution information measured on the inactive state of AC. Finally, due to a virtual screening study on another adenyl cyclase from Bacillus anthracis, weak inhibitors of AC have been discovered.

  17. A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels.

    Science.gov (United States)

    Velázquez, Encarna; Rivas, Raúl; del Villar, María; Valverde, Angel; Peix, Alvaro; Mateos, Pedro F; Velázquez, Enrique; Martínez-Molina, Eustoquio

    2006-05-01

    Low-molecular-weight (LMW) RNA profiles, which include ribosomal and transfer RNA molecules with similar small sizes, are molecular signatures of microorganisms with a great potential in microbial identification. The greatest resolution of these profiles was achieved by staircase electrophoresis in sequencing gels. Nevertheless, this technique is difficult to use because it takes 7 h, the gels have large sizes and it is necessary to heat the system and to recycle the buffer to maintain the denaturing conditions and avoid smile effects. Most available sequencing slabs have no internal temperature control or homogenizing devices, which by contrast are present in some newly designed non-sequencing slabs. Nevertheless, these slabs present two important problems for separating LMW RNA molecules, the size of gels is only 20 cm (instead of 40 cm) and the maximum voltage that can be reached is only 840 V (instead 2400 V). Staircase electrophoresis follows a model in which the external polarization is incrementally modified with a constant time step value. In the present work, we experimentally confirmed that by reducing the time step and increasing the total number of steps a suitable resolution is achieved. Under these conditions, despite the smaller size of the gels and the lower values of the electric field, the intensity reaches higher values than in sequencing gels and the LMW RNA profiles are correctly separated in 5 h. The resolution of these profiles obtained in non-sequencing gels is similar to that obtained in sequencing ones facilitating the analysis of large populations of microorganisms in any laboratory.

  18. Detection and molecular characterization of double-stranded RNA viruses in Philippine Trichomonas vaginalis isolates.

    Science.gov (United States)

    Rivera, Windell L; Justo, Christine Aubrey C; Relucio-San Diego, Mary Ann Cielo V; Loyola, Lorenz M

    2017-10-01

    The flagellated protozoon Trichomonas vaginalis that parasitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) viruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Trichomonasvirus of the family Totiviridae. Four species, formally recognized by the International Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by pairwise comparisons of the sequences of genes coding for major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the complimentary DNA of target virus genes coding for CP and RdRp. Sequence analyses confirmed the identity of the TVV isolates from T. vaginalis cultures. A total of 35 dsRNA viruses were identified from 18 (19%) T. vaginalis isolates. Multiple TVV species were observed in six of the 18 T. vaginalis cultures. Phylogenetic analyses show monophyly in TVV1 and TVV2 whereas TVV3 and TVV4 appear paraphyletic. The phylogeny of Philippine Trichomonasvirus reflects the global distribution of its host. This is the first study in the Philippines and one of the two reports worldwide to detect the four TVVs and their concurrent infection in a single T. vaginalis isolate. Copyright © 2015. Published by Elsevier B.V.

  19. Structure, dynamics, and elasticity of free 16S rRNA helix 44 studied by molecular dynamics simulations

    Czech Academy of Sciences Publication Activity Database

    Réblová, Kamila; Lankaš, F.; Rázga, Filip; Krasovská, Maryna V.; Koča, J.; Šponer, Jiří

    2006-01-01

    Roč. 82, č. 5 (2006), s. 504-520 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA203/05/0388; GA ČR(CZ) GA203/05/0009; GA ČR(CZ) GD204/03/H016; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040507 Keywords : molecular dynamics * helix 44 * 16S rRNA Subject RIV: BO - Biophysics Impact factor: 2.480, year: 2006

  20. A tumor mRNA-mediated bi-photosensitizer molecular beacon as an efficient imaging and photosensitizing agent.

    Science.gov (United States)

    Gao, Yuan; Qiao, Guangming; Zhuo, Linhai; Li, Na; Liu, Ying; Tang, Bo

    2011-05-14

    A bi-photosensitizer molecular beacon (bi-PS MB) is assembled by coupling two PS molecules, respectively, onto the opposite ends of a single MB. The MB can be triggered by a tumor marker-survivin mRNA. Fluorescence and cytotoxic (1)O(2) generation occur effectively in breast cancer cells, but not in normal cells. Compared with a single-PS MB, a bi-PS MB exhibits much-enhanced properties in the signal-to-background ratio and (1)O(2) generation simultaneously. © The Royal Society of Chemistry 2011

  1. Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

    Directory of Open Access Journals (Sweden)

    Jerome Deval

    2015-06-01

    Full Text Available Respiratory syncytial virus (RSV causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV, whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

  2. Catalytic devices

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ming; Zhang, Xiang

    2018-01-23

    This disclosure provides systems, methods, and apparatus related to catalytic devices. In one aspect, a device includes a substrate, an electrically insulating layer disposed on the substrate, a layer of material disposed on the electrically insulating layer, and a catalyst disposed on the layer of material. The substrate comprises an electrically conductive material. The substrate and the layer of material are electrically coupled to one another and configured to have a voltage applied across them.

  3. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

    Science.gov (United States)

    Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

    2013-07-01

    Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

  4. The human exosome and PNPase : molecular machines for controlled RNA degradation

    NARCIS (Netherlands)

    Raijmakers, Reinout

    2004-01-01

    The human exosome is a multi-protein complex present in all eukaryotic cells, which is involved in the processing and degradation of a large variety of RNA molecules. In patients suffering from an overlap syndrome of the autoimmune diseases polymyositis (PM) and scleroderma (Scl), autoantibodies are

  5. Exploring the molecular mechanism of action between drug and RNA polymerase based on partially-resolved spatial structures.

    Science.gov (United States)

    Sheng, Yajun; Zhang, Chen; Qiu, Xingye; Zheng, Wei; Ruan, Jishou; Shao, Yiming

    2013-12-01

    The RNA polymerase of Influenza A virus (IAV), which is comprised of three units PA, PB1 and PB2, is involved in transcription and replication of the influenza virus. In order to develop effective treatment for IAV, researchers have focused on designing drugs targeting IAV polymerase. Currently, crystal structures of the IAV polymerase PA-PB1, PB1-PB2 complexes and the PA subunit have been obtained by several groups, providing useful information regarding potential binding sites in drug design. However, to gain full understanding of the molecular mechanism of IAV polymerase in viral transcription and replication, thereby aiding drug development, a complete atomistic structure of the RNA polymerase is required. In this paper, we employed computer-aided drug design tools to describe the complete structure of the RNA polymerase and proposed a putative mechanism. We predict that the combination of Vancomycin and Oseltamivir will be an effective drug to universally treat IAVs with no resultant drug resistance if this putative mechanism is true.

  6. Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase.

    Science.gov (United States)

    Widderich, Nils; Pittelkow, Marco; Höppner, Astrid; Mulnaes, Daniel; Buckel, Wolfgang; Gohlke, Holger; Smits, Sander H J; Bremer, Erhard

    2014-02-06

    Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family. © 2013.

  7. MicroRNA Expression Profiling Identifies Molecular Diagnostic Signatures for Anaplastic Large Cell Lymphoma

    DEFF Research Database (Denmark)

    Liu, Cuiling; Iqbal, Javeed; Teruya-Feldstein, Julie

    2013-01-01

    Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9......) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway....... angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated...

  8. Ternary complexes of folate-PEG-appended dendrimer (G4)/α-cyclodextrin conjugate, siRNA and low-molecular-weight polysaccharide sacran as a novel tumor-selective siRNA delivery system.

    Science.gov (United States)

    Ohyama, Ayumu; Higashi, Taishi; Motoyama, Keiichi; Arima, Hidetoshi

    2017-06-01

    We previously developed a tumor-selective siRNA carrier by preparing polyamidoamine dendrimer (generation 4, G4) conjugates with α-cyclodextrin and folate-polyethylene glycol (Fol-PαC (G4)). In the present study, we developed ternary complexes of Fol-PαC (G4)/siRNA with low-molecular-weight-sacrans to achieve more effective siRNA transfer activity. Among the different molecular-weight sacrans, i.e. sacran 100, 1000 and 10,000 (MW 44,889Da, 943,692Da and 1,488,281Da, respectively), sacran 100 significantly increased the cellular uptake and the RNAi effects of Fol-PαC (G4)/siRNA binary complex with negligible cytotoxicity in KB cells (folate receptor-α positive cells). In addition, the ζ-potential and particle size of Fol-PαC (G4)/siRNA complex were decreased by the ternary complexation with sacran 100. Importantly, the in vivo RNAi effect of the ternary complex after the intravenous administration to tumor-bearing BALB/c mice was significantly higher than that of the binary complex. In conclusion, Fol-PαC (G4)/siRNA/sacran 100 ternary complex has a potential as a novel tumor-selective siRNA delivery system. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Catalytic oxidations with molecular oxygen

    NARCIS (Netherlands)

    Wentzel, B.B.

    2001-01-01

    How to make spearmint from oranges? This was the original idea behind this research project. The flavors and fragrances industry has access to a large supply of limonene, which is extracted from citrus fruit skins. A valuable product for the industry is carvone, which is chemically similar to

  10. Noncanonical structures and their thermodynamics of DNA and RNA under molecular crowding: beyond the Watson-Crick double helix.

    Science.gov (United States)

    Sugimoto, Naoki

    2014-01-01

    How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described. © 2014 Elsevier Inc. All rights reserved.

  11. Atomistic details of the molecular recognition of DNA-RNA hybrid ...

    Indian Academy of Sciences (India)

    transferase (NT) superfamily and is a prototypical member of a large family of enzymes that use two-metal ion (Mg2+ or Mn2+) catalysis to cleave nucleic acids. Long timescale molecular dynamics simulations have been performed on the ...

  12. An RNA molecular switch: Intrinsic flexibility of 23S rRNA helices 40 and 68 5’-UAA/5’-GAN internal loops studied by molecular dynamics methods

    Czech Academy of Sciences Publication Activity Database

    Réblová, Kamila; Střelcová, Z.; Kulhánek, P.; Beššeová, Ivana; Mathews, D.H.; Van Nostrand, K.; Yildirim, I.; Turner, D.H.; Šponer, Jiří

    2010-01-01

    Roč. 6, č. 3 (2010), s. 910-929 ISSN 1549-9618 R&D Projects: GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) IAA400040802; GA AV ČR(CZ) KJB400040901 Grant - others:GA MŠk(CZ) LC06030; GA ČR(CZ) GD203/09/H046; GA ČR(CZ) GA203/09/1476 Program:LC; GD; GA Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA * internal loop * molecular dynamics Subject RIV: AQ - Safety, Health Protection, Human - Machine Impact factor: 5.138, year: 2010

  13. Molecular basis for specific viral RNA recognition and 2'-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5).

    Science.gov (United States)

    Zhao, Yongqian; Soh, Tingjin Sherryl; Lim, Siew Pheng; Chung, Ka Yan; Swaminathan, Kunchithapadam; Vasudevan, Subhash G; Shi, Pei-Yong; Lescar, Julien; Luo, Dahai

    2015-12-01

    Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5' exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5' RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5' RNA cap: G0pppAG-RNA → (m7)G0pppAG-RNA ("cap-0")→(m7)G0pppAm2'-O-G-RNA ("cap-1"). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5'-(m7)G0pppA1G2U3U4G5U6U7-3'), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2'-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.

  14. Multivariate models from RNA-Seq SNVs yield candidate molecular targets for biomarker discovery: SNV-DA.

    Science.gov (United States)

    Paul, Matt R; Levitt, Nicholas P; Moore, David E; Watson, Patricia M; Wilson, Robert C; Denlinger, Chadrick E; Watson, Dennis K; Anderson, Paul E

    2016-03-31

    It has recently been shown that significant and accurate single nucleotide variants (SNVs) can be reliably called from RNA-Seq data. These may provide another source of features for multivariate predictive modeling of disease phenotype for the prioritization of candidate biomarkers. The continuous nature of SNV allele fraction features allows the concurrent investigation of several genomic phenomena, including allele specific expression, clonal expansion and/or deletion, and copy number variation. The proposed software pipeline and package, SNV Discriminant Analysis (SNV-DA), was applied on two RNA-Seq datasets with varying sample sizes sequenced at different depths: a dataset containing primary tumors from twenty patients with different disease outcomes in lung adenocarcinoma and a larger dataset of primary tumors representing two major breast cancer subtypes, estrogen receptor positive and triple negative. Predictive models were generated using the machine learning algorithm, sparse projections to latent structures discriminant analysis. Training sets composed of RNA-Seq SNV features limited to genomic regions of origin (e.g. exonic or intronic) and/or RNA-editing sites were shown to produce models with accurate predictive performances, were discriminant towards true label groupings, and were able to produce SNV rankings significantly different from than univariate tests. Furthermore, the utility of the proposed methodology is supported by its comparable performance to traditional models as well as the enrichment of selected SNVs located in genes previously associated with cancer and genes showing allele-specific expression. As proof of concept, we highlight the discovery of a previously unannotated intergenic locus that is associated with epigenetic regulatory marks in cancer and whose significant allele-specific expression is correlated with ER+ status; hereafter named ER+ associated hotspot (ERPAHS). The use of models from RNA-Seq SNVs to identify and

  15. RNA sequencing of Populus x canadensis roots identifies key molecular mechanisms underlying physiological adaption to excess zinc.

    Directory of Open Access Journals (Sweden)

    Andrea Ariani

    Full Text Available Populus x canadensis clone I-214 exhibits a general indicator phenotype in response to excess Zn, and a higher metal uptake in roots than in shoots with a reduced translocation to aerial parts under hydroponic conditions. This physiological adaptation seems mainly regulated by roots, although the molecular mechanisms that underlie these processes are still poorly understood. Here, differential expression analysis using RNA-sequencing technology was used to identify the molecular mechanisms involved in the response to excess Zn in root. In order to maximize specificity of detection of differentially expressed (DE genes, we consider the intersection of genes identified by three distinct statistical approaches (61 up- and 19 down-regulated and validate them by RT-qPCR, yielding an agreement of 93% between the two experimental techniques. Gene Ontology (GO terms related to oxidation-reduction processes, transport and cellular iron ion homeostasis were enriched among DE genes, highlighting the importance of metal homeostasis in adaptation to excess Zn by P. x canadensis clone I-214. We identified the up-regulation of two Populus metal transporters (ZIP2 and NRAMP1 probably involved in metal uptake, and the down-regulation of a NAS4 gene involved in metal translocation. We identified also four Fe-homeostasis transcription factors (two bHLH38 genes, FIT and BTS that were differentially expressed, probably for reducing Zn-induced Fe-deficiency. In particular, we suggest that the down-regulation of FIT transcription factor could be a mechanism to cope with Zn-induced Fe-deficiency in Populus. These results provide insight into the molecular mechanisms involved in adaption to excess Zn in Populus spp., but could also constitute a starting point for the identification and characterization of molecular markers or biotechnological targets for possible improvement of phytoremediation performances of poplar trees.

  16. Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Rommie E Amaro

    2007-11-01

    Full Text Available RNA editing ligase 1 (TbREL1 is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

  17. MicroRNA-15b Modulates Molecular Mediators of Blood Induced Arthropathy in Hemophilia Mice

    Directory of Open Access Journals (Sweden)

    Dwaipayan Sen

    2016-04-01

    Full Text Available The development of arthropathy is a major co-morbidity in patients with hemophilia. The present study was designed to study the role of a microRNA biomarker (miR-15b in the development of joint disease. To investigate the expression profile of miR-15b during the development of arthropathy, we first isolated and studied small RNA from the acute and chronic hemarthrosis model of hemophilia A mice. We observed that miR-15b was consistently repressed (~1- to 4-fold from the onset of joint bleeding (1, 3, 7 and 24 h until six bleeding episodes (up to 90 days. To test if reconstitution of miR-15b modulates biomarkers of joint damage in a chronic hemarthrosis model, we administered an adeno-associated virus (AAV 5-miR-15b vector intra-articularly alone or in combination with systemic administration of AAV2-factor VIII. miR-15b overexpression downregulated markers of angiogenesis and hypoxia (vascular epithelial growth factor α (VEGF-α and hypoxia inducing factor 2α (HIF-2α, ~70% and ~34%, respectively in the affected joints. In addition, the co-administration of miR-15b and factor VIII vectors reduced the levels of the chondrodegenerative matrix-metalloproteinases (MMPs 1, 3, 9 and 14 (~14% to 60% in the injured joints. These data demonstrate for the first time the role of a miR-15b in the development of hemophilic arthropathy and has implications in development of miR based therapies for joint disease.

  18. In vitro and in vivo siRNA delivery to hepatocyte utilizing ternary complexation of lactosylated dendrimer/cyclodextrin conjugates, siRNA and low-molecular-weight sacran.

    Science.gov (United States)

    Hayashi, Yuya; Higashi, Taishi; Motoyama, Keiichi; Jono, Hirofumi; Ando, Yukio; Arima, Hidetoshi

    2018-02-01

    In this study, we newly developed the ternary complexes consisting of lactosylated dendrimer (generation 3)/α-cyclodextrin conjugate (Lac-α-CDE), siRNA and the anionic polysaccharide sacrans, and evaluated their utility as siRNA transfer carriers. Three kinds of the low-molecular-weight sacrans, i.e. sacran (100) (Mw 44,889Da), sacran (1000) (Mw 943,692Da) and sacran (10,000) (Mw 1,488,281Da) were used. Lac-α-CDE/siRNA/sacran ternary complexes were prepared by adding the low-molecular-weight sacrans to the Lac-α-CDE/siRNA binary complex solution. Cellular uptake of the ternary complex with sacran (100) was higher than that of the binary complex or the other ternary complexes with sacran (1000) and sacran (10,000) in HepG2 cells. Additionally, the ternary complex possessed high serum resistance and endosomal escaping ability in HepG2 cells. High liver levels of siRNA and Lac-α-CDE were observed after the intravenous administration of the ternary complex rather than that of the binary complex. Moreover, intravenous administration of the ternary complex (siRNA 5mg/kg) induced the significant RNAi effect in the liver of mice with negligible change of blood chemistry values. Therefore, a ternary complexation of the Lac-α-CDE/siRNA binary complex with sacran is useful as a hepatocyte-specific siRNA delivery system. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Molecular characterization of the full-length 23S and 5S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis.

    Science.gov (United States)

    Tazumi, Akihiro; Saito, Satoru; Sekizuka, Tsuyoshi; Murayama, Ohoshi; Takamiya, Shinzaburo; Moore, John E; Millar, B Cherie; Matsuda, Motoo

    2007-08-01

    An approximately 4.2 kbp region encoding 23S and 5S rRNA genes was identified when recombinant plasmid DNAs from two genomic DNA libraries and an inverse PCR product of Taylorella asinigenitalis UK-1 isolate were analyzed. Full-length genes of 23S rRNA (3,225 bp) and 5S rRNA (117 bp) of T. asinigenitalis are described. The present sequence analysis identified a non-coding hypothetically intrinsic transcription terminator region downstream of the 5S rRNA gene. The sequence, however, downstream of the 5S rRNA gene did not show any distal tRNA genes. Surprisingly, an intervening sequence (IVS) of 270 bp in length, including two specific tandem repeat units of 80 bp and one partial unit of 48 bp with unknown functions was identified in the first quarter of the 23S rRNA gene sequence. A second IVS of 70 bp in length was also identified in the central region of the 23S rRNA gene. In addition, by using PCR and sequencing procedures, two T. asinigenitalis isolates, UK-1 and UK-2, carried multiple IVSs in the first quarter and central regions. Moreover, the 23S rRNA fragmentation occurred in the UK-1 isolate. A phylogenetic analysis was first carried out based on the 23S rRNA sequence data from T. asinigenitalis UK-1 and 13 other beta-Proteobacteria. This is the first report of IVSs in the 23S rRNA gene from the beta-Proteobacteria.

  20. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery

    Science.gov (United States)

    Lee, Hyukjin; Lytton-Jean, Abigail K. R.; Chen, Yi; Love, Kevin T.; Park, Angela I.; Karagiannis, Emmanouil D.; Sehgal, Alfica; Querbes, William; Zurenko, Christopher S.; Jayaraman, Muthusamy; Peng, Chang G.; Charisse, Klaus; Borodovsky, Anna; Manoharan, Muthiah; Donahoe, Jessica S.; Truelove, Jessica; Nahrendorf, Matthias; Langer, Robert; Anderson, Daniel G.

    2012-06-01

    Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ~ 24.2 min) than the parent siRNA (t1/2 ~ 6 min).

  1. Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington

    Science.gov (United States)

    Purcell, Maureen; Thompson, Rachel L.; Evered, Joy; Kerwin, John; Meyers, Ted R.; Stewart, Bruce; Winton, James

    2018-01-01

    This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.

  2. Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family.

    Science.gov (United States)

    Shiba, K; Motegi, H; Yoshida, M; Noda, T

    1998-11-15

    We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.

  3. Molecular characterization of a bipartite double-stranded RNA virus and its satellite-like RNA co-infecting the phytopathogenic fungus Sclerotinia sclerotiorum

    Directory of Open Access Journals (Sweden)

    Lijiang eLiu

    2015-05-01

    Full Text Available A variety of mycoviruses have been found in Sclerotinia sclerotiorum. In this study, we report a novel mycovirus Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1 that was originally isolated from the hypovirulent strain SCH941 of S. sclerotiorum. SsBRV1 has rigid spherical virions that are ~38 nm in diameter, and three dsRNA segments (dsRNA1, 2 and 3 with lengths of 6.4, 6.0 and 1.7 kbp, respectively were packaged in the virions. dsRNA1 encodes a cap-pol fusion protein, and dsRNA2 encodes a polyprotein with unknown functions but contributes to the formation of virus particles. The dsRNA3 is dispensable and may be a satellite-like RNA (SatlRNA of SsBRV1. Although phylogenetic analysis of the RdRp domain demonstrated that SsBRV1 is related to Botrytis porri RNA virus 1 (BpRV1 and Ustilago maydis dsRNA virus-H1 (UmV-H1, the structure proteins of SsBRV1 do not have any significant sequence similarities with other known viral proteins with the exception of those of BpRV1. SsBRV1 carrying dsRNA3 seems to have no obvious effects on the colony morphology, but can significantly reduce the growth rate and virulence of S. sclerotiorum. Notably, a growth hormone receptor binding domain (GHBP, Pfam12772 is detected in ORF2-encoded protein of SsBRV1, which have not been reported in any other viruses. These findings provide new insights into the virus taxonomy, virus evolution and the interactions between SsBRV1 and the fungal hosts.

  4. Separation of mouse testis cells on a Celsep (TM) apparatus and their usefulness as a source of high molecular weight DNA or RNA

    Science.gov (United States)

    Wolgemuth, D. J.; Gizang-Ginsberg, E.; Engelmyer, E.; Gavin, B. J.; Ponzetto, C.

    1985-01-01

    The use of a self-contained unit-gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep (TM), maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.

  5. Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis.

    Science.gov (United States)

    Tazumi, A; Sekizuka, T; Moore, J E; Millar, B C; Taneike, I; Matsuda, M

    2008-01-01

    Using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) of the 23S rRNA gene sequences of the genus Taylorella, none of the three representative T. equigenitalis strains NCTC11184(T), Kentucky 188 and EQ59 was shown to contain any IVSs in the first quarter region. In the central region, all three strains possessed one approximately 70 bp IVS (TeIVS2) different from any IVSs found in T. asinigenitalis. The predicted secondary structure model of the IVSs contained stem and loop structures. The central region of the IVS-stem structure contains an identical double-stranded consensus 15-bp sequence. The purified RNA fraction from the three strains contained 16S and 4-5S RNA species but no 23S rRNA species. Thus, the primary 23S rRNA transcripts from the three strains would be cleaved into approximately 1.2- and 1.6-kb rRNA fragments and approximately 70-bp IVS. In addition, 16 other T. equigenitalis isolates were found to carry a similar 70-bp IVS in the central region and to produce fragmented 23S rRNA.

  6. Molecular characterization of the non-coding promoter and leader regions and full-length 16S ribosomal RNA (rRNA) gene of Taylorella asinigenitalis.

    Science.gov (United States)

    Tazumi, A; Saito, S; Sekizuka, T; Murayama, O; Moore, J E; Millar, B C; Matsuda, M

    2007-06-01

    The 3,339 base pair (bp) sequences encoding a putative open reading frame (ORF), non-coding promoter and leader regions (approximately 320 bp), full-length 16S ribosomal RNA (rRNA) gene (approximate 1,540 bp) and part of the 16S-23S rDNA internal spacer region (ISR) were determined from genome DNA libraries of the Taylorella asinigenitalis (UK-1) isolate. The non-coding promoter and leader regions included antiterminators (boxB, boxA and boxC) immediately upstream of the 16S rRNA gene sequence. An approximately 680 bp region upstream of the non-coding promoter region appears to contain a putative ORF with high sequence similarity to GTP cyclohydrolase I. In addition, a typical order of intercistronic tRNA genes with the 48 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in a part of the 16S-23S rDNA ISR. The antiterminators of boxB and boxA were also identified in the ISR.A phylogenetic analysis based on the 16S rRNA gene sequence information clearly demonstrated that the five T. asinigenitalis isolates formed a cluster together with the three T. equigenitalis strains, more similar to Pelistega europaea than the other beta-Proteobacteria from the 13 species of 11 genera.

  7. SOX2 and OCT4 mRNA-Expressing Cells, Detected by Molecular Beacons, Localize to the Center of Neurospheres during Differentiation

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba; Dufva, Martin

    2013-01-01

    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......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...

  8. microRNA-mediated R gene regulation: molecular scabbards for double-edged swords.

    Science.gov (United States)

    Deng, Yingtian; Liu, Minglei; Li, Xiaofei; Li, Feng

    2018-02-01

    Plant resistance (R) proteins are immune receptors that recognize pathogen effectors and trigger rapid defense responses, namely effector-triggered immunity. R protein-mediated pathogen resistance is usually race specific. During plant-pathogen coevolution, plant genomes accumulated large numbers of R genes. Even though plant R genes provide important natural resources for breeding disease-resistant crops, their presence in the plant genome comes at a cost. Misregulation of R genes leads to developmental defects, such as stunted growth and reduced fertility. In the past decade, many microRNAs (miRNAs) have been identified to target various R genes in plant genomes. miRNAs reduce R gene levels under normal conditions and allow induction of R gene expression under various stresses. For these reasons, we consider R genes to be double-edged "swords" and miRNAs as molecular "scabbards". In the present review, we summarize the contributions and potential problems of these "swords" and discuss the features and production of the "scabbards", as well as the mechanisms used to pull the "sword" from the "scabbard" when needed.

  9. Molecular docking and molecular dynamics simulation studies on Thermus thermophilus leucyl-tRNA synthetase complexed with different amino acids and pre-transfer editing substrates

    Directory of Open Access Journals (Sweden)

    Rayevsky A. V.

    2016-02-01

    Full Text Available Aim. To investigate the structural bases for the amino acid selectivity of the Thermus thermophilus leucyl-tRNA synthetase (LeuRSTT aminoacylation site and to disclose the binding pattern of pre-transfer editing substrates. Methods. Eight amino acids proposed as semi-cognate substrates for aminoacylation and eight aminoacyl-adenylates (formed from AMP and eight amino acids were prepared in zwitterions form. The protein structure with a co-crystallized substrate in the aminoacylation site [PDBID: 1OBH] was taken from RCSB. Docking settings and evaluation of substrate efficiency were followed by twofold docking function analysis for each conformation with Gold CCDC. The molecular dynamics simulation was performed using Gromacs. The procedures of relaxation and binding study were separated in two different subsequent simulations for 50ns and 5ns. Results. The evaluation of substrate efficiency for 8 amino acids by twofold docking function analysis, based on score values,has shown that the ligands of LeuRSTT can be positioned in the following order: Leu>Nva>Hcy>Nle>Met>Cys>Ile >Val. MD simulation has revealed lower electrostatic interactions of isoleucine with the active site of the enzyme compared with those for norvaline and leucine. In the case of aminoacyl-adenylates no significant differences were found based on score values for both GoldScore and Asp functions. Molecular dynamics of leucyl-, isoleucyl- and norvalyl-adenylates showed that the most stable and conformationally favorable is leucine, then follow norvaline and isoleucine. It has been also found that the TYR43 of the active site covers carboxyl group of leucine and norvaline like a shield and deflected towards isoleucine, allowing water molecules to come closer. Conclusions. In this study we revealed some structural basis for screening unfavorable substrates by shape, size and flexibility of a radical. The results obtained for different amino acids by molecular docking and MD studies

  10. Interstitial contacts in an RNA-dependent RNA polymerase lattice

    Science.gov (United States)

    Tellez, Andres B.; Wang, Jing; Tanner, Elizabeth J.; Spagnolo, Jeannie F.; Kirkegaard, Karla; Bullitt, Esther

    2011-01-01

    Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the ‘thumb’ domain of one polymerase and the back of the ‘palm’ domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, a hybrid approach of both electron microscopic and biochemical evaluation of wild-type and mutant viral polymerases was used to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality, or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should faciliate antiviral drug design and provide a precedent for other positive-strand RNA viruses. PMID:21839092

  11. Catalytic activity of superconducting ceramics of Y-Ba-Cu-O type in reaction of H-D exchange of molecular hydrogen

    International Nuclear Information System (INIS)

    Parbuzin, V.S.; Gul'yants, V.V.

    1989-01-01

    Catalytic activity of high-temperature superconducting oxide ceramics of Y-Ba-Cu-O type in reaction of deuterium-hydrogen exchange was investigated under chromatographic conditions. Rate constants of the reaction and activation energy of the process, equal to 58 ± 2 kJ/mol, were determined in 350 - 450 K range and at 18250Pa hydrogen pressure. Assumption about applicability of Bonhoeffer-Farkas mechanism with adsorption on copper atoms, adjoinig oxygen vacancies, was made

  12. Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

    Science.gov (United States)

    Li, Chia-Lung; Yang, Wei-Zen; Shi, Zhonghao; Yuan, Hanna S

    2018-02-13

    Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1 to SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I-U and U-I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca 2+ -dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I-U and U-I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca 2+ -binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  13. RNA Polymerase II Second Largest Subunit Molecular Identification of Boletus griseipurpureus Corner From Thailand and Antibacterial Activity of Basidiocarp Extracts.

    Science.gov (United States)

    Aung-Aud-Chariya, Amornrat; Bangrak, Phuwadol; Lumyong, Saisamorn; Phupong, Worrapong; Aggangan, Nelly Siababa; Kamlangdee, Niyom

    2015-03-01

    Boletus griseipurpureus Corner, an edible mushroom, is a putative ectomycorrhizal fungus. Currently, the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties. The purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities. Basidiocarps were collected from 2 north-eastern provinces, Roi Et and Ubon Ratchathani, and from 2 southern provinces, Songkhla and Surat Thani, in Thailand. Genomic DNA was extracted and molecular structure was examined using the RNA polymerase II (RPB2) analysis. Antibacterial activities of basidiocarp extracts were conducted with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523 and methicillin-resistant Staphylococcus aureus (MRSA) 189 using the agar-well diffusion method. All the samples collected for this study constituted a monophyletic clade, which was closely related with the Boletus group of polypore fungi. For the antibacterial study, it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract. Basidomes collected from four locations in Thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria. The health benefits of this edible species should be evaluated further.

  14. Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

    Science.gov (United States)

    Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M; Hoshika, Shuichi; Frye, Carole B; Benner, Steven A

    2015-06-15

    Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

    Science.gov (United States)

    Gritsun, Dmitri J; Jones, Ian M; Gould, Ernest A; Gritsun, Tamara S

    2014-01-01

    RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

  16. Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

    Directory of Open Access Journals (Sweden)

    Dmitri J Gritsun

    Full Text Available RNA secondary structures in the 3'untranslated regions (3'UTR of the viruses of the family Flaviviridae, previously identified as essential (promoters or beneficial (enhancers for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV. RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

  17. Molecular Archaeology of Flaviviridae Untranslated Regions: Duplicated RNA Structures in the Replication Enhancer of Flaviviruses and Pestiviruses Emerged via Convergent Evolution

    Science.gov (United States)

    Gritsun, Dmitri J.; Jones, Ian M.; Gould, Ernest A.; Gritsun, Tamara S.

    2014-01-01

    RNA secondary structures in the 3′untranslated regions (3′UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3′UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3′UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3′UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3′UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3′UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs. PMID:24647143

  18. Non-Watson-Crick base pairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E

    Czech Academy of Sciences Publication Activity Database

    Réblová, K.; Špačková, Naďa; Koča, J.; Leontis, N. B.; Šponer, Jiří

    2003-01-01

    Roč. 20, č. 6 (2003), s. 986 ISSN 0739-1102. [Albany 2003: Conversation 13. 17.06.2003-21.06.2003, Albany] Institutional research plan: CEZ:AV0Z5004920 Keywords : non- Watson -Crick base pairs * Loop E * RNA Subject RIV: BO - Biophysics

  19. Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR

    Science.gov (United States)

    Bonner, Eric R.; D’Elia, John N.; Billips, Benjamin K.; Switzer, Robert L.

    2001-01-01

    The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5′-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4–5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure. PMID:11726695

  20. Culture-dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Nabeel M. Alikunhi

    2017-09-01

    Full Text Available Fish contamination has been extensively investigated along the Saudi coasts, but studies pertaining to bacterial pathogens are scarce. We conducted qualitative assessment and molecular identification of culture-dependent bacteria in 13 fish species from three coastal sites and a local fish market in Jeddah, Saudi Arabia. Bacterial counts of gills, skin, gut and muscle were examined on agar plates of Macconkey’s (Mac, Eosin Methylene Blue (EMB and Thiosulfate Citrate Bile Salts (TCBS culture media. Bacterial counts significantly differed between species, sources and feeding habits of examined fishes. Mugil cephalus exhibited higher counts on TCBS (all body parts, Mac (gills, muscle and gut and EMB (gills and muscle. Fishes from Area I had higher bacterial loads, coinciding with those in seawater and sediment from the same site, indicating direct association between habitat conditions and the levels of bacterial contamination. By feeding habit, detritivorous fish harbored higher counts than herbivorous and carnivorous species. Bacterial counts of skin were higher in fish from market than field sites, and positively correlated with other body parts indicating the relation of surface bacterial load on the overall quality of fish. Rahnella aquatilis (Enterobacteriaceae and Photobacterium damselae (Vibrionaceae were among the dominant species from fish muscle based on 16S rRNA sequencing. These species are known human pathogens capable of causing foodborne illness with severe antibiotic resistance. Opportunistic pathogens, e.g. Hafnia sp. (Enterobacteriaceae and Pseudomonas stutzeri (Pseudomonadaceae also occurred in fish muscle. The inclusion of bacterial contamination in future monitoring efforts is thus crucial.

  1. Culture dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing

    KAUST Repository

    Alikunhi, Nabeel M.

    2016-05-27

    Fish contaminations have been extensively investigated in Saudi coasts, but studies pertaining to bacterial pathogens are meager. We conducted qualitative assessment and molecular identification of culture dependent bacteria in 13 fish species collected from three fishing sites and a local fish market in Jeddah, Saudi Arabia. The bacterial counts of gills, skin, gut and muscle were examined on agar plates of Macconkey’s (Mac), Eosin methylene blue (EMB) and Thiosulfate Citrate Bile Salts (TCBS) culture media. Bacterial counts exhibited interspecific, locational and behavioral differences. Mugil cephalus exhibited higher counts on TCBS (all body-parts), Mac (gills, muscle and gut) and EMB (gills and muscle). Samples of Area I were with higher counts, concurrent to seawater and sediment samples, revealing the influence of residing environment on fish contamination. Among feeding habits, detritivorous fish harbored higher bacterial counts, while carnivorous group accounted for lesser counts. Counts were higher in skin of fish obtained from market compared to field samples, revealing market as a major source of contamination. Bacterial counts of skin were positively correlated with other body-parts indicating influence of surface bacterial biota in overall quality of fish. Hence, hygienic practices and proper storage facilities in the Jeddah fish market is recommended to prevent adverse effect of food-borne illness in consumers. Rahnella aquatilis (Enterobacteriaceae) and Photobacterium damselae (Vibrionaceae) were among the dominant species identified from fish muscle samples using Sanger sequencing of 16S rRNA. This bacterial species are established human pathogens capable of causing foodborne illness with severe antibiotic resistance. Opportunistic pathogens such as Hafnia sp. (Enterobacteriaceae) and Pseudomonas stutzeri (Pseudomonadaceae) were also identified from fish muscle. These findings indicate bacterial contamination risk in commonly consumed fish of

  2. A novel method for room temperature distribution and conservation of RNA and DNA reference materials for guaranteeing performance of molecular diagnostics in onco-hematology: A GBMHM study.

    Science.gov (United States)

    Cayuela, Jean-Michel; Mauté, Carole; Fabre, Anne-Lise; Nibourel, Olivier; Dulucq, Stéphanie; Delabesse, Eric; Villarèse, Patrick; Hayette, Sandrine; Mozziconacci, Marie-Joelle; Macintyre, Elizabeth

    2015-10-01

    Performance of methods used for molecular diagnostics must be closely controlled by regular analysis of internal quality controls. However, conditioning, shipping and long lasting storage of nucleic acid controls remain problematic. Therefore, we evaluated the minicapsule-based innovative process developed by Imagene (Evry, France) for implementing DNA and RNA controls designed for clonality assessment of lymphoproliferations and BCR-ABL1 mRNA quantification, respectively. DNA samples were extracted from 12 cell lines selected for giving specific amplifications with most BIOMED-2 PCR tubes. RNA samples were extracted from 8 cell line mixtures expressing various BCR-ABL1 transcript levels. DNA and RNA were encapsulated by Imagene and shipped at room temperature to participating laboratories. Biologists were asked to report quality data of recovered nucleic acids as well as PCR results. Encapsulated nucleic acids samples were easily and efficiently recovered from minicapsules. The expected rearrangements at immunoglobulin, T-cell receptor and BCL2 loci were detected in DNA samples by all laboratories. Quality of RNA was consistent between laboratories and met the criteria requested for quantification of BCR-ABL1 transcripts. Expression levels measured by the 5 laboratories were within ±2 fold interval from the corresponding pre-encapsulation reference value. Moreover aging studies of encapsulated RNA simulating up to 100 years storage at room temperature show no bias in quantitative outcome. Therefore, Imagene minicapsules are suitable for storage and distribution at room temperature of genetic material designed for proficiency control of molecular diagnostic methods based on end point or real-time quantitative PCR. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  3. An effect of 16S rRNA intercistronic variability on coevolutionary analysis in symbiotic bacteria: Molecular phylogeny of Arsenophonus triatominarum

    Czech Academy of Sciences Publication Activity Database

    Šorfová, Pavlína; Škeříková, Andrea; Hypša, Václav

    2008-01-01

    Roč. 31, č. 2 (2008), s. 88-100 ISSN 0723-2020 R&D Projects: GA ČR GA206/04/0520; GA AV ČR IAA601410708 Institutional research plan: CEZ:AV0Z60220518 Keywords : intragenomic heterogeneity * 16S rRNA * coevolution * insect symbionts * molecular phylogeny Subject RIV: EE - Microbiology, Virology Impact factor: 2.582, year: 2008

  4. Conformational transitions of flanking purines in HIV-1 RNA dimerization initiation site kissing complexes studied by charmm explicit solvent molecular dynamics

    Czech Academy of Sciences Publication Activity Database

    Sarzynska, J.; Réblová, Kamila; Šponer, Jiří; Kulinski, T.

    2008-01-01

    Roč. 89, č. 9 (2008), s. 732-746 ISSN 0006-3525 R&D Projects: GA MŠk(CZ) LC06030; GA AV ČR(CZ) 1QS500040581 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : molecular dynamics * RNA * force field Subject RIV: BO - Biophysics Impact factor: 2.823, year: 2008

  5. Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

    Directory of Open Access Journals (Sweden)

    Bryan E. Dutton

    2002-12-01

    Full Text Available We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.

  6. A focus on polarity: Investigating the role of orientation cues in mediating student performance on mRNA synthesis tasks in an introductory cell and molecular biology course.

    Science.gov (United States)

    Olimpo, Jeffrey T; Quijas, Daniel A; Quintana, Anita M

    2017-11-01

    The central dogma has served as a foundational model for information flow, exchange, and storage in the biological sciences for several decades. Despite its continued importance, however, recent research suggests that novices in the domain possess several misconceptions regarding the aforementioned processes, including those pertaining specifically to the formation of messenger ribonucleic acid (mRNA) transcripts. In the present study, we sought to expand upon these observations through exploration of the influence of orientation cues on students' aptitude at synthesizing mRNAs from provided deoxyribonucleic acid (DNA) template strands. Data indicated that participants (n = 45) were proficient at solving tasks of this nature when the DNA template strand and the mRNA molecule were represented in an antiparallel orientation. In contrast, participants' performance decreased significantly on items in which the mRNA was depicted in a parallel orientation relative to the DNA template strand. Furthermore, participants' Grade Point Average, self-reported confidence in understanding the transcriptional process, and spatial ability were found to mediate their performance on the mRNA synthesis tasks. Collectively, these data reaffirm the need for future research and pedagogical interventions designed to enhance students' comprehension of the central dogma in a manner that makes transparent its relevance to real-world scientific phenomena. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):501-508, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  7. Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold.

    Science.gov (United States)

    Wang, Peng; Chen, Xiu-Lan; Li, Chun-Yang; Gao, Xiang; Zhu, De-yu; Xie, Bin-Bin; Qin, Qi-Long; Zhang, Xi-Ying; Su, Hai-Nan; Zhou, Bai-Cheng; Xun, Lu-ying; Zhang, Yu-Zhong

    2015-10-01

    The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds, but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO4 (3-) and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  8. Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

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    Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T.; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.

    2016-08-02

    Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

  9. Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA

    Science.gov (United States)

    Caton, Evan A; Kelly, Erin K; Kamalampeta, Rajashekhar

    2018-01-01

    Abstract H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p–Nop10p–Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs. PMID:29177505

  10. Structural map of Kaposi sarcoma-associated herpesvirus RNA provides clues to molecular interactions | Center for Cancer Research

    Science.gov (United States)

    Scientists from CCR have generated a comprehensive structural map of Kaposi sarcoma-associated herpesvirus polyadenylated nuclear (PAN) RNA, a long non-coding RNA that helps the virus evade detection by its host’s immune system. The findings open new oppportunites to study the life cycle of this cancer-causing virus.  Learn more...

  11. Long-term administration of a small molecular weight catalytic metalloporphyrin antioxidant, AEOL 10150, protects lungs from radiation-induced injury

    International Nuclear Information System (INIS)

    Rabbani, Zahid N.; Batinic-Haberle, Ines; Anscher, Mitchell S.; Huang Jie; Day, Brian J.; Alexander, Elaine; Dewhirst, Mark W.; Vujaskovic, Zeljko

    2007-01-01

    Purpose: To determine whether administration of a catalytic antioxidant, Mn(III) tetrakis(N,N'-diethylimidazolium-2-yl) porphyrin, AEOL 10150, with superoxide dismutase (SOD) mimetic properties, reduces the severity of radiation-induced injury to the lung from single-dose irradiation (RT) of 28 Gy. Methods and Materials: Rats were randomly divided into four different dose groups (0, 1, 10, and 30 mg/kg/day of AEOL 10150), receiving either short-term (1 week) or long-term (10 weeks) drug administration via osmotic pumps. Rats received single-dose irradiation (RT) of 28 Gy to the right hemithorax. Breathing rates, body weights, blood samples, histopathology, and immunohistochemistry were used to assess lung damage. Results: There was no significant difference in any of the study endpoints between the irradiated controls and the three groups receiving RT and short-term administration of AEOL 10150. For the long-term administration, functional determinants of lung damage 20 weeks postradiation were significantly worse for RT + phosphate-buffered saline (PBS) and RT + 1 mg/kg/day of AEOL 10150 as compared with the irradiated groups treated with higher doses of AEOL 10150 (10 or 30 mg/kg/day). Lung histology at 20 weeks revealed a significant decrease in structural damage and collagen deposition in rats receiving 10 or 30 mg/kg/day after radiation in comparison to the RT + PBS and 1 mg/kg/day groups. Immunohistochemistry demonstrated a significant reduction in macrophage accumulation, oxidative stress, and hypoxia in rats receiving AEOL 10150 (10 or 30 mg/kg/day) after lung irradiation compared with the RT + PBS and 1 mg/kg/day groups. Conclusions: The chronic administration of a novel catalytic antioxidant, AEOL 10150, demonstrates a significant protective effect from radiation-induced lung injury. AEOL 10150 has its primary impact on the cascade of events after irradiation, and adding the drug before irradiation and its short-term administration have no significant

  12. Molecular phylogenetics and systematics of the bivalve family Ostreidae based on rRNA sequence-structure models and multilocus species tree.

    Directory of Open Access Journals (Sweden)

    Daniele Salvi

    Full Text Available The bivalve family Ostreidae has a worldwide distribution and includes species of high economic importance. Phylogenetics and systematic of oysters based on morphology have proved difficult because of their high phenotypic plasticity. In this study we explore the phylogenetic information of the DNA sequence and secondary structure of the nuclear, fast-evolving, ITS2 rRNA and the mitochondrial 16S rRNA genes from the Ostreidae and we implemented a multi-locus framework based on four loci for oyster phylogenetics and systematics. Sequence-structure rRNA models aid sequence alignment and improved accuracy and nodal support of phylogenetic trees. In agreement with previous molecular studies, our phylogenetic results indicate that none of the currently recognized subfamilies, Crassostreinae, Ostreinae, and Lophinae, is monophyletic. Single gene trees based on Maximum likelihood (ML and Bayesian (BA methods and on sequence-structure ML were congruent with multilocus trees based on a concatenated (ML and BA and coalescent based (BA approaches and consistently supported three main clades: (i Crassostrea, (ii Saccostrea, and (iii an Ostreinae-Lophinae lineage. Therefore, the subfamily Crassostreinae (including Crassostrea, Saccostreinae subfam. nov. (including Saccostrea and tentatively Striostrea and Ostreinae (including Ostreinae and Lophinae taxa are recognized [corrected]. Based on phylogenetic and biogeographical evidence the Asian species of Crassostrea from the Pacific Ocean are assigned to Magallana gen. nov., whereas an integrative taxonomic revision is required for the genera Ostrea and Dendostrea. This study pointed out the suitability of the ITS2 marker for DNA barcoding of oyster and the relevance of using sequence-structure rRNA models and features of the ITS2 folding in molecular phylogenetics and taxonomy. The multilocus approach allowed inferring a robust phylogeny of Ostreidae providing a broad molecular perspective on their systematics.

  13. RNA-Seq transcriptomics and pathway analyses reveal potential regulatory genes and molecular mechanisms in high- and low-residual feed intake in Nordic dairy cattle.

    Science.gov (United States)

    Salleh, M S; Mazzoni, G; Höglund, J K; Olijhoek, D W; Lund, P; Løvendahl, P; Kadarmideen, H N

    2017-03-24

    The selective breeding of cattle with high-feed efficiencies (FE) is an important goal of beef and dairy cattle producers. Global gene expression patterns in relevant tissues can be used to study the functions of genes that are potentially involved in regulating FE. In the present study, high-throughput RNA sequencing data of liver biopsies from 19 dairy cows were used to identify differentially expressed genes (DEGs) between high- and low-FE groups of cows (based on Residual Feed Intake or RFI). Subsequently, a profile of the pathways connecting the DEGs to FE was generated, and a list of candidate genes and biomarkers was derived for their potential inclusion in breeding programmes to improve FE. The bovine RNA-Seq gene expression data from the liver was analysed to identify DEGs and, subsequently, identify the molecular mechanisms, pathways and possible candidate biomarkers of feed efficiency. On average, 57 million reads (short reads or short mRNA sequences cows, respectively. The interaction analysis (high vs. low RFI x control vs. high concentrate diet) showed no interaction effects in the Holstein cows, while two genes showed interaction effects in the Jersey cows. The analyses showed that DEGs act through certain pathways to affect or regulate FE, including steroid hormone biosynthesis, retinol metabolism, starch and sucrose metabolism, ether lipid metabolism, arachidonic acid metabolism and drug metabolism cytochrome P450. We used RNA-Seq-based liver transcriptomic profiling of high- and low-RFI dairy cows in two breeds and identified significantly DEGs, their molecular mechanisms, their interactions with other genes and functional enrichments of different molecular pathways. The DEGs that were identified were the CYP's and GIMAP genes for the Holstein and Jersey cows, respectively, which are related to the primary immunodeficiency pathway and play a major role in feed utilization and the metabolism of lipids, sugars and proteins.

  14. Molecular cloning and characterization of an inducible RNA-dependent RNA polymerase gene, GhRdRP, from cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Gao, Qiuqiang; Liu, Yan; Wang, Meimei; Zhang, Jiedao; Gai, Yingping; Zhu, Changxiang; Guo, Xingqi

    2009-01-01

    The RNA-dependent RNA polymerase (RdRP) cDNA, designated as Gossypium hirsutum RdRP (GhRdRP) was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 3,672 bp in size and encoded an open reading frame (ORF) of 1,110 amino acids which contained the RdRP conserved functional domain and the signature motif DbDGD. Amino acid sequence alignment indicated that GhRdRP shared the highest identity (66.37%) with AtRdRP1 and had homology with other plant, fungal, yeast and nematode RdRPs. The corresponding genomic DNA containing five exons and four introns, was isolated and analyzed. Also a 5'-flanking region was cloned, and a group of putative cis-acting elements were identified. Southern blot analysis revealed a single copy of the GhRdRP gene in cotton genome. The expression analysis by semi-quantitative RT-PCR showed that GhRdRP was induced by salicylic acid (SA), 5-chloroSA (5-CSA) and fungal infection of Rhizoctonia solani Kuhn. The cloning and characterization of the GhRdRP gene will be useful for further studies of biological roles of GhRdRP in plants.

  15. Identification and molecular characterization of a new nonsegmented double-stranded RNA virus isolated from Culex mosquitoes in Japan.

    Science.gov (United States)

    Isawa, Haruhiko; Kuwata, Ryusei; Hoshino, Keita; Tsuda, Yoshio; Sakai, Kouji; Watanabe, Shumpei; Nishimura, Miho; Satho, Tomomitsu; Kataoka, Michiyo; Nagata, Noriyo; Hasegawa, Hideki; Bando, Hisanori; Yano, Kazuhiko; Sasaki, Toshinori; Kobayashi, Mutsuo; Mizutani, Tetsuya; Sawabe, Kyoko

    2011-01-01

    Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations.

    Directory of Open Access Journals (Sweden)

    Hafiz Muhammad Ali

    Full Text Available RET/PTC3 junction oncogene is typical of radiation-induced childhood papillary thyroid carcinoma (PTC with a short latency period. Since, RET/PTC3 is only present in the tumour cells, thus represents an interesting target for specific therapy by small interfering RNA (siRNA. Our aim is to demonstrate in vitro and in vivo molecular and cellular effects of siRNA on RET/PTC3 knockdown for therapeutic application.First, we established a novel cell line stably expressing RET/PTC3 junction oncogene, named RP3 which was found tumorigenic in nude mice compared to NIH/3T3 mouse fibroblasts. Among four siRNAs and five concentrations tested against RET/PTC3, an efficient siRNA RET/PTC3 and an appropriate dose (50 nM were selected which showed significant inhibition (p<0.001 of gene (RT-qPCR and protein (Western blot expressions. This siRNA was found efficient in RP3 cells (harbouring RET/PTC3 but non-efficient in BHP10-3 SCmice cell line (harbouring RET/PTC1 showing that a specific siRNA against fusion sequence is required to target the junction oncogene. In vitro siRNA RET/PTC3 showed significant (p<0.001 inhibitory effects on RP3 cell viability (MTT assay and on invasion/migration (IncuCyte scratch test with blockage of cell cycle at G0/G1 phase (flow cytometry and induced apoptosis by caspase-3 and PARP1 cleavage (WB. After intravenous injection in nude mice, respective squalene (SQ nanoparticles (NPs of siRNA RET/PTC3 significantly (p<0.001 reduced RP3 tumour growth, oncogene and oncoprotein expressions, induced apoptosis and partially restored differentiation (decrease in Ki67. Hence, our findings highly support the use of siRNA RET/PTC3-SQ NPs as a new promising treatment for patients affected by PTC expressing RET/PTC3.

  17. Single Cell Analysis Linking Ribosomal (r)DNA and rRNA Copy Numbers to Cell Size and Growth Rate Provides Insights into Molecular Protistan Ecology.

    Science.gov (United States)

    Fu, Rao; Gong, Jun

    2017-11-01

    Ribosomal (r)RNA and rDNA have been golden molecular markers in microbial ecology. However, it remains poorly understood how ribotype copy number (CN)-based characteristics are linked with diversity, abundance, and activity of protist populations and communities observed at organismal levels. Here, we applied a single-cell approach to quantify ribotype CNs in two ciliate species reared at different temperatures. We found that in actively growing cells, the per-cell rDNA and rRNA CNs scaled with cell volume (CV) to 0.44 and 0.58 powers, respectively. The modeled rDNA and rRNA concentrations thus appear to be much higher in smaller than in larger cells. The observed rRNA:rDNA ratio scaled with CV 0.14 . The maximum growth rate could be well predicted by a combination of per-cell ribotype CN and temperature. Our empirical data and modeling on single-cell ribotype scaling are in agreement with both the metabolic theory of ecology and the growth rate hypothesis, providing a quantitative framework for linking cellular rDNA and rRNA CNs with body size, growth (activity), and biomass stoichiometry. This study also demonstrates that the expression rate of rRNA genes is constrained by cell size, and favors biomass rather than abundance-based interpretation of quantitative ribotype data in population and community ecology of protists. © 2017 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

  18. Disease Control in Animals Using Molecular Technology by Inactivation of ASO, RNAi and ss-siRNA Genes

    Directory of Open Access Journals (Sweden)

    Muhamad Ali

    2014-03-01

    Full Text Available Globalization causes high mobility of human and livestock, hence increase the transmission of infectious diseases, including avian influenza, severe acute respiratory syndrome (SARS, and swine influenza. Therefore, prevention of those diseases is required. Vaccines are effective to prevent infectious diseases; however, their development takes a long time and they cannot provide immediate protection in pandemic cases. This paper describes several gene silencing technologies including antisense oligonucleotide (ASO, RNA interference (RNAi and single strand-small interfering RNA (ss-siRNA for controlling diseases. The primary mechanism of these technologies is inhibition of gene expression, typically by causing the destruction of specific RNA molecule of the pathogen. The use of gene silencing technologies is expected to give new alternative that is more effective in eradication of infectious diseases in animals before threaten human being.

  19. Marker-Assisted Molecular Profiling, Deletion Mutant Analysis, and RNA-Seq Reveal a Disease Resistance Cluster Associated with Uromyces appendiculatus Infection in Common Bean Phaseolus vulgaris L.

    Science.gov (United States)

    Todd, Antonette R; Donofrio, Nicole; Sripathi, Venkateswara R; McClean, Phillip E; Lee, Rian K; Pastor-Corrales, Marcial; Kalavacharla, Venu Kal

    2017-05-23

    Common bean ( Phaseolus vulgaris L.) is an important legume, useful for its high protein and dietary fiber. The fungal pathogen Uromyces appendiculatus (Pers.) Unger can cause major loss in susceptible varieties of the common bean. The Ur-3 locus provides race specific resistance to virulent strains or races of the bean rust pathogen along with Crg , (Complements resistance gene), which is required for Ur-3 -mediated rust resistance. In this study, we inoculated two common bean genotypes (resistant "Sierra" and susceptible crg) with rust race 53 of U. appendiculatus , isolated leaf RNA at specific time points, and sequenced their transcriptomes. First, molecular markers were used to locate and identify a 250 kb deletion on chromosome 10 in mutant crg (which carries a deletion at the Crg locus). Next, we identified differential expression of several disease resistance genes between Mock Inoculated (MI) and Inoculated (I) samples of "Sierra" leaf RNA within the 250 kb delineated region. Both marker assisted molecular profiling and RNA-seq were used to identify possible transcriptomic locations of interest regarding the resistance in the common bean to race 53. Identification of differential expression among samples in disease resistance clusters in the bean genome may elucidate significant genes underlying rust resistance. Along with preserving favorable traits in the crop, the current research may also aid in global sustainability of food stocks necessary for many populations.

  20. 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.

  1. Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M. [NIH; (UNC); (Columbia)

    2014-08-06

    Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

  2. Molecular evolution of adiponectin in Carnivora and its mRNA expression in relation to hepatic lipidosis.

    Science.gov (United States)

    Nieminen, Petteri; Rouvinen-Watt, Kirsti; Kapiainen, Suvi; Harris, Lora; Mustonen, Anne-Mari

    2010-09-15

    Adiponectin is a novel adipocyte-derived hormone with low circulating concentrations and/or mRNA expression in obesity and non-alcoholic fatty liver disease (NAFLD). The adiponectin mRNA of several Carnivora species was sequenced to enable further gene expression studies in this clade with potential experimental species to examine the connections of hypoadiponectinemia to hepatic lipidosis. In addition, adiponectin mRNA expression was studied in the retroperitoneal fat of the American mink (Neovison vison), as hepatic lipidosis with close similarities to NAFLD can be rapidly induced to the species by fasting. The mRNA expression was determined after overnight-7d of food deprivation and 28d of re-feeding and correlated to the liver fat %. The homologies between the determined carnivoran mRNA sequences and that of the domestic dog were 92.2-99.1%. As the mRNA expression was not affected by short-term fasting and did not correlate with the liver fat %, there seems to be no clear connection between adiponectin and the development of lipidosis in the American mink. In the future, the obtained sequences can be utilized in further studies of adiponectin expression in comparative endocrinology. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  3. Molecular basis of arabinobio-hydrolase activity in phytopathogenic fungi: crystal structure and catalytic mechanism of Fusarium graminearum GH93 exo-alpha-L-arabinanase.

    Science.gov (United States)

    Carapito, Raphaël; Imberty, Anne; Jeltsch, Jean-Marc; Byrns, Simon C; Tam, Pui-Hang; Lowary, Todd L; Varrot, Annabelle; Phalip, Vincent

    2009-05-01

    The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. alpha-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-alpha-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear alpha-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases alpha-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with alpha-1,5-l-arabinobiose, a degradation product of alpha-Me-1,5-l-arabinotetraose, at 1.85 and 2.05A resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed beta-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases.

  4. Catalytic ethanolysis of Kraft lignin into high-value small-molecular chemicals over a nanostructured α-molybdenum carbide catalyst.

    Science.gov (United States)

    Ma, Rui; Hao, Wenyue; Ma, Xiaolei; Tian, Ye; Li, Yongdan

    2014-07-07

    We report the complete ethanolysis of Kraft lignin over an α-MoC1-x /AC catalyst in pure ethanol at 280 °C to give high-value chemicals of low molecular weight with a maximum overall yield of the 25 most abundant liquid products (LP25) of 1.64 g per gram of lignin. The LP25 products consisted of C6 -C10 esters, alcohols, arenes, phenols, and benzyl alcohols with an overall heating value of 36.5 MJ kg(-1) . C6 alcohols and C8 esters predominated and accounted for 82 wt % of the LP25 products. No oligomers or char were formed in the process. With our catalyst, ethanol is the only effective solvent for the reaction. Supercritical ethanol on its own degrades Kraft lignin into a mixture of small molecules and molecular fragments of intermediate size with molecular weights in the range 700-1400, differing in steps of 58 units, which is the weight of the branched-chain linkage C3 H6 O in lignin. Hydrogen was found to have a negative effect on the formation of the low-molecular-weight products. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Understanding the Catalytic Mechanism and the Nature of the Transition State of an Attractive Drug-Target Enzyme (Shikimate Kinase) by Quantum Mechanical/Molecular Mechanical (QM/MM) Studies.

    Science.gov (United States)

    Yao, Jianzhuang; Wang, Xia; Luo, Haixia; Gu, Pengfei

    2017-11-16

    Shikimate kinase (SK) is the fifth bacterial enzyme involved in the shikimate pathway for biosynthesis of life-indispensable components, such as aromatic amino acids. The absence of the shikimate pathway in humans makes SK an attractive target for the rational design of drugs aimed at pathogenesis bacteria, such as Mycobacterium tuberculosis and Helicobacter pylori. However, an effective inhibitor of SK (e.g., a transition-state analogue) is still not available on the market due, at least in part, to a lack of knowledge on the catalytic mechanism and the nature of the rate-limiting transition state. Herein, quantum mechanical/molecular mechanical (QM/MM) reaction coordinate, molecular dynamics (MD), and free-energy simulations have been performed to answer these questions. The results presented herein demonstrate that the phosphoryl-transfer process, which is the rate-limiting step of SK-catalyzed phosphorylation of shikimic acid (SKM), is a concerted one-step reaction proceeding through a loose transition state. The computational results agree well with those of experimental studies, specifically NMR results, X-ray crystal structure observation, and activation free-energy barrier. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. RNA-Seq transcriptomics and pathway analyses reveal potential regulatory genes and molecular mechanisms in high- and low-residual feed intake in Nordic dairy cattle

    DEFF Research Database (Denmark)

    Salleh, M. S.; Mazzoni, G.; Höglund, J. K.

    2017-01-01

    -throughput RNA sequencing data of liver biopsies from 19 dairy cows were used to identify differentially expressed genes (DEGs) between high- and low-FE groups of cows (based on Residual Feed Intake or RFI). Subsequently, a profile of the pathways connecting the DEGs to FE was generated, and a list of candidate......The selective breeding of cattle with high-feed efficiencies (FE) is an important goal of beef and dairy cattle producers. Global gene expression patterns in relevant tissues can be used to study the functions of genes that are potentially involved in regulating FE. In the present study, high...... genes and biomarkers was derived for their potential inclusion in breeding programmes to improve FE. The bovine RNA-Seq gene expression data from the liver was analysed to identify DEGs and, subsequently, identify the molecular mechanisms, pathways and possible candidate biomarkers of feed efficiency...

  7. Calculating the Na⁺ translocating V-ATPase catalytic site affinity for substrate binding by homology modeled NtpA monomer using molecular dynamics/free energy calculation.

    Science.gov (United States)

    Muhammed, Zahed; Arai, Satoshi; Saijo, Shinya; Yamato, Ichiro; Murata, Takeshi; Suenaga, Atsushi

    2012-07-01

    Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and two peripheral stalks (NtpC, NtpD-G and NtpE-F). Recently nucleotide binding of catalytic NtpA monomer has been reported (Arai et al.). In the present study, we calculated the nucleotide binding affinity of NtpA by molecular dynamics (MD) simulation/free energy calculation using MM-GBSA approach based on homology modeled structure of NtpA monomer docked with ATP analogue, adenosine 5'-[β, γ-imido] triphosphate (AMP-PNP). The calculated binding free energies showed qualitatively good agreement with experimental data. The calculation was cross-validated further by the rigorous method, thermodynamic integration (TI) simulation. Finally, the interaction between NtpA and nucleotides at the atomic level was investigated by the analyses of components of free energy and the optimized model structures obtained from MD simulations, suggesting that electrostatic contribution is responsible for the difference in nucleotide binding to NtpA monomer. This is the first observation and suggestion to explain the difference of nucleotide binding properties in V-ATPase NtpA subunit, and our method can be a valuable primary step to predict nucleotide binding affinity to other subunits (NtpAB, NtpA₃B₃) and to explore subunit interactions and eventually may help to understand energy transduction mechanism of E. hirae V-ATPase. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Catalytic Oligopeptide Synthesis.

    Science.gov (United States)

    Liu, Zijian; Noda, Hidetoshi; Shibasaki, Masakatsu; Kumagai, Naoya

    2018-02-02

    Waste-free catalytic assembly of α-amino acids is fueled by a multiboron catalyst that features a characteristic B 3 NO 2 heterocycle, providing a versatile catalytic protocol wherein functionalized natural α-amino acid units are accommodated and commonly used protecting groups are tolerated. The facile dehydrative conditions eliminate the use of engineered peptide coupling reagents, exemplifying a greener catalytic alternative for peptide coupling. The catalysis is sufficiently robust to enable pentapeptide synthesis, constructing all four amide bond linkages in a catalytic fashion.

  9. Molecular identification and characterization of the intervening sequences (IVSs) within 23S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis isolated in France.

    Science.gov (United States)

    Tazumi, Akihiro; Petry, Sandrine; Hayashi, Kyohei; Moore, John E; Millar, Beverley C; Matsuda, Motoo

    2012-02-01

    In the helix 25 region, 32 French Taylorella asinigenitalis isolates carried at least one 23S rRNA gene not containing intervening sequences (IVSs). No IVSs in the region were identified in three isolates and the other remaining 29 isolates carried one or more IVSs (UCD-1(T)IVS1A, UCD-1(T)IVS1B and UK-1IVS1B) described already and two new kinds of IVS (TaIVS1C and TaIVS1D). In the helix 45 region, no T. asinigenitalis isolates not carrying any IVSs were identified. UK-1IVS2B was identified in the region from 26 isolates. Five new kinds of IVSs (TaIVS2D, E, F, G and H) occurred in the region in the 13 isolates. Distinctly different tandem repeat units (RS48 and RS32 and RS-A, -B and -C) were evident in both regions, respectively, from the French (n=32) and American (n=3) T. asinigenitalis isolates. Thus, several different kinds of tandem repeat units and their combinations in IVSs in both regions within the gene were shown in 32 French isolates. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Self-assembled 3D heterometallic Cu(II)/Fe(II) coordination polymers with octahedral net skeletons: structural features, molecular magnetism, thermal and oxidation catalytic properties.

    Science.gov (United States)

    Karabach, Yauhen Y; Guedes da Silva, M Fátima C; Kopylovich, Maximilian N; Gil-Hernández, Beatriz; Sanchiz, Joaquin; Kirillov, Alexander M; Pombeiro, Armando J L

    2010-12-06

    The new three-dimensional (3D) heterometallic Cu(II)/Fe(II) coordination polymers [Cu(6)(H(2)tea)(6)Fe(CN)(6)](n)(NO(3))(2n)·6nH(2)O (1) and [Cu(6)(Hmdea)(6)Fe(CN)(6)](n)(NO(3))(2n)·7nH(2)O (2) have been easily generated by aqueous-medium self-assembly reactions of copper(II) nitrate with triethanolamine or N-methyldiethanolamine (H(3)tea or H(2)mdea, respectively), in the presence of potassium ferricyanide and sodium hydroxide. They have been isolated as air-stable crystalline solids and fully characterized including by single-crystal X-ray diffraction analyses. The latter reveal the formation of 3D metal-organic frameworks that are constructed from the [Cu(2)(μ-H(2)tea)(2)](2+) or [Cu(2)(μ-Hmdea)(2)](2+) nodes and the octahedral [Fe(CN)(6)](4-) linkers, featuring regular (1) or distorted (2) octahedral net skeletons. Upon dehydration, both compounds show reversible escape and binding processes toward water or methanol molecules. Magnetic susceptibility measurements of 1 and 2 reveal strong antiferromagnetic [J = -199(1) cm(-1)] or strong ferromagnetic [J = +153(1) cm(-1)] couplings between the copper(II) ions through the μ-O-alkoxo atoms in 1 or 2, respectively. The differences in magnetic behavior are explained in terms of the dependence of the magnetic coupling constant on the Cu-O-Cu bridging angle. Compounds 1 and 2 also act as efficient catalyst precursors for the mild oxidation of cyclohexane by aqueous hydrogen peroxide to cyclohexanol and cyclohexanone (homogeneous catalytic system), leading to maximum total yields (based on cyclohexane) and turnover numbers (TONs) up to about 22% and 470, respectively.

  11. In silico molecular docking analysis of the human Argonaute 2 PAZ domain reveals insights into RNA interference

    Science.gov (United States)

    Kandeel, Mahmoud; Kitade, Yukio

    2013-07-01

    RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3' end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.

  12. 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...

  13. Classifications within molecular subtypes enables identification of BRCA1/BRCA2 mutation carriers by RNA tumor profiling

    DEFF Research Database (Denmark)

    Larsen, Martin J; Kruse, Torben A; Tan, Qihua

    2013-01-01

    Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants...... of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic...... tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority...

  14. Catalytic synthesis of ammonia using vibrationally excited nitrogen molecules

    DEFF Research Database (Denmark)

    Hansen, Flemming Yssing; Henriksen, Niels Engholm; Billing, Gert D.

    1992-01-01

    The dissociation of nitrogen is the rate-limiting step in the catalytic synthesis of ammonia. Theoretical calculations have shown that the dissociative sticking probability of molecular nitrogen on catalytic active metal surfaces is enhanced by orders of magnitude when the molecules...

  15. Free-Energy Landscape of Reverse tRNA Translocation through the Ribosome Analyzed by Electron Microscopy Density Maps and Molecular Dynamics Simulations

    Science.gov (United States)

    Ishida, Hisashi; Matsumoto, Atsushi

    2014-01-01

    To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA. PMID:24999999

  16. Raman crystallography of RNA.

    Science.gov (United States)

    Gong, Bo; Chen, Jui-Hui; Yajima, Rieko; Chen, Yuanyuan; Chase, Elaine; Chadalavada, Durga M; Golden, Barbara L; Carey, Paul R; Bevilacqua, Philip C

    2009-10-01

    Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.

  17. Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens

    Czech Academy of Sciences Publication Activity Database

    Brabec, Jan; Kostadinova, Aneta; Scholz, Tomáš; Littlewood, D. T. J.

    2015-01-01

    Roč. 8, JUN 19 2015 (2015), s. 336 ISSN 1756-3305 R&D Projects: GA MŠk(CZ) EE2.3.30.0032; GA ČR(CZ) GA15-14198S Grant - others:GA MŠk(CZ) LM2010005 Institutional support: RVO:60077344 Keywords : Diplostomum (Platyhelminthes: Trematoda) * fish pathogens * mitochondrial genome * ribosomal RNA * illumina next- gene ration sequencing * phylogeny Subject RIV: EG - Zoology Impact factor: 3.234, year: 2015

  18. INTEGRATIVE MICRO-RNA AND PROTEOMIC APPROACHES IDENTIFY MOLECULAR MARKERS PREDICTIVE OF MUSCLE ATROPHY IN RAINBOW TROUT

    Science.gov (United States)

    Background: MicroRNAs (miRNA) are small, highly conserved, non-coding RNAs. MiRNAs are the most significant regulators in gene expression which negatively regulate target mRNAs at the posttranscriptional levels. Recently, we have cloned and characterized a “miRNAome” in rainbow trout. In addition, w...

  19. lsosteric and Nonisosteric Base Pairs in RNA Motifs: Molecular Dynamics and Bioinformatics Study of the Sarcin Ricin Internal Loop

    Czech Academy of Sciences Publication Activity Database

    Havrila, Marek; Réblová, Kamila; Zirbel, C.L.; Leontis, B. N.; Šponer, Jiří

    2013-01-01

    Roč. 117, č. 46 (2013), s. 14302-14319 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68081707 Keywords : 23S RIBOSOMAL-RNA * PARTICLE MESH EWALD * NUCLEIC-ACIDS Subject RIV: BO - Biophysics Impact factor: 3.377, year: 2013

  20. Molecular phylogenetic studies on an unnamed bovine Babesia sp. based on small subunit ribosomal RNA gene sequences.

    Science.gov (United States)

    Luo, Jianxun; Yin, Hong; Liu, Zhijie; Yang, Dongying; Guan, Guiquan; Liu, Aihong; Ma, Miling; Dang, Shengzhi; Lu, Bingyi; Sun, Caiqin; Bai, Qi; Lu, Wenshun; Chen, Puyan

    2005-10-10

    The 18S small subunit ribosomal RNA (18S rRNA) gene of an unnamed Babesia species (designated B. U sp.) was sequenced and analyzed in an attempt to distinguish it from other Babesia species in China. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was ligated to the pGEM-T Easy vector for sequencing. It was found that the length of the 18S rRNA gene of all B. U sp. Kashi 1 and B. U sp. Kashi 2 was 1699 bp and 1689 bp. Two phylogenetic trees were, respectively, inferred based on 18S rRNA sequence of the Chinese bovine Babesia isolates and all of Babesia species available in GenBank. The first tree showed that B. U sp. was situated in the branch between B. major Yili and B. bovis Shannxian, and the second tree revealed that B. U sp. was confined to the same group as B. caballi. The percent identity of B. U sp. with other Chinese Babesia species was between 74.2 and 91.8, while the percent identity between two B. U sp. isolates was 99.7. These results demonstrated that this B. U sp. is different from other Babesia species, but that two B. U sp. isolates obtained with nymphal and adultal Hyalomma anatolicum anatolicum tick belong to the same species.

  1. Classifications within Molecular Subtypes Enables Identification of BRCA1/BRCA2 Mutation Carriers by RNA Tumor Profiling

    Science.gov (United States)

    Larsen, Martin J.; Kruse, Torben A.; Tan, Qihua; Lænkholm, Anne-Vibeke; Bak, Martin; Lykkesfeldt, Anne E.; Sørensen, Kristina P.; Hansen, Thomas v. O.; Ejlertsen, Bent; Gerdes, Anne-Marie; Thomassen, Mads

    2013-01-01

    Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement (“BRCAness”) by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors. PMID:23704984

  2. Comparative MiRNA Expressional Profiles and Molecular Networks in Human Small Bowel Tissues of Necrotizing Enterocolitis and Spontaneous Intestinal Perforation.

    Directory of Open Access Journals (Sweden)

    Pak Cheung Ng

    Full Text Available Necrotizing enterocolitis (NEC and spontaneous intestinal perforation (SIP are acute intestinal conditions which could result in mortality and severe morbidity in preterm infants. Our objective was to identify dysregulated micro-RNAs (miRNAs in small bowel tissues of NEC and SIP, and their possible roles in disease pathophysiology.We performed differential miRNA arrays on tissues of NEC (n = 4, SIP (n = 4 and surgical-control (Surg-CTL; n = 4, and validated target miRNAs by qPCR (n = 10 each group. The association of target miRNAs with 52 dysregulated mRNAs was investigated by bioinformatics on functional and base-pair sequence algorithms, and correlation in same tissue samples.We presented the first miRNA profiles of NEC, SIP and Surg-CTL intestinal tissues in preterm infants. Of 28 validated miRNAs, 21 were significantly different between NEC or SIP and Surg-CTL. Limited overlapping in the aberrant expression of miRNAs between NEC and SIP indicated their distinct molecular mechanisms. A proposed network of dysregulated miRNA/mRNA pairs in NEC suggested interaction at bacterial receptor TLR4 (miR-31, miR-451, miR-203, miR-4793-3p, mediated via key transcription factors NFKB2 (miR-203, AP-1/FOSL1 (miR-194-3p, FOXA1 (miR-21-3p, miR-431 and miR-1290 and HIF1A (miR-31, and extended downstream to pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia/oxidative stress, inflammation and muscle contraction. In contrast, upregulation of miR-451 and miR-223 in SIP suggested modulation of G-protein-mediated muscle contraction.The robust response of miRNA dysregulation in NEC and SIP, and concerted involvement of specific miRNAs in the molecular networks indicated their crucial roles in mucosa integrity and disease pathophysiology.

  3. RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests.

    Directory of Open Access Journals (Sweden)

    Laetitia Ninove

    Full Text Available Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs. In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i to design specific ECs for both DNA and RNA viruses and (ii to use T4 (DNA or MS2 (RNA phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%, broncho-alveolar liquid (41% and stools (36%. The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

  4. RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests.

    Science.gov (United States)

    Ninove, Laetitia; Nougairede, Antoine; Gazin, Celine; Thirion, Laurence; Delogu, Ilenia; Zandotti, Christine; Charrel, Remi N; De Lamballerie, Xavier

    2011-02-09

    Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

  5. Deadenylation of mRNA by the CCR4-NOT complex in Drosophila: molecular and developmental aspects

    Directory of Open Access Journals (Sweden)

    Claudia eTemme

    2014-05-01

    Full Text Available Controlled shortening of the poly(A tail of mRNAs is the first step in eukaryotic mRNA decay and can also be used for translational inactivation of mRNAs. The CCR4-NOT complex is the most important among a small number of deadenylases, enzymes catalyzing poly(A tail shortening. Rates of poly(A shortening differ between mRNAs as the CCR4-NOT complex is recruited to specific mRNAs by means of either sequence-specific RNA binding proteins or miRNAs. This review summarizes our current knowledge concerning the subunit composition and deadenylation activity of the Drosophila CCR4-NOT complex and the mechanisms by which the complex is recruited to particular mRNAs. We discuss genetic data implicating the complex in the regulation of specific mRNAs, in particular in the context of development.

  6. Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

    Science.gov (United States)

    Choubey, Amit

    Biological cell membranes provide mechanical stability to cells and understanding their structure, dynamics and mechanics are important biophysics problems. Experiments coupled with computational methods such as molecular dynamics (MD) have provided insight into the physics of membranes. We use long-time and large-scale MD simulations to study the structure, dynamics and mechanical behavior of membranes. We investigate shock-induced collapse of nanobubbles in water using MD simulations based on a reactive force field. We observe a focused jet at the onset of bubble shrinkage and a secondary shock wave upon bubble collapse. The jet length scales linearly with the nanobubble radius, as observed in experiments on micron-to-millimeter size bubbles. Shock induces dramatic structural changes, including an ice-VII-like structural motif at a particle velocity of 1 km/s. The incipient ice VII formation and the calculated Hugoniot curve are in good agreement with experimental results. We also investigate molecular mechanisms of poration in lipid bilayers due to shock-induced collapse of nanobubbles. Our multimillion-atom MD simulations reveal that the jet impact generates shear flow of water on bilayer leaflets and pressure gradients across them. This transiently enhances the bilayer permeability by creating nanopores through which water molecules translocate rapidly across the bilayer. Effects of nanobubble size and temperature on the porosity of lipid bilayers are examined. The second research project focuses on cholesterol (CHOL) dynamics in phospholipid bilayers. Several experimental and computational studies have been performed on lipid bilayers consisting of dipalmitoylphosphatidylcholine (DPPC) and CHOL molecules. CHOL interleaflet transport (flip-flop) plays an important role in interleaflet coupling and determining CHOL flip-flop rate has been elusive. Various studies report that the rate ranges between milliseconds to seconds. We calculate CHOL flip-flop rates by

  7. Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing

    Directory of Open Access Journals (Sweden)

    Dorothee Pflueger

    2013-11-01

    Full Text Available TFE3 translocation renal cell carcinoma (tRCC is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study,we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNAread-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2 point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs. The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P<.001 and 22 papillaryRCCs (P<.05-.07. Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2 and Contactin 3 (CNTN3 expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both.

  8. Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNAseq, RNA interference and irradiation approach.

    Science.gov (United States)

    Solana, Jordi; Kao, Damian; Mihaylova, Yuliana; Jaber-Hijazi, Farah; Malla, Sunir; Wilson, Ray; Aboobaker, Aziz

    2012-01-01

    Planarian stem cells, or neoblasts, drive the almost unlimited regeneration capacities of freshwater planarians. Neoblasts are traditionally described by their morphological features and by the fact that they are the only proliferative cell type in asexual planarians. Therefore, they can be specifically eliminated by irradiation. Irradiation, however, is likely to induce transcriptome-wide changes in gene expression that are not associated with neoblast ablation. This has affected the accurate description of their specific transcriptomic profile. We introduce the use of Smed-histone-2B RNA interference (RNAi) for genetic ablation of neoblast cells in Schmidtea mediterranea as an alternative to irradiation. We characterize the rapid, neoblast-specific phenotype induced by Smed-histone-2B RNAi, resulting in neoblast ablation. We compare and triangulate RNA-seq data after using both irradiation and Smed-histone-2B RNAi over a time course as means of neoblast ablation. Our analyses show that Smed-histone-2B RNAi eliminates neoblast gene expression with high specificity and discrimination from gene expression in other cellular compartments. We compile a high confidence list of genes downregulated by both irradiation and Smed-histone-2B RNAi and validate their expression in neoblast cells. Lastly, we analyze the overall expression profile of neoblast cells. Our list of neoblast genes parallels their morphological features and is highly enriched for nuclear components, chromatin remodeling factors, RNA splicing factors, RNA granule components and the machinery of cell division. Our data reveal that the regulation of planarian stem cells relies on posttranscriptional regulatory mechanisms and suggest that planarians are an ideal model for this understudied aspect of stem cell biology.

  9. lsosteric and Nonisosteric Base Pairs in RNA Motifs: Molecular Dynamics and Bioinformatics Study of the Sarcin Ricin Internal Loop

    Czech Academy of Sciences Publication Activity Database

    Havrila, Marek; Réblová, K.; Zirbel, C.L.; Leontis, N. B.

    2013-01-01

    Roč. 117, č. 46 (2013), s. 14302-14319 ISSN 1520-6106 R&D Projects: GA ČR GBP305/12/G034; GA MŠk ED1.1.00/02.0068 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : 23S RIBOSOMAL-RNA * PARTICLE MESH EWALD * NUCLEIC-ACIDS Subject RIV: BO - Biophysics Impact factor: 3.377, year: 2013

  10. Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis

    Czech Academy of Sciences Publication Activity Database

    Liebl, K.; Dršata, Tomáš; Lankaš, Filip; Lipfert, J.; Zacharias, M.

    2015-01-01

    Roč. 43, č. 21 (2015), s. 10143-10156 ISSN 0305-1048 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : double stranded RNA * B-DNA * nucleic acids Subject RIV: BO - Biophysics Impact factor: 9.202, year: 2015 http://nar.oxfordjournals.org/content/43/21/10143.full.pdf+html

  11. Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis

    Directory of Open Access Journals (Sweden)

    Zhen Li

    2016-12-01

    Full Text Available The reproductive function of G-protein subunit Galphaq (GNAQ, a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR. The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01 and testis (p<0.05. Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.

  12. Catalytic conversion of light alkanes

    Energy Technology Data Exchange (ETDEWEB)

    Lyons, J.E.

    1992-06-30

    The second Quarterly Report of 1992 on the Catalytic Conversion of Light Alkanes reviews the work done between April 1, 1992 and June 31, 1992 on the Cooperative Agreement. The mission of this work is to devise a new catalyst which can be used in a simple economic process to convert the light alkanes in natural gas to oxygenate products that can either be used as clean-burning, high octane liquid fuels, as fuel components or as precursors to liquid hydrocarbon uwspomdon fuel. During the past quarter we have continued to design, prepare, characterize and test novel catalysts for the mild selective reaction of light hydrocarbons with air or oxygen to produce alcohols directly. These catalysts are designed to form active metal oxo (MO) species and to be uniquely active for the homolytic cleavage of the carbon-hydrogen bonds in light alkanes producing intermediates which can form alcohols. We continue to investigate three molecular environments for the active catalytic species that we are trying to generate: electron-deficient macrocycles (PHASE I), polyoxometallates (PHASE II), and regular oxidic lattices including zeolites and related structures as well as other molecular surface structures having metal oxo groups (PHASE I).

  13. Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

    Directory of Open Access Journals (Sweden)

    Shu-ichi Nakano

    2017-11-01

    Full Text Available Catalytic nucleic acids are regarded as potential therapeutic agents and biosensors. The catalytic activities of nucleic acid enzymes are usually investigated in dilute aqueous solutions, although the physical properties of the reaction environment inside living cells and that in the area proximal to the surface of biosensors in which they operate are quite different from those of pure water. The effect of the molecular environment is also an important focus of research aimed at improving and expanding nucleic acid function by addition of organic solvents to aqueous solutions. In this study, the catalytic activities of RNA and DNA enzymes (hammerhead ribozyme, 17E DNAzyme, R3C ribozyme, and 9DB1 DNAzyme were investigated using 21 different mixed aqueous solutions comprising organic compounds. Kinetic measurements indicated that these enzymes can display enhanced catalytic activity in mixed solutions with respect to the solution containing no organic additives. Correlation analyses revealed that the turnover rate of the reaction catalyzed by hammerhead ribozyme increased in a medium with a lower dielectric constant than water, and the turnover rate of the reaction catalyzed by 17E DNAzyme increased in conditions that increased the strength of DNA interactions. On the other hand, R3C ribozyme and 9DB1 DNAzyme displayed no significant turnover activity, but their single-turnover rates increased in many mixed solutions. Our data provide insight into the activity of catalytic nucleic acids under various conditions that are applicable to the medical and technology fields, such as in living cells and in biosensors.

  14. Stepwise dissection and visualization of the catalytic mechanism of haloalkane dehalogenase LinB using molecular dynamics simulations and computer graphics.

    Science.gov (United States)

    Negri, Ana; Marco, Esther; Damborsky, Jiri; Gago, Federico

    2007-10-01

    The different steps of the dehalogenation reaction carried out by LinB on three different substrates have been characterized using a combination of quantum mechanical calculations and molecular dynamics simulations. This has allowed us to obtain information in atomic detail about each step of the reaction mechanism, that is, substrate entrance and achievement of the near-attack conformation, transition state stabilization within the active site, halide stabilization, water molecule activation and subsequent hydrolytic attack on the ester intermediate with formation of alcohol, and finally product release. Importantly, no bias or external forces were applied during the whole procedure so that both intermediates and products were completely free to sample configuration space in order to adapt to the plasticity of the active site and/or search for an exit. Differences in substrate reactivity were found to be correlated with the ease of adopting the near-attack conformation and two different exit pathways were found for product release that do not interfere with substrate entrance. Additional support for the different entry and exit pathways was independently obtained from an examination of the enzyme's normal modes.

  15. Role of Au-C Interactions on the Catalytic Activity of Au Nanoparticles Supported on TiC(001) toward Molecular Oxygen Dissociation

    International Nuclear Information System (INIS)

    Rodriguez, J.; Feria, L.; Jirsak, T.; Takahashi, Y.; Nakamura, K.; Illas, F.

    2010-01-01

    High-resolution photoemission and density functional calculations on realistic slab surface models were used to study the interaction and subsequent dissociation of O 2 with Au nanoparticles supported on TiC(001). The photoemission results indicate that at 150 K O 2 adsorbs molecularly on the supported gold nanoparticles, and upon heating to temperatures above 200 K the O 2 → 2O reaction takes place with migration of atomic oxygen to the TiC(001) substrate. The addition of Au to TiC(001) substantially enhances the rate of O 2 dissociation at room temperature. The reactivity of Au nanoparticles supported on TiC(001) toward O 2 dissociation is much larger than that of similar nanoparticles supported either on TiO 2 (110) or MgO(001) surfaces, where the cleavage of O-O bonds is very difficult. Density functional calculations carried out on large supercells show that the contact of Au with TiC(001) is essential for charge polarization and an enhancement in the chemical activity of Au. Small two-dimensional particles which expose Au atoms in contact with TiC(001) are the most reactive. While O 2 prefers binding to Au sites, the O atoms interact more strongly with the TiC(001) surface. The oxygen species active during the low-temperature ( 2 . Once atomic O binds to TiC(001), the chemisorption bond is so strong that temperatures well above 400 K are necessary to remove the O adatoms from the TiC(001) substrate by direct reaction with CO. The high reactivity of Au/TiC(001) toward O 2 at low-temperature opens the route for the transformation of alcohols and amines on the supported Au nanoparticles.

  16. Molecular characterization of rockfish (Sebastes schlegeli) gonadotropin subunits and their mRNA expression profiles during oogenesis.

    Science.gov (United States)

    Kim, Dae-Jung; Cho, Yong Chul; Sohn, Young Chang

    2005-05-01

    In an attempt to understand the reproductive regulation in viviparous teleosts, gonadotropin (GTH) subunit cDNAs were characterized and the expression levels of GTH subunit mRNAs in the pituitary glands of the rockfish, Sebastes schlegeli (Scorpaeniformes, Scorpaenidae), were examined by Northern blot analysis. The complete sequences of rockfish GTH subunits (GTHalpha, FSHbeta, and LHbeta) were determined by rapid amplification of cDNA ends (RACE) and nucleotide sequencing. Based on the RACE analysis, the cDNAs of GTHalpha, FSHbeta, and LHbeta consisted of 655, 540, and 529 nucleotides encoding peptides of 132, 130, and 143 amino acids, respectively. The mature peptides of rockfish FSHbeta, LHbeta, and common GTHalpha showed high sequence identities (FSHbeta, 58-62%; LHbeta, 86-94%; and GTHalpha, 87-88%) to those of other perciforme (e.g., orange spotted grouper, red seabream, stripped bass, and sea bass). In a sequence alignment of the mature peptides, rockfish FSHbeta exhibited a unique feature, the lack of a conserved N-glycosylation site. This is in contrast to both LHbeta and GTHalpha which contain one and two N-glycosylation sites, respectively, and is consistent with those of other teleosts. The mRNA levels of the GTHalpha subunit increased at the vitellogenic stage and remained steady from ovulation to post-parturition. FSHbeta mRNA levels increased abruptly during the vitellogenic stages and then decreased during ovulation, embryonic development, and post-parturition. LHbeta mRNA levels were observed to rapidly increase during the vitellogenic stage, reached its highest levels during ovulation and was then followed by a decrease at post-parturition. These results suggest that in the female rockfish FSH and LH syntheses are predominant during vitellogenesis and oocyte maturation, respectively.

  17. Rift Valley Fever Virus: Molecular Biologic Studies of the M Segment RNA(Ribonucleic Acids) for Application in Disease Prevention

    Science.gov (United States)

    1989-02-01

    ORGANIZATION Molecular Genetica , Inc. S6- ADDRESS (City, Stat., and ZIP Code) 7b. ADO~tESS (City, SWOat.dd ZIP Cod.) 10320 Bren Road East Minnetonka...adhered to the "Guide for the Care and Use of Laboratory Arimals" of the Institute of Laboratory Animal Resources, National Research Council (DHEW

  18. Catalytic distillation process

    Science.gov (United States)

    Smith, L.A. Jr.

    1982-06-22

    A method is described for conducting chemical reactions and fractionation of the reaction mixture comprising feeding reactants to a distillation column reactor into a feed zone and concurrently contacting the reactants with a fixed bed catalytic packing to concurrently carry out the reaction and fractionate the reaction mixture. For example, a method for preparing methyl tertiary butyl ether in high purity from a mixed feed stream of isobutene and normal butene comprising feeding the mixed feed stream to a distillation column reactor into a feed zone at the lower end of a distillation reaction zone, and methanol into the upper end of said distillation reaction zone, which is packed with a properly supported cationic ion exchange resin, contacting the C[sub 4] feed and methanol with the catalytic distillation packing to react methanol and isobutene, and concurrently fractionating the ether from the column below the catalytic zone and removing normal butene overhead above the catalytic zone.

  19. Catalytic Functions of Standards

    NARCIS (Netherlands)

    K. Blind (Knut)

    2009-01-01

    textabstractThe three different areas and the examples have illustrated several catalytic functions of standards for innovation. First, the standardisation process reduces the time to market of inventions, research results and innovative technologies. Second, standards themselves promote the

  20. Catalytic distillation structure

    Science.gov (United States)

    Smith, L.A. Jr.

    1984-04-17

    Catalytic distillation structure is described for use in reaction distillation columns, and provides reaction sites and distillation structure consisting of a catalyst component and a resilient component intimately associated therewith. The resilient component has at least about 70 volume % open space and is present with the catalyst component in an amount such that the catalytic distillation structure consists of at least 10 volume % open space. 10 figs.

  1. miRNA, siRNA, piRNA - Kleiner Wiener Ribonukleinsauren

    Czech Academy of Sciences Publication Activity Database

    Svoboda, Petr; Grosshans, H.

    2007-01-01

    Roč. 29, č. 9 (2007), s. 940-943 ISSN 0265-9247 Grant - others:EMBO(XE) EMBO Installation Grant 1483 Institutional research plan: CEZ:AV0Z50520514 Keywords : small RNA * siRNA * miRNA * piRNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.402, year: 2007

  2. RNA profiling reveals familial aggregation of molecular subtypes in non-BRCA1/2 breast cancer families

    DEFF Research Database (Denmark)

    Larsen, Martin J; Thomassen, Mads; Tan, Qihua

    2014-01-01

    and provide evidence for epigenetic inactivation of BRCA1 in three of the tumors. In addition, 7 BRCA2-like tumors were found. CONCLUSIONS: Our finding indicates involvement of hereditary factors in non-BRCA1/2 breast cancer families in which family members may carry genetic susceptibility not just to breast...... cancer but to a particular subtype of breast cancer. This is the first study to provide a biological link between breast cancers from family members of high-risk non-BRCA1/2 families in a systematic manner, suggesting that future genetic analysis may benefit from subgrouping families into molecularly......BACKGROUND: In more than 70% of families with a strong history of breast and ovarian cancers, pathogenic mutation in BRCA1 or BRCA2 cannot be identified, even though hereditary factors are expected to be involved. It has been proposed that tumors with similar molecular phenotypes also share similar...

  3. Molecular profiling of short-term and long-term surviving patients identifies CD34 mRNA level as prognostic for glioblastoma survival

    DEFF Research Database (Denmark)

    Michaelsen, Signe Regner; Urup, Thomas; Olsen, Lars Rønn

    2018-01-01

    Despite extensive treatment, overall survival (OS) for glioblastoma (GBM) remains poor. A small proportion of patients present long survival over 3 years, but the underlying molecular background separating these long-term survivors (LTS) from short-term survivors (STS) are insufficiently understood......-type. RNA expression levels in diagnostic tumor specimen for 792 genes were analyzed by NanoString technology. While no differences were found with regard to GBM subtype between LTS versus STS, comparative analysis of individual genes identified 14 significantly differently expressed candidate genes...... analyses in an independent dataset from the AVAglio study confirmed CD34 as significant in comparative analysis between STS and LTS patients and as an independent prognostic factor. Analysis of this dataset further supported CD34 expression to be associated with improved bevacizumab efficacy, while CD34...

  4. How Amino Acids and Peptides Shaped the RNA World

    NARCIS (Netherlands)

    P.T.S. van der Gulik (Peter); D. Speijer (Dave)

    2015-01-01

    htmlabstractThe “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein

  5. How amino acids and peptides shaped the RNA world

    NARCIS (Netherlands)

    van der Gulik, Peter T. S.; Speijer, Dave

    2015-01-01

    The "RNA world" hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this

  6. Development of RNA aptamers as molecular probes for HER2+ breast cancer study using cell-SELEX

    Directory of Open Access Journals (Sweden)

    Seyedeh Alia Moosavian

    2015-06-01

    Full Text Available Objective(s: Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2 is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line. Materials and Methods: Panel of aptamers was selected using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX. Results: Binding studies showed that selected aptamers can identify TUBO cell line with high affinity and selectivity. Our preliminary investigation of the target of aptamers suggested that aptamers bind with HER2 proteins on the surface of TUBO cells. Conclusion: We believe the selected aptamers could be useful ligands for targeted breast cancer therapy.

  7. Molecular cloning, mRNA expression and alternative splicing of a ryanodine receptor gene from the citrus whitefly, Dialeurodes citri (Ashmead).

    Science.gov (United States)

    Yuan, Guo-Rui; Wang, Ke-Yi; Mou, Xing; Luo, Ruo-Yu; Dou, Wei; Wang, Jin-Jun

    2017-10-01

    Insect ryanodine receptors are the main targets of diamide insecticides that have highly selective insecticidal activity but are less toxic to mammals. Therefore, these insecticides are ideal for pest control. Ryanodine receptors (RyRs) play a critical role in Ca 2+ signaling in muscle and non-muscle cells. In this study, we cloned the complete cDNA (DcRyR) of the RyR from the citrus whitefly, Dialeurodes citri, a serious pest of citrus orchards in China. The open reading frame of RyR is 15,378bp long and encodes a protein with 5126 amino acids with a computed molecular weight of 579.523kDa. DcRyR shows a high amino acid sequence identity to RyRs from other insects (76%-95%) and low identity to those from nematodes and mammals (44%-52%). DcRyR shares many features of insect and vertebrate RyRs, including a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeat domain, RIH-associated domain at the N-terminus, two consensus calcium-binding EF-hands and six transmembrane domains at the C-terminus. The expression of DcRyR mRNA was the highest in the nymphs and lowest in eggs; DcRyR mRNA was 1.85-fold higher in the nymphs than in the eggs. Among the tissues, DcRyR mRNA expression was 4.18- and 4.02-fold higher in the adult head and thorax than in the abdomen. DcRyR had three alternative splice sites and the splice variants showed body part-specific expression and were developmentally regulated. These results may help investigate target-based resistance to diamide insecticides in D. citri. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Molecular dynamics and MM/GBSA-integrated protocol probing the correlation between biological activities and binding free energies of HIV-1 TAR RNA inhibitors.

    Science.gov (United States)

    Peddi, Saikiran Reddy; Sivan, Sree Kanth; Manga, Vijjulatha

    2018-02-01

    The interaction of HIV-1 transactivator protein Tat with its cognate transactivation response (TAR) RNA has emerged as a promising target for developing antiviral compounds and treating HIV infection, since it is a crucial step for efficient transcription and replication. In the present study, molecular dynamics (MD) simulations and MM/GBSA calculations have been performed on a series of neamine derivatives in order to estimate appropriate MD simulation time for acceptable correlation between ΔG bind and experimental pIC 50 values. Initially, all inhibitors were docked into the active site of HIV-1 TAR RNA. Later to explore various conformations and examine the docking results, MD simulations were carried out. Finally, binding free energies were calculated using MM/GBSA method and were correlated with experimental pIC 50 values at different time scales (0-1 to 0-10 ns). From this study, it is clear that in case of neamine derivatives as simulation time increased the correlation between binding free energy and experimental pIC 50 values increased correspondingly. Therefore, the binding energies which can be interpreted at longer simulation times can be used to predict the bioactivity of new neamine derivatives. Moreover, in this work, we have identified some plausible critical nucleotide interactions with neamine derivatives that are responsible for potent inhibitory activity. Furthermore, we also provide some insights into a new class of oxadiazole-based back bone cyclic peptides designed by incorporating the structural features of neamine derivatives. On the whole, this approach can provide a valuable guidance for designing new potent inhibitors and modify the existing compounds targeting HIV-1 TAR RNA.

  9. Molecular characterization of double-stranded RNA virus in Trichomonas vaginalis Egyptian isolates and its association with pathogenicity.

    Science.gov (United States)

    El-Gayar, Eman K; Mokhtar, Amira B; Hassan, Wael A

    2016-10-01

    Trichomoniasis is a common human sexually transmitted infection caused by Trichomonas vaginalis. The parasite can be infected with double-stranded RNA viruses (TVV). This viral infection may have important implications on trichomonal virulence and disease pathogenesis. This study aimed to determine the prevalence of T. vaginalis virus among isolates obtained from infected (symptomatic and asymptomatic) women in Ismailia City, Egypt, and to correlate the virus-infected isolates with the clinical manifestations of patients. In addition, the pathogenicity of TVV infected isolates on mice was also evaluated. T. vaginalis isolates were obtained from symptomatic and asymptomatic female patients followed by axenic cultivation in Diamond's TYM medium. The presence of T. vaginalis virus was determined from total extraction of nucleic acids (DNA-RNA) followed by reverse transcriptase-PCR. Representative samples were inoculated intraperitoneally in female albino/BALB mice to assess the pathogenicity of different isolates. A total of 110 women were examined; 40 (36.3 %) samples were positive for T. vaginalis infection. Of these 40 isolates, 8 (20 %) were infected by TVV. Five isolates contained TVV-2 virus species, and the remaining three isolates were infected withTVV-4 variant. A significant association was found between the presence of TVV and particular clinical manifestations of trichomoniasis. Experimental mice infection showed varying degrees of pathogenicity. This is the first report on T. vaginalis infection by TVV in Egypt. The strong association detected between TVV and particular clinical features of trichomoniasis and also the degree of pathogenicity in experimentally infected mice may indicate a possible clinical significance of TVV infection of T. vaginalis isolates.

  10. Molecular identification of Azospirillum spp.: Limitations of 16S rRNA and qualities of rpoD as genetic markers.

    Science.gov (United States)

    Maroniche, Guillermo A; García, Julia E; Salcedo, Florencia; Creus, Cecilia M

    2017-01-01

    Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been subjected to intensive research due to their biotechnological potential as crop inoculants. Phylogenetic analysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsistencies and low confidence often arise when working with close species. In this work, it was observed that these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list of housekeeping genes, the correlation between pairwise gene and whole-genome similarities was examined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for the PCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 type strains of different species. The sequences obtained were used for inferring a phylogenetic tree of the genus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla isolated from many different locations of Argentine. In addition, several strains that might represent novel species were detected. The results indicate that the sequencing of rpoD is a suitable alternative method for a confident molecular identification in Azospirillum spp. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Molecular phylogeny of the neritidae (Gastropoda: Neritimorpha) based on the mitochondrial genes cytochrome oxidase I (COI) and 16S rRNA

    International Nuclear Information System (INIS)

    Quintero Galvis, Julian Fernando; Castro, Lyda Raquel

    2013-01-01

    The family Neritidae has representatives in tropical and subtropical regions that occur in a variety of environments, and its known fossil record dates back to the late Cretaceous. However there have been few studies of molecular phylogeny in this family. We performed a phylogenetic reconstruction of the family Neritidae using the COI (722 bp) and the 16S rRNA (559 bp) regions of the mitochondrial genome. Neighbor-joining, maximum parsimony and Bayesian inference were performed. The best phylogenetic reconstruction was obtained using the COI region, and we consider it an appropriate marker for phylogenetic studies within the group. Consensus analysis (COI +16S rRNA) generally obtained the same tree topologies and confirmed that the genus Nerita is monophyletic. The consensus analysis using parsimony recovered a monophyletic group consisting of the genera Neritina, Septaria, Theodoxus, Puperita, and Clithon, while in the Bayesian analyses Theodoxus is separated from the other genera. The phylogenetic status of the species from the genus Nerita from the Colombian Caribbean generated in this study was consistent with that reported for the genus in previous studies. In the resulting consensus tree obtained using maximum parsimony, we included information on habitat type for each species, to map the evolution by habitat. Species of the family Neritidae possibly have their origin in marine environments, which is consistent with conclusions from previous reports based on anatomical studies.

  12. Synthesis and electrochemistry of β-pyrrole nitro-substituted cobalt(II) porphyrins. The effect of the NO₂ group on redox potentials, the electron transfer mechanism and catalytic reduction of molecular oxygen in acidic media.

    Science.gov (United States)

    Sun, Bin; Ou, Zhongping; Yang, Shuibo; Meng, Deying; Lu, Guifen; Fang, Yuanyuan; Kadish, Karl M

    2014-07-28

    Four cobalt(II) porphyrins, two of which contain a β-pyrrole nitro substituent, were synthesized and characterized by electrochemistry and spectroelectrochemistry. The investigated compounds are represented as (TRPP)Co and (NO2TRPP)Co, where TRPP is the dianion of a substituted tetraphenylporphyrin and R is a CH3 or OCH3 substituent on the four phenyl rings of the macrocycle. Two reductions and three oxidations are observed for each compound in CH2Cl2 containing 0.10 M tetra-n-butylammonium perchlorate. The first reduction of the compounds without a nitro substituent is metal-centered and leads to formation of a Co(I) porphyrin which then reacts with the CH2Cl2 solvent to generate a carbon σ-bonded Co(III)-R complex. A further reduction then occurs at more negative potentials to generate an unstable Co(II) σ-bonded compound. In contrast to these reactions, the first reduction of the nitro-substituted porphyrins is macrocycle-centered under the same solution conditions and gives a Co(II) porphyrin π-anion radical product. This reversible electron transfer is then followed at more negative potentials by a second reversible one-electron addition to give a Co(II) dianion. Three reversible one-electron oxidations are also seen for each compound. The first is metal-centered and the next two involve the conjugated π-system of the macrocycle. Each neutral Co(II) porphyrin was also examined as to its catalytic activity for electroreduction of molecular oxygen when coated on an edge-plane pyrolytic graphite electrode in 1.0 M HClO4. The β-pyrrole nitro-substituted derivatives were shown to be better catalysts than the non-nitro substituted compounds under the utilized experimental conditions.

  13. A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

    Directory of Open Access Journals (Sweden)

    Jiqin Wu

    2015-06-01

    Full Text Available RNA-dependent RNA polymerases (RdRPs from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

  14. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers.

    Science.gov (United States)

    Lacerenza, D; Aneli, S; Omedei, M; Gino, S; Pasino, S; Berchialla, P; Robino, C

    2016-05-01

    "Touch DNA" refers to the DNA that is left behind when a person touches or comes into contact with an item. However, the source of touch DNA is still debated and the large variability in DNA yield from casework samples suggests that, besides skin, various body fluids can be transferred through contact. Another important issue concerning touch DNA is the possible occurrence of secondary transfer, but the data published in the literature in relation to the background levels of foreign DNA present on the hand surfaces of the general population are very limited. As the present study aimed at better understanding the nature and characteristics of touch DNA, samples were collected from the palmar surface of the hands and fingers ("PHF" samples) of 30 male and 30 female donors by tape-lifting/swabbing and subjected to DNA/RNA co-extraction. Multiplex mRNA profiling showed that cellular material different from skin could be observed in 15% of the PHF samples. The total amount of DNA recovered from these samples (median 5.1 ng) was significantly higher than that obtained from samples containing skin cells only (median 1.6 ng). The integrity of the DNA isolated from the donors' hands and fingers as well as the prevalence of DNA mixtures were evaluated by STR typing and compared with reference STR profiles from buccal swabs. DNA integrity appeared significantly higher in the male rather than in the female subsample, as the average percentage of the donors' alleles effectively detected in PHF profiles was 75.1% and 60.1%, respectively. The prevalence of mixtures with a foreign DNA contribution ≥20% was 19.2% (30.0% in the female PHF samples and 8.3% in the male PHF samples). The obtained results support the hypothesis that transfer of cellular material different from skin may underlie the occasional recovery of quality STR profiles from handled items. These results also suggest that gender may represent an important factor influencing the propensity of individuals to carry

  15. Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach.

    Science.gov (United States)

    Zhang, Kaiming; Keane, Sarah C; Su, Zhaoming; Irobalieva, Rossitza N; Chen, Muyuan; Van, Verna; Sciandra, Carly A; Marchant, Jan; Heng, Xiao; Schmid, Michael F; Case, David A; Ludtke, Steven J; Summers, Michael F; Chiu, Wah

    2018-03-06

    Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS] 2 ; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2 H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene.

    Science.gov (United States)

    Ragan, M A; Bird, C J; Rice, E L; Gutell, R R; Murphy, C A; Singh, R K

    1994-01-01

    A phylogeny of marine Rhodophyta has been inferred by a number of methods from nucleotide sequences of nuclear genes encoding small subunit rRNA from 39 species in 15 orders. Sequence divergences are relatively large, especially among bangiophytes and even among congeners in this group. Subclass Bangiophycidae appears polyphyletic, encompassing at least three lineages, with Porphyridiales distributed between two of these. Subclass Florideophycidae is monophyletic, with Hildenbrandiales, Corallinales, Ahnfeltiales, and a close association of Nemaliales, Acrochaetiales, and Palmariales forming the four deepest branches. Cermiales may represent a convergence of vegetative and reproductive morphologies, as family Ceramiaceae is at best weakly related to the rest of the order, and one of its members appears to be allied to Gelidiales. Except for Gigartinales, for which more data are required, the other florideophyte orders appear distinct and taxonomically justified. A good correlation was observed with taxonomy based on pit-plug ultrastructure. Tests under maximum-likelihood and parsimony of alternative phylogenies based on structure and chemistry refuted suggestions that Acrochaetiales is the most primitive florideophyte order and that Gelidiales and Hildenbrandiales are sister groups. PMID:8041780

  17. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, Ronald C. [Case Western Reserve Univ., Cleveland, OH (United States)

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese`s group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  18. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, R.C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese's group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  19. [Molecular phylogeny of Turbellaria, based on data from comparing the nucleotide sequences of 18S ribosomal RNA genes].

    Science.gov (United States)

    Kuznedelov, K D; Timoshkin, O A

    1995-01-01

    Polymerase chain reaction and direct sequencing of the 5'-end region of the 18S ribosomal RNA gene were used to infer phylogenetic relationship among turbellarian flatworms from Lake Baikal. Representatives of 5 orders (Tricladida--10 spp., Lecithoepitheliata--5 spp., Prolecithophora--3 spp., Proseriata and Kalyptorhynchia one for each) were studied; nucleotide sequence of more than 340 nucleotides was determined for each species. Consensus sequence for each order having more than one representative species was determined. Distance matrix and maximum parsimony approaches were applied to infer phylogenies. Bootstrap procedure was used to estimate confidence limits, at the 100% level by bootstrapping, the group of three orders: Kalyptorhynchia, Proseriata and Lecithoepitheliata was found to be monophyletic. However, subsets inside the group had no significant support to be preferred or rejected. Our data do not support traditional systematics which joins two suborders Tricladida and Proseriata into the single order Seriata, and also do not support comparative anatomical data which show close relationship of Lecithoepitheliata and lower Prolecithophora.

  20. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

    Directory of Open Access Journals (Sweden)

    Yuping Zhang

    Full Text Available Heat shock proteins (Hsps are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78, Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h, were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

  1. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

    Science.gov (United States)

    Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2015-01-01

    Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

  2. Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

    Science.gov (United States)

    Ahmed, W; Staley, C; Sadowsky, M J; Gyawali, P; Sidhu, J P S; Palmer, A; Beale, D J; Toze, S

    2015-10-01

    In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Determinants for association and gRNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei

    OpenAIRE

    Hernandez, Alfredo; Panigrahi, Aswini; Cifuentes-Rojas, Catherine; Sacharidou, Anastasia; Stuart, Kenneth; Cruz-Reyes, Jorge

    2008-01-01

    U-insertion/deletion RNA editing in the single mitochondrion of ancient kinetoplastids is a unique mRNA maturation process needed for translation. Multi-subunit editing complexes recognize many pre-mRNA sites and modify them via cycles of three catalytic steps: guide-RNA (gRNA) mediated cleavage, insertion or deletion of uridylates at the 3’ terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have...

  4. Steam reformer with catalytic combustor

    Science.gov (United States)

    Voecks, Gerald E. (Inventor)

    1990-01-01

    A steam reformer is disclosed having an annular steam reforming catalyst bed formed by concentric cylinders and having a catalytic combustor located at the center of the innermost cylinder. Fuel is fed into the interior of the catalytic combustor and air is directed at the top of the combustor, creating a catalytic reaction which provides sufficient heat so as to maintain the catalytic reaction in the steam reforming catalyst bed. Alternatively, air is fed into the interior of the catalytic combustor and a fuel mixture is directed at the top. The catalytic combustor provides enhanced radiant and convective heat transfer to the reformer catalyst bed.

  5. Molecular catalytic system for efficient water splitting

    NARCIS (Netherlands)

    Joya, Khurram Saleem

    2011-01-01

    The aim of this dissertation is to construct and explore artificial oxygen evolving complexes that are synthetically accessible, stable, functionally robust and efficient. To achieve this, a class of mono metal water splitting catalysts is introduced in this manuscript and exploitation of these

  6. RNA-seq of Rice Yellow Stem Borer Scirpophaga incertulas Reveals Molecular Insights During Four Larval Developmental Stages

    Directory of Open Access Journals (Sweden)

    Pichili Renuka

    2017-09-01

    Full Text Available The yellow stem borer (YSB, Scirpophaga incertulas, is a prominent pest in rice cultivation causing serious yield losses. The larval stage is an important stage in YSB, responsible for maximum infestation. However, limited knowledge exists on the biology and mechanisms underlying the growth and differentiation of YSB. To understand and identify the genes involved in YSB development and infestation, so as to design pest control strategies, we performed de novo transcriptome analysis at the first, third, fifth, and seventh larval developmental stages employing Illumina Hi-seq. High-quality reads (HQR of ∼229 Mb were assembled into 24,775 transcripts with an average size of 1485 bp. Genes associated with various metabolic processes, i.e., detoxification mechanism [CYP450, GSTs, and carboxylesterases (CarEs], RNA interference (RNAi machinery (Dcr-1, Dcr-2, Ago-1, Ago-2, Sid-1, Sid-2, Sid-3, and Sid-1-related gene, chemoreception (CSPs, GRs, OBPs, and ORs, and regulators [transcription factors (TFs and hormones] were differentially regulated during the developmental stages. Identification of stage-specific transcripts made it possible to determine the essential processes of larval development. Comparative transcriptome analysis revealed that YSB has not evolved much with respect to the detoxification mechanism, but showed the presence of distinct RNAi machinery. The presence of strong specific visual recognition coupled with chemosensory mechanisms supports the monophagous nature of YSB. Designed expressed sequenced tags-simple-sequence repeats (EST-SSRs will facilitate accurate estimation of the genetic diversity of YSB. This is the first report on characterization of the YSB transcriptome and the identification of genes involved in key processes, which will help researchers and industry to devise novel pest control strategies. This study also opens up a new avenue to develop next-generation resistant rice using RNAi or genome editing approaches.

  7. Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function.

    Science.gov (United States)

    Umasuthan, Navaneethaiyer; Bathige, S D N K; Kasthuri, Saranya Revathy; Wan, Qiang; Whang, Ilson; Lee, Jehee

    2013-08-01

    Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 °C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. CATALYTIC KINETIC SPECTROPHOTOMETRIC DETERMINATION ...

    African Journals Online (AJOL)

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    acetylchlorophosphonazo(CPApA) by hydrogen peroxide in 0.10 M phosphoric acid. A novel catalytic kinetic-spectrophotometric method is proposed for the determination of copper based on this principle. Copper(II) can be determined spectrophotometrically ...

  9. CATALYTIC KINETIC SPECTROPHOTOMETRIC DETERMINATION ...

    African Journals Online (AJOL)

    Based on the property that in 0.12 M sulfuric acid medium titanium(IV) catalyzes the discoloring reaction of DBS-arsenazo oxidized by potassium bromate, a new catalytic kinetic spectrophotometric method for the determination of trace titanium (IV) was developed. The linear range of the determination of titanium is

  10. Trim65: a cofactor for regulation of the microRNA pathway.

    Science.gov (United States)

    Li, Shitao; Wang, Lingyan; Fu, Bishi; Dorf, Martin E

    2014-01-01

    MicroRNA (miRNA) comprise a large family of non-protein coding transcripts which regulate gene expression in diverse biological pathways of both plants and animals. We recently used a systematic proteomic approach to generate a protein interactome map of the human miRNA pathway involved in miRNA biogenesis and processing. The interactome expands the number of candidate proteins in the miRNA pathway and connects the network to other cellular processes. Functional analyses identified TRIM65 and at least 3 other proteins as novel regulators of the miRNA pathway. Biochemical studies established that TRIM65 forms stable complexes with TNRC6 proteins and these molecules co-localize in P-body-like structures. Gain of function and RNAi analyses reveal that TRIM65 negatively regulates miRNA-driven suppression of mRNA translation by targeting TNRC6 proteins for ubiquitination and degradation. The potential molecular mechanisms which regulate TRIM65 catalytic activity are discussed.

  11. Molecular cloning of cytochrome P450 side-chain cleavage and changes in its mRNA expression during gonadal development of brown hagfish, Paramyxine atami.

    Science.gov (United States)

    Nishiyama, Maki; Uchida, Katsuhisa; Abe, Nozomi; Nozaki, Masumi

    2015-02-01

    Since hagfishes are considered the most primitive vertebrate known, extant or extinct, studies on their reproduction are indispensable for understanding phylogenetic aspects of vertebrate reproduction. However, little information is available on the endocrine regulation of the gonadal function in the hagfish. Based on EST analysis of the testis of the brown hagfish (Paramyxine atami), P450 side chain cleavage (CYP11A), which is the first and essential enzyme for steroidogenesis in jawed vertebrates, was cloned. The deduced amino acid sequence of hagfish CYP11A shows high identity to other animal forms especially in two functional domains, adrenodoxin binding domain and heme-binding domain. In the phylogenetic analysis, hagfish CYP11A forms a clade with the vertebrate CYP11A. Following the real-time PCR analysis, CYP11A mRNA expression levels were clearly correlated to the developmental stages of gonads in both sexes of the brown hagfish. By in situ hybridization, CYP11A mRNA signals were found in the theca cells of the ovarian follicles and Leydig cells and the tubule-boundary cells of the testis. These molecular and histological evidences are suggesting that CYP11A plays functional roles as a steroidogenic enzyme in gonadal development. Moreover, native GTH purified from hagfish pituitary stimulated the transcriptional levels of CYP11A in the organ-cultured testis in vitro, clearly suggesting that the steroidogenic activity of the hagfish is under the control of the pituitary GTH. It is suggested that vertebrates, during their early evolution, have established the pituitary-gonadal reproductive system. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Molecular confirmation of Trichomonas gallinae and other parabasalids from Brazil using the 5.8S and ITS-1 rRNA regions.

    Science.gov (United States)

    Ecco, Roselene; Preis, Ingred S; Vilela, Daniel A R; Luppi, Marcela M; Malta, Marcelo C C; Beckstead, Robert B; Stimmelmayr, Raphaela; Stimmelmayer, Raphaela; Gerhold, Richard W

    2012-11-23

    Clinical, gross, and histopathology lesions and molecular characterization of Trichomonas spp. infection were described in two striped owls (Asio (Rhinoptynx) clamator), one American kestrel (Falco sparverius), two green-winged saltators (Saltator similis), and in a toco toucan (Ramphastos toco) from Brazil. These birds presented clinical signs including emaciation, ruffled feathers, abundant salivation and open mouth breathing presumably due to abundant caseous material. Gross lesions were characterized by multifocal yellow friable plaques on the surface of the tongue, pharynx and/or caseous masses partially occluding the laryngeal entrance. In the owls, the caseous material extended into the mandibular muscles and invaded the sinuses of the skull. Histopathologically, marked necrotic and inflammatory lesions were associated with numerous round to oval, pale eosinophilic structures (6-10μm) with basophilic nuclei, consistent with trichomonads. Organisms similar to those described above also were found in the liver of the two green-winged saltators. To the authors' knowledge, this is the first report of trichomonosis in a striped owl and a toco toucan. Sequence analysis of the Trichomonas spp. internal transcribed spacer 1 (ITS-1) region and partial 5.8S of the ribosomal RNA (rRNA) disclosed significant genetic diversity. Two sequences had 100% identity to Trichomonas gallinae, whereas two sequences had a 99% and 92% identity to a Trichomonas vaginalis-like sequence, respectively. One sequence (green-winged saltator 502-08) had a 100% identity to a newly recognized genus Simplicomonas. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state.

    Directory of Open Access Journals (Sweden)

    Becka M Warfield

    Full Text Available RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are

  14. Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation.

    Science.gov (United States)

    Bai, Hongkun; Qiao, Hui; Li, Fajun; Fu, Hongtuo; Sun, Shengming; Zhang, Wenyi; Jin, Shubo; Gong, Yongsheng; Jiang, Sufei; Xiong, Yiwei

    2015-05-10

    Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid amplification of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor (VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process of vitellogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Point -of -care testing (POCT) in molecular diagnostics: Performance evaluation of GeneXpert HCV RNA test in diagnosing and monitoring of HCV infection.

    Science.gov (United States)

    Gupta, Ekta; Agarwala, Pragya; Kumar, Guresh; Maiwall, Rakhi; Sarin, Shiv Kumar

    2017-03-01

    Molecular testing at the point-of-care may turn out to be game changer for HCV diagnosis and treatment monitoring, through increased sensitivity, reduced turnaround time, and ease of performance. One such assay GeneXpert ® has recently been released. Comparative analysis between performances of GeneXpert ® and Abbott HCV-RNA was done. 174 HCV infected patients were recruited and, one time plasma samples from 154 patients and repeated samples from 20 patients, obtained at specific treatment time-points (0, 4, 12 and 24) weeks were serially re-tested on Xpert ® . Genotype 3 was the commonest, seen in 80 (66%) of the cases, genotype 1 in 34 (28.3%), genotype 4 in 4 (3.3%) and genotypes 2 and 5 in 1 (0.8%) each. Median HCV RNA load was 4.69 log 10 (range: 0-6.98log 10 ) IU/ml. Overall a very good correlation was seen between the two assays (R 2 =0.985), concordance of the results between the assays was seen in 138 samples (89.6%). High and low positive standards were tested ten times on Xpert ® to evaluate the precision and the coefficient of variation was 0.01 for HPC and 0.07 for the LPC. Monitoring of patients on two different regimes of treatment, pegylated interferon plus ribavirin and sofosbuvir plus ribavirin was done by both the systems at baseline, 4, 12 and 24 weeks. Perfect correlation between the assays in the course of therapy at different treatment time- point in genotypes 3 and 1 was seen. The study demonstrates excellent performance of the Xpert ® HCV assay in viral load assessment and in treatment course monitoring consistency. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. RNA topology

    OpenAIRE

    Frank-Kamenetskii, Maxim D.

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.

  17. Chemical fidelity of an RNA polymerase ribozyme

    DEFF Research Database (Denmark)

    Attwater, J.; Tagami, S.; Kimoto, M.

    2013-01-01

    The emergence of catalytically active RNA enzymes (ribozymes) is widely believed to have been an important transition in the origin of life. In the context of a likely heterogeneous chemical environment, substrate specificity and selectivity of these primordial enzymes would have been critical...... for function. Here we have explored the chemical fidelity, i.e. substrate selectivity and specificity for both single and multiple catalytic steps of the Z RNA polymerase ribozyme-a modern day analogue of the primordial RNA replicase. Using a wide range of nucleotide analogues and ionic conditions, we observe...

  18. Molecular response to imatinib & its correlation with mRNA expression levels of imatinib influx & efflux transporters in patients with chronic myeloid leukaemia in chronic phase

    Directory of Open Access Journals (Sweden)

    Hemant Malhotra

    2015-01-01

    Full Text Available Background & objectives: Imatinib is the standard first-line treatment for chronic myeloid leukaemia (CML patients. About 20 to 30 per cent patients develop resistance to imatinib and fail imatinib treatment. One of the mechanisms proposed is varying expression levels of the drug transporters. This study was aimed to determine the expression levels of imatinib transporter genes (OCT1, ABCB1, ABCG2 in CML patients and to correlate these levels with molecular response. Methods: Sixty three CML chronic phase patients who were on 400 mg/day imatinib for more than two years were considered for gene expression analysis study for OCT1, ABCB1 and ABCG2 genes. These were divided into responders and non-responders. The relative transcript expression levels of the three genes were compared between these two categories. The association between the expression values of these three genes was also determined. Results: No significant difference in the expression levels of OCT1, ABCB1 and ABCG2 was found between the two categories. The median transcript expression levels of OCT1, ABCB1 and ABCG2 genes in responders were 26.54, 10.78 and 0.64 versus 33.48, 7.09 and 0.53 in non-responders, respectively. A positive association was observed between the expression of the ABCB1 and ABCG2 transporter genes (r=0.407, P<0.05 while no association was observed between the expression of either of the ABC transporter genes with the OCT1 gene. Interpretation & conclusions: Our findings demonstrated that the mRNA expression levels of imatinib transporter genes were not correlated with molecular response in CML patients. Further studies need to be done on a large sample of CML patients to confirm these findings.

  19. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy

    Directory of Open Access Journals (Sweden)

    Ke-Yi Wang

    2015-07-01

    Full Text Available Ryanodine receptors (RyRs play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR, an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.

  20. Identification of Two Distinct Molecular Subtypes of Non-Invasive Follicular Neoplasm with Papillary-Like Nuclear Features by Digital RNA Counting.

    Science.gov (United States)

    Giannini, Riccardo; Ugolini, Clara; Poma, Anello Marcello; Urpì, Maria; Niccoli, Cristina; Elisei, Rossella; Chiarugi, Massimo; Vitti, Paolo; Miccoli, Paolo; Basolo, Fulvio

    2017-10-01

    The follicular variant (FV) of papillary thyroid cancer (PTC) is one of the most common variants of PTC. Clinically, non-infiltrative FVPTC is considered a low-risk variant of PTC, and the non-invasive encapsulated forms of FVPTC represent a group of thyroid tumors with a particularly good prognosis. Consequently, these neoplasms have been very recently reclassified as non-invasive follicular neoplasms with papillary-like nuclear features (NIFTP). From a molecular standpoint, NIFTP appears to be similar to follicular neoplasms. However, only limited data are currently available regarding their gene expression profile. The aim of this study was to identify specific molecular signatures of 26 NIFTPs compared to those of 19 follicular adenomas (FAs) and 18 infiltrative FVPTCs (IFVPTCs). A nanoString custom assay was used to perform mRNA expression analysis. All cases were also genotyped for BRAF, N-, H-, and K-RAS mutations. Samples were grouped on the basis of gene expression profiles by Pearson's correlation and non-negative matrix factorization clustering analysis. Finally, the uncorrelated shrunken centroid machine-learning algorithm was used to classify the samples. The results revealed distinct expression profiles of FAs and IFVPTCs. NIFTP samples can exhibit different expression profiles, more similar to FAs (FA-like) or to IFVPTCs (IFVPTC-like), and these different expression profiles largely depend on the presence of different mutations (RAS or BRAF). In conclusion, although further validation of the model is required by using a larger group of prospective cases, these data reinforce the hypothesis that IFVPTC-like NIFTPs might represent precursors of IFVPTC.

  1. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...... pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways....

  2. Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

    Science.gov (United States)

    Chu, Wuying; Fu, Guihong; Bing, Shiyu; Meng, Tao; Zhou, Ruixue; Cheng, Jia; Zhao, Falan; Zhang, Hongfang; Zhang, Jianshe

    2010-03-01

    The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%-76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%-80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

  3. Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure

    International Nuclear Information System (INIS)

    Henriksen, Niel M.; Davis, Darrell R.; Cheatham, Thomas E. III

    2012-01-01

    Restrained molecular dynamics simulations are a robust, though perhaps underused, tool for the end-stage refinement of biomolecular structures. We demonstrate their utility—using modern simulation protocols, optimized force fields, and inclusion of explicit solvent and mobile counterions—by re-investigating the solution structures of two RNA hairpins that had previously been refined using conventional techniques. The structures, both domain 5 group II intron ribozymes from yeast ai5γ and Pylaiella littoralis, share a nearly identical primary sequence yet the published 3D structures appear quite different. Relatively long restrained MD simulations using the original NMR restraint data identified the presence of a small set of violated distance restraints in one structure and a possibly incorrect trapped bulge nucleotide conformation in the other structure. The removal of problematic distance restraints and the addition of a heating step yielded representative ensembles with very similar 3D structures and much lower pairwise RMSD values. Analysis of ion density during the restrained simulations helped to explain chemical shift perturbation data published previously. These results suggest that restrained MD simulations, with proper caution, can be used to “update” older structures or aid in the refinement of new structures that lack sufficient experimental data to produce a high quality result. Notable cautions include the need for sufficient sampling, awareness of potential force field bias (such as small angle deviations with the current AMBER force fields), and a proper balance between the various restraint weights.

  4. Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure.

    Science.gov (United States)

    Henriksen, Niel M; Davis, Darrell R; Cheatham, Thomas E

    2012-08-01

    Restrained molecular dynamics simulations are a robust, though perhaps underused, tool for the end-stage refinement of biomolecular structures. We demonstrate their utility-using modern simulation protocols, optimized force fields, and inclusion of explicit solvent and mobile counterions-by re-investigating the solution structures of two RNA hairpins that had previously been refined using conventional techniques. The structures, both domain 5 group II intron ribozymes from yeast ai5γ and Pylaiella littoralis, share a nearly identical primary sequence yet the published 3D structures appear quite different. Relatively long restrained MD simulations using the original NMR restraint data identified the presence of a small set of violated distance restraints in one structure and a possibly incorrect trapped bulge nucleotide conformation in the other structure. The removal of problematic distance restraints and the addition of a heating step yielded representative ensembles with very similar 3D structures and much lower pairwise RMSD values. Analysis of ion density during the restrained simulations helped to explain chemical shift perturbation data published previously. These results suggest that restrained MD simulations, with proper caution, can be used to "update" older structures or aid in the refinement of new structures that lack sufficient experimental data to produce a high quality result. Notable cautions include the need for sufficient sampling, awareness of potential force field bias (such as small angle deviations with the current AMBER force fields), and a proper balance between the various restraint weights.

  5. Catalytic thermal barrier coatings

    Science.gov (United States)

    Kulkarni, Anand A.; Campbell, Christian X.; Subramanian, Ramesh

    2009-06-02

    A catalyst element (30) for high temperature applications such as a gas turbine engine. The catalyst element includes a metal substrate such as a tube (32) having a layer of ceramic thermal barrier coating material (34) disposed on the substrate for thermally insulating the metal substrate from a high temperature fuel/air mixture. The ceramic thermal barrier coating material is formed of a crystal structure populated with base elements but with selected sites of the crystal structure being populated by substitute ions selected to allow the ceramic thermal barrier coating material to catalytically react the fuel-air mixture at a higher rate than would the base compound without the ionic substitutions. Precious metal crystallites may be disposed within the crystal structure to allow the ceramic thermal barrier coating material to catalytically react the fuel-air mixture at a lower light-off temperature than would the ceramic thermal barrier coating material without the precious metal crystallites.

  6. Understanding the molecular mechanisms underlying the effects of light intensity on flavonoid production by RNA-seq analysis in Epimedium pseudowushanense B.L.Guo.

    Directory of Open Access Journals (Sweden)

    Junqian Pan

    Full Text Available Epimedium pseudowushanense B.L.Guo, a light-demanding shade herb, is used in traditional medicine to increase libido and strengthen muscles and bones. The recognition of the health benefits of Epimedium has increased its market demand. However, its resource recycling rate is low and environmentally dependent. Furthermore, its natural sources are endangered, further increasing prices. Commercial culture can address resource constraints of it.Understanding the effects of environmental factors on the production of its active components would improve the technology for cultivation and germplasm conservation. Here, we studied the effects of light intensities on the flavonoid production and revealed the molecular mechanism using RNA-seq analysis. Plants were exposed to five levels of light intensity through the periods of germination to flowering, the flavonoid contents were measured using HPLC. Quantification of epimedin A, epimedin B, epimedin C, and icariin showed that the flavonoid contents varied with different light intensity levels. And the largest amount of epimedin C was produced at light intensity level 4 (I4. Next, the leaves under the treatment of three light intensity levels ("L", "M" and "H" with the largest differences in the flavonoid content, were subjected to RNA-seq analysis. Transcriptome reconstruction identified 43,657 unigenes. All unigene sequences were annotated by searching against the Nr, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes (KEGG databases. In total, 4008, 5260, and 3591 significant differentially expressed genes (DEGs were identified between the groups L vs. M, M vs. H and L vs. H. Particularly, twenty-one full-length genes involved in flavonoid biosynthesis were identified. The expression levels of the flavonol synthase, chalcone synthase genes were strongly associated with light-induced flavonoid abundance with the highest expression levels found in the H group. Furthermore, 65 transcription factors

  7. Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells.

    Science.gov (United States)

    Arami, Sanam; Mahdavi, Majid; Rashidi, Mohammad-Reza; Yekta, Reza; Rahnamay, Mohammad; Molavi, Leila; Hejazi, Mohammad-Saeid; Samadi, Nasser

    2017-08-05

    Delivery of small interfering RNAs (siRNAs) into cells still remains a challenge in gene delivery studies. Here, we investigated the ability of synthesized Fe 3 O 4 -PEG-LAC-chitosan-PEI nanoparticles for siRNA delivery of survivin as the model gene into cells. The cellular uptake of survivin siRNA carried by synthesized nanoparticles into MCF-7 breast cancer cell line was evaluated by florescent microscopy and flowcytometry, both proving the efficacy of nanoparticles in delivery of up to 64.7% in comparison with lipofectamine 2000. Furthermore, the delivery of survivin siRNA by the nanoparticles (nanoplex) induced apoptosis that was assessed through DAPI staining and Annexin V/PI assays. In addition, we evaluated the efficacy of treatment with nanoplexes in the presence of mitoxantrone, as a chemotherapeutic agent. Our data indicated that inhibition of survivin expression increased the cell sensitivity to mitoxantrone. Real-time PCR and western blotting analysis revealed a significant reduction in mRNA and protein levels of survivin upon delivery of siRNA. Molecular docking studies showed that nanoparticles can bind to centeral BIR domain of survivin, exactly above zinc ion location with high affinity (ΔG: -10.3Kcal/mol). Also, thermodynamic studies proved the experimental results theoretically, revealing that the siRNA-loaded nanoparticles have a suppressing effect on survivin mRNA. Therefore, delivery of survivin siRNA into MCF-7 cells using Fe 3 O 4 -PEG-LAC-chitosan-PEI nanoparticles as a carrier enhances the cell death. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

    Science.gov (United States)

    Fujimura, Tsutomu; Esteban, Rosa

    2016-10-01

    The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm. © 2016 John Wiley & Sons Ltd.

  9. Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference.

    Science.gov (United States)

    Tants, Jan-Niklas; Fesser, Stephanie; Kern, Thomas; Stehle, Ralf; Geerlof, Arie; Wunderlich, Christoph; Juen, Michael; Hartlmüller, Christoph; Böttcher, Romy; Kunzelmann, Stefan; Lange, Oliver; Kreutz, Christoph; Förstemann, Klaus; Sattler, Michael

    2017-12-01

    RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. 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

  11. Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions

    Directory of Open Access Journals (Sweden)

    Guelorget Amandine

    2011-12-01

    Full Text Available Abstract Background tRNA m1A58 methyltransferases (TrmI catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m1A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers. Results In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges. Conclusions The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.

  12. Molecular characterization and mRNA expression of hypoxia inducible factor-1 and cognate inhibiting factor in Macrobrachium nipponense in response to hypoxia.

    Science.gov (United States)

    Sun, Shengming; Xuan, Fujun; Fu, Hongtuo; Ge, Xianping; Zhu, Jian; Qiao, Hui; Jin, Shubo; Zhang, Wenyi

    2016-01-01

    Hypoxia inducible factors (HIFs) are considered to be the master switches of oxygen-dependent gene expression in mammalian species. Currently, very little is known about the function of this important pathway or the molecular structures of key players in the hypoxia-sensitive Oriental River Prawn Macrobrachium nipponense. In this study, HIFs-1α (HIF-1α), -1β (HIF-1β) and HIF 1-alpha inhibitor (FIH-1) from M. nipponense were cloned. The 4903-bp cDNA of M. nipponense HIF-1α (MnHIF-1α) encodes a protein of 1088 aa, M. nipponense HIF-1β (MnHIF-1β) spans 2042bp encoding 663 aa and the 1163bp M. nipponense FIH-1 (MnFIH-1) specifies a polypeptide of 345 aa. MnHIF-1 and MnFIH-1 homologs exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate isoforms. Phylogenetic analysis identifies that genetic diversification of HIF-1 and FIH-1 occurred within the invertebrate lineage, indicating functional specialization of the oxygen sensing pathways in this group. Quantitative real-time RT-PCR demonstrated that MnHIF-1 and MnFIH-1 mRNA are expressed in different tissues and exhibit transcriptional responses to severe hypoxia in gill and muscle tissue, consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. The role of HIF-1α in response to hypoxia was further investigated in the gills and muscles of prawns using in situ hybridization. These results suggested that HIF-1α plays an important role in oxygen sensing and homeostasis in M. nipponense. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. MOLECULAR PHYLOGENY OF THE NERITIDAE (GASTROPODA: NERITIMORPHA BASED ON THE MITOCHONDRIAL GENES CYTOCHROME OXIDASE I (COI AND 16S rRNA

    Directory of Open Access Journals (Sweden)

    Julián Fernando Quintero Galvis

    2013-05-01

    La familia Neritidae cuenta con representantes en regiones tropicales y subtropicales adaptadas a diferentes ambientes, con un registro fósil que data para finales del Cretáceo. Sin embargo no se han realizado estudios de filogenia molecular en la familia. En este estudio se realizó una reconstrucción filogenética de la familia Neritidae utilizando las regiones COI (722 pb y 16S rRNA (559 pb del genoma mitocondrial. Se realizaron análisis de distancias de Neighbor-Joining, Máxima Parsimonia e Inferencia Bayesiana. La mejor reconstrucción filogenética fue mediante la región COI, considerándola un marcador apropiado para realizar estudios filogenéticos dentro del grupo. El consenso de las relaciones filogenéticas (COI+16S rRNA permitió confirmar que el género Nerita es monofilético. El consenso del análisis de parsimonia reveló un grupo monofilético formado por los géneros Neritina, Septaria, Theodoxus, Puperita y Clithon, mientras que en el análisis bayesiano Theodoxus se encuentra separado de los otros géneros. El resultado en las especies del género Nerita del Caribe colombiano fue consistente con lo reportado para el género en estudios previos. En el árbol resultante del análisis de parsimonia se sobrepuso la

  14. Genes 16S RNA ribossomal e pld como marcadores moleculares para identificação genotípica de amostras clínicas de Corynebacterium spp.

    OpenAIRE

    Ericsson de Oliveira Xavier, Alessandra Rejane; de Moura Freitas, Angélica Alves; de almeida, Anna Christina; de Carvalho Azevedo, Vasco Ariston; Brandi, Igor Viana

    2016-01-01

    The genetic characterization of species is essential when you want to elect a vaccine strain or control the spread of outbreaks of a disease in a region. The 16S rRNA and pld Corynebacterium pseudotuberculosis genes play an important role as markers for bacterial genotype identification. The aim of this study was to validate an “in house” molecular methodology for genetic identification of Corynebacterium spp. isolated from clinical samples maintained in the laboratory. For this purpose, elev...

  15. Catalytic reforming methods

    Science.gov (United States)

    Tadd, Andrew R; Schwank, Johannes

    2013-05-14

    A catalytic reforming method is disclosed herein. The method includes sequentially supplying a plurality of feedstocks of variable compositions to a reformer. The method further includes adding a respective predetermined co-reactant to each of the plurality of feedstocks to obtain a substantially constant output from the reformer for the plurality of feedstocks. The respective predetermined co-reactant is based on a C/H/O atomic composition for a respective one of the plurality of feedstocks and a predetermined C/H/O atomic composition for the substantially constant output.

  16. Multiple RNA processing defects and impaired chloroplast function in plants deficient in the organellar protein-only RNase P enzyme.

    Directory of Open Access Journals (Sweden)

    Wenbin Zhou

    Full Text Available Transfer RNA (tRNA precursors undergo endoribonucleolytic processing of their 5' and 3' ends. 5' cleavage of the precursor transcript is performed by ribonuclease P (RNase P. While in most organisms RNase P is a ribonucleoprotein that harbors a catalytically active RNA component, human mitochondria and the chloroplasts (plastids and mitochondria of seed plants possess protein-only RNase P enzymes (PRORPs. The plant organellar PRORP (PRORP1 has been characterized to some extent in vitro and by transient gene silencing, but the molecular, phenotypic and physiological consequences of its down-regulation in stable transgenic plants have not been assessed. Here we have addressed the function of the dually targeted organellar PRORP enzyme in vivo by generating stably transformed Arabidopsis plants in which expression of the PRORP1 gene was suppressed by RNA interference (RNAi. PRORP1 knock-down lines show defects in photosynthesis, while mitochondrial respiration is not appreciably affected. In both plastids and mitochondria, the effects of PRORP1 knock-down on the processing of individual tRNA species are highly variable. The drastic reduction in the levels of mature plastid tRNA-Phe(GAA and tRNA-Arg(ACG suggests that these two tRNA species limit plastid gene expression in the PRORP1 mutants and, hence, are causally responsible for the mutant phenotype.

  17. Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA

    Energy Technology Data Exchange (ETDEWEB)

    Wasmuth, Elizabeth V.; Januszyk, Kurt; Lima, Christopher D. [MSKCC

    2014-08-20

    The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3' to 5' exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 Å crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3' end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome.

  18. Novel Catalytic Membrane Reactors

    Energy Technology Data Exchange (ETDEWEB)

    Stuart Nemser, PhD

    2010-10-01

    There are many industrial catalytic organic reversible reactions with amines or alcohols that have water as one of the products. Many of these reactions are homogeneously catalyzed. In all cases removal of water facilitates the reaction and produces more of the desired chemical product. By shifting the reaction to right we produce more chemical product with little or no additional capital investment. Many of these reactions can also relate to bioprocesses. Given the large number of water-organic compound separations achievable and the ability of the Compact Membrane Systems, Inc. (CMS) perfluoro membranes to withstand these harsh operating conditions, this is an ideal demonstration system for the water-of-reaction removal using a membrane reactor. Enhanced reaction synthesis is consistent with the DOE objective to lower the energy intensity of U.S. industry 25% by 2017 in accord with the Energy Policy Act of 2005 and to improve the United States manufacturing competitiveness. The objective of this program is to develop the platform technology for enhancing homogeneous catalytic chemical syntheses.

  19. Catalytic membrane-installed microchannel reactors for one-second allylic arylation.

    Science.gov (United States)

    Yamada, Yoichi M A; Watanabe, Toshihiro; Torii, Kaoru; Uozumi, Yasuhiro

    2009-10-07

    A variety of catalytic membranes of palladium-complexes with linear polymer ligands were prepared inside a microchannel reactor via coordinative and ionic molecular convolution to provide catalytic membrane-installed microdevices, which were applied to the instantaneous allylic arylation reaction of allylic esters and aryl boron reagents under microflow conditions to afford the corresponding coupling products within 1 second of residence time.

  20. 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.

  1. Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets

    NARCIS (Netherlands)

    Farazi, Thalia A.; ten Hoeve, Jelle J.; Brown, Miguel; Mihailovic, Aleksandra; Horlings, Hugo M.; van de Vijver, Marc J.; Tuschl, Thomas; Wessels, Lodewyk F. A.

    2014-01-01

    Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to

  2. The microRNA molecular signature of atypic and common acquired melanocytic nevi: differential expression of miR-125b and let-7c

    DEFF Research Database (Denmark)

    Holst, Line Marie Broksø; Kaczkowski, Bogumil; Glud, Martin

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression through base pairing with mRNA and which are crucially involved in carcinogenesis (the so-called oncomiRs). We compared the miRNA signature between acquired melanocytic nevi showing clinical atypia (atypic nevi, AN...

  3. Catalytic pyrolysis of olive mill wastewater sludge

    Science.gov (United States)

    Abdellaoui, Hamza

    From 2008 to 2013, an average of 2,821.4 kilotons/year of olive oil were produced around the world. The waste product of the olive mill industry consists of solid residue (pomace) and wastewater (OMW). Annually, around 30 million m3 of OMW are produced in the Mediterranean area, 700,000 m3 year?1 in Tunisia alone. OMW is an aqueous effluent characterized by an offensive smell and high organic matter content, including high molecular weight phenolic compounds and long-chain fatty acids. These compounds are highly toxic to micro-organisms and plants, which makes the OMW a serious threat to the environment if not managed properly. The OMW is disposed of in open air evaporation ponds. After evaporation of most of the water, OMWS is left in the bottom of the ponds. In this thesis, the effort has been made to evaluate the catalytic pyrolysis process as a technology to valorize the OMWS. The first section of this research showed that 41.12 wt. % of the OMWS is mostly lipids, which are a good source of energy. The second section proved that catalytic pyrolysis of the OMWS over red mud and HZSM-5 can produce green diesel, and 450 °C is the optimal reaction temperature to maximize the organic yields. The last section revealed that the HSF was behind the good fuel-like properties of the OMWS catalytic oils, whereas the SR hindered the bio-oil yields and quality.

  4. LincRNA-p21 inhibits invasion and metastasis of hepatocellular carcinoma through miR-9/E-cadherin cascade signaling pathway molecular mechanism

    Directory of Open Access Journals (Sweden)

    Ding G

    2017-06-01

    Full Text Available Gangqiang Ding, Zhen Peng, Jia Shang, Yi Kang, Huibin Ning, Chongshan Mao Department of Infectious Diseases, People’s Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, China Abstract: In the previous study, it was found that long intergenic noncoding RNA-p21 (lincRNA-p21 was downregulated in hepatocellular carcinoma (HCC and lincRNA-p21 overexpression inhibited tumor invasion through inducing epithelial–mesenchymal transition. However, the underlying mechanism was not fully elaborated. In this study, lincRNA-p21 expression was measured in 12 paired HCC and nontumor adjacent normal tissues by ­quantitative real-time polymerase chain reaction. The effects of lincRNA-p21 on HCC cells were studied using lentivirus expressing lincRNA-p21 vector in vitro. The association between lincRNA-p21 level and miR-9 level was tested with the Spearman rank correlation. The effects of miR-9 on HCC cells were studied by using miR-9 inhibitor in vitro. Luciferase assay was used to validate the target of miR-9. The results showed that lincRNA-p21 was downregulated in human HCC tissues and cell lines. LincRNA-p21 overexpression significantly inhibited HCC cell migration and invasion in vitro. Besides, lincRNA-p21 negatively regulated miR-9 expression level, and miR-9 was upregulated in human HCC tissues and cells. MiR-9 knockdown inhibited HCC cell migration and invasion in vitro. Finally, the luciferase assay results showed that E-cadherin was a direct target of miR-9. The expression level of E-cadherin was found to be regulated by lincRNA-p21 and miR-9. Altogether, the results suggested that lincRNA-p21 inhibits migration and invasion of HCC cells through regulating miR-9-mediated E-cadherin cascade signaling pathway. Keywords: hepatocellular carcinoma, lincRNA-p21, miR-9, E-cadherin, epithelial–mesenchymal transition

  5. Catalytic Antibodies: Concept and Promise

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 11. Catalytic Antibodies: Concept and Promise. Desirazu N Rao Bharath Wootla. General Article Volume 12 Issue ... Keywords. Catalytic antibodies; abzymes; hybridome technology; Diels– Alder reaction; Michaelis– Menten kinetics; Factor VIII.

  6. Catalytic aspects of a copper (II) complex: biological oxidase to ...

    Indian Academy of Sciences (India)

    This copper complex displays excellent catalytic efficiency, kcat /KM (h⁻¹) = 6.17 × 10⁵ towards the oxidative coupling of 2-aminophenol (2-AP) to aminophenoxazin-3-one. Further, upon stoichiometric addition of copper(II) complex to 3,5-DTBC in presence of molecular oxygen in ethanol medium, the copper complex ...

  7. Key steps from the “RNA World” to the “DNA World”

    Directory of Open Access Journals (Sweden)

    Renard B.-L.

    2014-02-01

    Full Text Available In the « RNA World » hypothesis of the origin of life, RNAs are assumed to be the central macromolecules able to self-replicate, conserve information and catalyze the reactions necessary for a primitive metabolism and many enzymatic cofactors may be regarded as molecular fossils of the “RNA World”. In the key steps involved in the transition from the RNA World to the DNA World, two main steps can be distinguished: (i the synthesis of 2’-deoxyribonucleotides from ribonucleotides catalyzed nowadays by the enzyme ribonucleotide reductase and (ii the synthesis of thymine, a base specific for DNA, from uracil which is a base specific for RNA, catalyzed today by the enzyme thymidylate synthase. In regard to the chemistry of sulfur used by both enzymes for achieving their respective catalysis, we were interested in the search for simple sulfur reactions able to catalyze such transformations and report here on first results in an approach from thionucleosides to the catalysis involved in the conversion of uracil to thymine. In the RNA World, the recruitment of cofactors was crucial to expand the catalytic repertoire of RNA and we also describe interesting preliminary results obtained in the prebiotic synthesis of pyridoxal (vitamin B6 that is the precursor of the key coenzyme pyridoxal phosphate (PLP able to catalyze nowadays seven different enzymatic reactions.

  8. tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry

    Science.gov (United States)

    Carter, Charles W.; Wolfenden, Richard

    2016-01-01

    abstract The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology. PMID:26595350

  9. Catalytic interface erosion

    International Nuclear Information System (INIS)

    Meng, H.; Cohen, E.G.D.

    1995-01-01

    We study interface erosion processes: catalytic erosions. We present two cases. (1) The erosion of a completely occupied lattice by one single moving particle starting from somewhere inside the lattice, considering deterministic as well as probabilistic erosion rules. In the latter case, the eroded regions appear to have interfaces with continuously tunable fractal dimensions. (2) The kinetic roughening of an initially flat surface, where ballistic or diffusion-limited particles, which remain intact themselves, erode the surface coming from the outside, using the same erosion rules as in (1). Many features resembling realistic interfaces, for example, islands and inlets, are generated. The dependence of the surface width on the system size is due to both the erosion mechanism and the way particles move before reaching the surface

  10. Catalytic detritiation of water

    International Nuclear Information System (INIS)

    Rogers, M.L.; Lamberger, P.H.; Ellis, R.E.; Mills, T.K.

    1977-01-01

    A pilot-scale system has been used at Mound Laboratory to investigate the catalytic detritiation of water. A hydrophobic, precious metal catalyst is used to promote the exchange of tritium between liquid water and gaseous hydrogen at 60 0 C. Two columns are used, each 7.5 m long by 2.5 cm ID and packed with catalyst. Water flow is 5-10 cm 3 /min and countercurrent hydrogen flow is 9,000-12,000 cm 3 /min. The equipment, except for the columns, is housed in an inert atmosphere glovebox and is computer controlled. The hydrogen is obtained by electrolysis of a portion of the water stream. Enriched gaseous tritium is withdrawn for further enrichment. A description of the system is included along with an outline of its operation. Recent experimental data are discussed

  11. 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...

  12. Mapping the active site of vaccinia virus RNA triphosphatase

    International Nuclear Information System (INIS)

    Gong Chunling; Shuman, Stewart

    2003-01-01

    The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

  13. The microRNA molecular signature of atypic and common acquired melanocytic nevi: differential expression of miR-125b and let-7c

    DEFF Research Database (Denmark)

    Holst, Line Marie Broksø; Kaczkowski, Bogumil; Glud, Martin

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression through base pairing with mRNA and which are crucially involved in carcinogenesis (the so-called oncomiRs). We compared the miRNA signature between acquired melanocytic nevi showing clinical atypia (atypic nevi, AN......) and common acquired nevi (common nevi, CN). We obtained miRNA profiles from 41 biopsies (22 AN and 19 CN) and showed that AN could be differentiated from CN on the basis of the expression of 36 miRNAs (false discovery rate...

  14. Catalytic Combustion of Gasified Waste

    Energy Technology Data Exchange (ETDEWEB)

    Kusar, Henrik

    2003-09-01

    This thesis concerns catalytic combustion for gas turbine application using a low heating-value (LHV) gas, derived from gasified waste. The main research in catalytic combustion focuses on methane as fuel, but an increasing interest is directed towards catalytic combustion of LHV fuels. This thesis shows that it is possible to catalytically combust a LHV gas and to oxidize fuel-bound nitrogen (NH{sub 3}) directly into N{sub 2} without forming NO{sub x} The first part of the thesis gives a background to the system. It defines waste, shortly describes gasification and more thoroughly catalytic combustion. The second part of the present thesis, paper I, concerns the development and testing of potential catalysts for catalytic combustion of LHV gases. The objective of this work was to investigate the possibility to use a stable metal oxide instead of noble metals as ignition catalyst and at the same time reduce the formation of NO{sub x} In paper II pilot-scale tests were carried out to prove the potential of catalytic combustion using real gasified waste and to compare with the results obtained in laboratory scale using a synthetic gas simulating gasified waste. In paper III, selective catalytic oxidation for decreasing the NO{sub x} formation from fuel-bound nitrogen was examined using two different approaches: fuel-lean and fuel-rich conditions. Finally, the last part of the thesis deals with deactivation of catalysts. The various deactivation processes which may affect high-temperature catalytic combustion are reviewed in paper IV. In paper V the poisoning effect of low amounts of sulfur was studied; various metal oxides as well as supported palladium and platinum catalysts were used as catalysts for combustion of a synthetic gas. In conclusion, with the results obtained in this thesis it would be possible to compose a working catalytic system for gas turbine application using a LHV gas.

  15. ResBoost: characterizing and predicting catalytic residues in enzymes

    Directory of Open Access Journals (Sweden)

    Freund Yoav

    2009-06-01

    Full Text Available Abstract Background Identifying the catalytic residues in enzymes can aid in understanding the molecular basis of an enzyme's function and has significant implications for designing new drugs, identifying genetic disorders, and engineering proteins with novel functions. Since experimentally determining catalytic sites is expensive, better computational methods for identifying catalytic residues are needed. Results We propose ResBoost, a new computational method to learn characteristics of catalytic residues. The method effectively selects and combines rules of thumb into a simple, easily interpretable logical expression that can be used for prediction. We formally define the rules of thumb that are often used to narrow the list of candidate residues, including residue evolutionary conservation, 3D clustering, solvent accessibility, and hydrophilicity. ResBoost builds on two methods from machine learning, the AdaBoost algorithm and Alternating Decision Trees, and provides precise control over the inherent trade-off between sensitivity and specificity. We evaluated ResBoost using cross-validation on a dataset of 100 enzymes from the hand-curated Catalytic Site Atlas (CSA. Conclusion ResBoost achieved 85% sensitivity for a 9.8% false positive rate and 73% sensitivity for a 5.7% false positive rate. ResBoost reduces the number of false positives by up to 56% compared to the use of evolutionary conservation scoring alone. We also illustrate the ability of ResBoost to identify recently validated catalytic residues not listed in the CSA.

  16. RNA self-assembly and RNA nanotechnology.

    Science.gov (United States)

    Grabow, Wade W; Jaeger, Luc

    2014-06-17

    CONSPECTUS: Nanotechnology's central goal involves the direct control of matter at the molecular nanometer scale to build nanofactories, nanomachines, and other devices for potential applications including electronics, alternative fuels, and medicine. In this regard, the nascent use of nucleic acids as a material to coordinate the precise arrangements of specific molecules marked an important milestone in the relatively recent history of nanotechnology. While DNA served as the pioneer building material in nucleic acid nanotechnology, RNA continues to emerge as viable alternative material with its own distinct advantages for nanoconstruction. Several complementary assembly strategies have been used to build a diverse set of RNA nanostructures having unique structural attributes and the ability to self-assemble in a highly programmable and controlled manner. Of the different strategies, the architectonics approach uniquely endeavors to understand integrated structural RNA architectures through the arrangement of their characteristic structural building blocks. Viewed through this lens, it becomes apparent that nature routinely uses thermodynamically stable, recurrent modular motifs from natural RNA molecules to generate unique and more complex programmable structures. With the design principles found in natural structures, a number of synthetic RNAs have been constructed. The synthetic nanostructures constructed to date have provided, in addition to affording essential insights into RNA design, important platforms to characterize and validate the structural self-folding and assembly properties of RNA modules or building blocks. Furthermore, RNA nanoparticles have shown great promise for applications in nanomedicine and RNA-based therapeutics. Nevertheless, the synthetic RNA architectures achieved thus far consist largely of static, rigid particles that are still far from matching the structural and functional complexity of natural responsive structural elements such

  17. Catalytic production of biodiesel

    Energy Technology Data Exchange (ETDEWEB)

    Theilgaard Madsen, A.

    2011-07-01

    The focus of this thesis is the catalytic production of diesel from biomass, especially emphasising catalytic conversion of waste vegetable oils and fats. In chapter 1 an introduction to biofuels and a review on different catalytic methods for diesel production from biomass is given. Two of these methods have been used industrially for a number of years already, namely the transesterification (and esterification) of oils and fats with methanol to form fatty acid methyl esters (FAME), and the hydrodeoxygenation (HDO) of fats and oils to form straight-chain alkanes. Other possible routes to diesel include upgrading and deoxygenation of pyrolysis oils or aqueous sludge wastes, condensations and reductions of sugars in aqueous phase (aqueous-phase reforming, APR) for monofunctional hydrocarbons, and gasification of any type of biomass followed by Fischer-Tropsch-synthesis for alkane biofuels. These methods have not yet been industrialised, but may be more promising due to the larger abundance of their potential feedstocks, especially waste feedstocks. Chapter 2 deals with formation of FAME from waste fats and oils. A range of acidic catalysts were tested in a model fat mixture of methanol, lauric acid and trioctanoin. Sulphonic acid-functionalised ionic liquids showed extremely fast convertion of lauric acid to methyl laurate, and trioctanoate was converted to methyl octanoate within 24 h. A catalyst based on a sulphonated carbon-matrix made by pyrolysing (or carbonising) carbohydrates, so-called sulphonated pyrolysed sucrose (SPS), was optimised further. No systematic dependency on pyrolysis and sulphonation conditions could be obtained, however, with respect to esterification activity, but high activity was obtained in the model fat mixture. SPS impregnated on opel-cell Al{sub 2}O{sub 3} and microporous SiO{sub 2} (ISPS) was much less active in the esterification than the original SPS powder due to low loading and thereby low number of strongly acidic sites on the

  18. DbpA is a region-specific RNA helicase.

    Science.gov (United States)

    Moore, Anthony F T; Gentry, Riley C; Koculi, Eda

    2017-03-01

    DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site-specific enzyme or region-specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region-specific or a site-specific enzyme. Our data suggest that DbpA is a region-specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C-terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double-helices in its vicinity. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core's grasp. © 2016 Wiley Periodicals, Inc.

  19. Metallocene Catalytic Insertion Polymerization of 1-Silene to Polycarbosilanes

    Science.gov (United States)

    Tian, Yuelong; Ge, Min; Zhang, Weigang; Lv, Xiaoxu; Yu, Shouquan

    2015-11-01

    Metallocene of zirconium were used as a catalyst for an insertion polymerization of 1-methylsilene directly into pre-ceramic precursor polyzirconocenecarbosilane (PZCS) during dechlorination of dichlorodimethylesilane by sodium, which exhibits high catalytic effectiveness with the maximum conversion ratio of polycarbosilane up to 91%. The average molecular weights of polymers synthesized are less than 1400, all with very narrow polymolecularities. The mechanism of catalytic polymerization was assumed to be similar to a coordination insertion polymerization of 1-olefins by metallocenes. The obtained PZCS show high ceramic yields with formation of composite ceramics of ZrC-SiC, which are novel polymeric precursors of ultra-high temperature ceramic (UHTC) fiber and composite.

  20. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... of molecular biology were thinking on gene expression and genetic code. In a famous letter send in 1955 to the “RNA Tie. Club” Francis Crick predicted the existence of small adaptor. RNA molecules that would carry their own amino acids and. The early history of tRNA recognition by aminoacyl-tRNA.

  1. Catalytic Membrane Sensors

    Energy Technology Data Exchange (ETDEWEB)

    Boyle, T.J.; Brinker, C.J.; Gardner, T.J.; Hughes, R.C.; Sault, A.G.

    1998-12-01

    The proposed "catalytic membrane sensor" (CMS) was developed to generate a device which would selectively identify a specific reagent in a complex mixture of gases. This was to be accomplished by modifying an existing Hz sensor with a series of thin films. Through selectively sieving the desired component from a complex mixture and identifying it by decomposing it into Hz (and other by-products), a Hz sensor could then be used to detect the presence of the select component. The proposed "sandwich-type" modifications involved the deposition of a catalyst layered between two size selective sol-gel layers on a Pd/Ni resistive Hz sensor. The role of the catalyst was to convert organic materials to Hz and organic by-products. The role of the membraneo was to impart both chemical specificity by molecukir sieving of the analyte and converted product streams, as well as controlling access to the underlying Pd/Ni sensor. Ultimately, an array of these CMS elements encompassing different catalysts and membranes were to be developed which would enable improved selectivity and specificity from a compiex mixture of organic gases via pattern recognition methodologies. We have successfully generated a CMS device by a series of spin-coat deposited methods; however, it was determined that the high temperature required to activate the catalyst, destroys the sensor.

  2. Catalytic cracking of lignites

    Energy Technology Data Exchange (ETDEWEB)

    Seitz, M.; Nowak, S.; Naegler, T.; Zimmermann, J. [Hochschule Merseburg (Germany); Welscher, J.; Schwieger, W. [Erlangen-Nuernberg Univ. (Germany); Hahn, T. [Halle-Wittenberg Univ., Halle (Germany)

    2013-11-01

    A most important factor for the chemical industry is the availability of cheap raw materials. As the oil price of crude oil is rising alternative feedstocks like coal are coming into focus. This work, the catalytic cracking of lignite is part of the alliance ibi (innovative Braunkohlenintegration) to use lignite as a raw material to produce chemicals. With this new one step process without an input of external hydrogen, mostly propylene, butenes and aromatics and char are formed. The product yield depends on manifold process parameters. The use of acid catalysts (zeolites like MFI) shows the highest amount of the desired products. Hydrogen rich lignites with a molar H/C ratio of > 1 are to be favoured. Due to primary cracking and secondary reactions the ratio between catalyst and lignite, temperature and residence time are the most important parameter to control the product distribution. Experiments at 500 C in a discontinuous rotary kiln reactor show yields up to 32 wt-% of hydrocarbons per lignite (maf - moisture and ash free) and 43 wt-% char, which can be gasified. Particularly, the yields of propylene and butenes as main products can be enhanced four times to about 8 wt-% by the use of catalysts while the tar yield decreases. In order to develop this innovative process catalyst systems fixed on beads were developed for an easy separation and regeneration of the used catalyst from the formed char. (orig.)

  3. Understanding in-line probing experiments by modeling cleavage of nonreactive RNA nucleotides.

    Science.gov (United States)

    Mlýnský, Vojtěch; Bussi, Giovanni

    2017-05-01

    Ribonucleic acid (RNA) is involved in many regulatory and catalytic processes in the cell. The function of any RNA molecule is intimately related with its structure. In-line probing experiments provide valuable structural data sets for a variety of RNAs and are used to characterize conformational changes in riboswitches. However, the structural determinants that lead to differential reactivities in unpaired nucleotides have not been investigated yet. In this work, we used a combination of theoretical approaches, i.e., classical molecular dynamics simulations, multiscale quantum mechanical/molecular mechanical calculations, and enhanced sampling techniques in order to compute and interpret the differential reactivity of individual residues in several RNA motifs, including members of the most important GNRA and UNCG tetraloop families. Simulations on the multinanosecond timescale are required to converge the related free-energy landscapes. The results for uGAAAg and cUUCGg tetraloops and double helices are compared with available data from in-line probing experiments and show that the introduced technique is able to distinguish between nucleotides of the uGAAAg tetraloop based on their structural predispositions toward phosphodiester backbone cleavage. For the cUUCGg tetraloop, more advanced ab initio calculations would be required. This study is the first attempt to computationally classify chemical probing experiments and paves the way for an identification of tertiary structures based on the measured reactivity of nonreactive nucleotides. © 2017 Mlýnský and Bussi; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

    Science.gov (United States)

    Hayashi, K; Tazumi, A; Nakanishi, S; Nakajima, T; Matsubara, K; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

    2012-06-01

    Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

  5. Molecular interactions and immune responses between Maize fine streak virus and the leafhopper vector Graminella nigrifrons through differential expression and RNA interference.

    Science.gov (United States)

    Chen, Y; Redinbaugh, M G; Michel, A P

    2015-06-01

    Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in G. nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G. nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G. nigrifrons transcripts will facilitate the study of gene function and pathogen transmission, and may provide approaches for developing novel targets of RNAi-based pest control. © 2015 The Royal Entomological Society.

  6. Molecular Characterization of the 16S rRNA Gene of Helicobacter fennelliae Isolated from Stools and Blood Cultures from Paediatric Patients in South Africa

    Directory of Open Access Journals (Sweden)

    Heidi E. M. Smuts

    2011-01-01

    Full Text Available Forty strains of H. fennelliae collected from paediatric blood and stool samples over an 18 year period at a children's hospital in Cape Town, South Africa, were amplified by PCR of the 16S rRNA. Two distinct genotypes of H. fennelliae were identified based on the phylogenetic analysis. This was confirmed by sequencing a portion of the beta subunit of the RNA polymerase (rpoB gene. All isolates from South Africa clustered with a proposed novel Helicobacter strain (accession number AF237612 isolated in Australia, while three H. fennelliae type strains from the northern hemisphere, NCTC 11612, LMG 7546 and CCUG 18820, formed a separate branch. A large (355bp highly conserved intervening sequence (IVS in the 16S rRNA was found in all isolates. Predicted secondary structures of the IVS from the 16S rRNA and 23S rRNA were characterised by a primary stem structure formed by base pairing of the 3′ and 5′ ends and internal loops and stems. This phylogenetic analysis is the largest undertaken of H. fennelliae. The South African H. fennelliae isolates are closely related to an Australian isolate previously reported to be a possible novel species of Helicobacter. This study suggests that the latter is strain of H. fennelliae.

  7. RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression.

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J

    2015-03-20

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Catalytic pyrolysis of lignocellulosic biomass

    NARCIS (Netherlands)

    Seshan, Kulathuiyer; Sa, Jacinto

    2014-01-01

    This chapter reports on the latest developments of biomass catalytic pyrolysis for the production of fuels. The primary focus is on the role of catalysts in the process, namely, their influence in the liquefaction of lignocellulosic biomass.

  9. Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Dutta, Summi; Kumar, Dhananjay; Jha, Shailendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

    2017-11-01

    A novel leaf rust responsive ta-siRNA-producing locus was identified in wheat showing similarity to 28S rRNA and generated four differentially expressing ta-siRNAs by phasing which targeted stress responsive genes. Trans-acting-small interfering RNAs (Ta-siRNAs) are plant specific molecules generally involved in development and are also stress responsive. Ta-siRNAs identified in wheat till date are all responsive to abiotic stress only. Wheat cultivation is severely affected by rusts and leaf rust particularly affects grain filling. This study reports a novel ta-siRNA producing locus (TAS) in wheat which is a segment of 28S ribosomal RNA but shows differential expression during leaf rust infestation. Four small RNA libraries prepared from wheat Near Isogenic Lines were treated with leaf rust pathogen and compared with untreated controls. A TAS with the ability to generate four ta-siRNAs by phasing events was identified along with the microRNA TamiR16 as the phase initiator. The targets of the ta-siRNAs included α-gliadin, leucine rich repeat, trans-membrane proteins, glutathione-S-transferase, and fatty acid desaturase among others, which are either stress responsive genes or are essential for normal growth and development of plants. Expression of the TAS, its generated ta-siRNAs, and their target genes were profiled at five different time points after pathogen inoculation of susceptible and resistant wheat isolines and compared with mock-inoculated controls. Comparative analysis of expression unveiled differential and reciprocal relationship as well as discrete patterns between susceptible and resistant isolines. The expression profiles of the target genes of the identified ta-siRNAs advocate more towards effector triggered susceptibility favouring pathogenesis. The study helps in discerning the functions of wheat genes regulated by ta-siRNAs in response to leaf rust.

  10. Molecular characterization of branchial aquaporin 1aa and effects of seawater acclimation, emersion or ammonia exposure on its mRNA expression in the gills, gut, kidney and skin of the freshwater climbing perch, Anabas testudineus.

    Directory of Open Access Journals (Sweden)

    Yuen K Ip

    Full Text Available We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH3 transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.

  11. The porcine skin associated T-cell homing chemokine CCL27: molecular cloning and mRNA expression in piglets infected experimentally with Staphylococcus hyicus

    DEFF Research Database (Denmark)

    Johnsen, C. K.; Jensen, Annette Nygaard; Ahrens, P.

    2003-01-01

    . In this paper, we report the cloning of porcine CCL27 cDNA and investigation of CCL27 mRNA expression in Staphylococcus hyicus infected piglets. At the protein level, 77 and 74% homology was found to human and mouse CCL27 sequences, respectively. The results of the expression analyses show that CCL27 m...

  12. Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains*

    Science.gov (United States)

    Barnes, Mark; van Rensburg, Gerrit; Li, Wai-Ming; Mehmood, Kashif; Mackedenski, Sebastian; Chan, Ching-Man; King, Dustin T.; Miller, Andrew L.; Lee, Chow H.

    2015-01-01

    The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function. PMID:25389298

  13. Relations between DNA- and RNA-based molecular methods for cyanobacteria and microcystin concentration at Maumee Bay State Park Lakeside Beach, Oregon, Ohio, 2012

    Science.gov (United States)

    Stelzer, Erin A.; Loftin, Keith A.; Struffolino, Pamela

    2013-01-01

    Water samples were collected from Maumee Bay State Park Lakeside Beach, Oregon, Ohio, during the 2012 recreational season and analyzed for selected cyanobacteria gene sequences by DNA-based quantitative polymerase chain reaction (qPCR) and RNA-based quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results from the four DNA assays (for quantifying total cyanobacteria, total Microcystis, and Microcystis and Planktothrix strains that possess the microcystin synthetase E (mcyE) gene) and two RNA assays (for quantifying Microcystis and Planktothrix genera that are expressing the microcystin synthetase E (mcyE) gene) were compared to microcystin concentration results determined by an enzyme-linked immunosorbent assay (ELISA). Concentrations of the target in replicate analyses were log10 transformed. The average value of differences in log10 concentrations for the replicates that had at least one detection were found to range from 0.05 to >0.37 copy per 100 milliliters (copy/100 mL) for DNA-based methods and from >0.04 to >0.17 copy/100 mL for RNA-based methods. RNA has a shorter half-life than DNA; consequently, a 24-hour holding-time study was done to determine the effects of holding time on RNA concentrations. Holding-time comparisons for the RNA-based Microcystis toxin mcyE assay showed reductions in the number of copies per 100 milliliters over 24 hours. The log difference between time 2 hours and time 24 hours was >0.37 copy/100 mL, which was higher than the analytical variability (log difference of >0.17 copy/100 mL). Spearman’s correlation analysis indicated that microcystin toxin concentrations were moderately to highly related to DNA-based assay results for total cyanobacteria (rho=0.69), total Microcystis (rho=0.74), and Microcystis strains that possess the mcyE gene (rho=0.81). Microcystin toxin concentrations were strongly related with RNA-based assay results for Microcystis mcyE gene expression (rho=0.95). Correlation analysis could

  14. Molecular analysis of methylmalonic acidemia: Identification of novel mutations in the methylmalonyl-CoA mutase gene with decreased level of mutant mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Ogasawara, M.; Matsubara, Y.; Mikami, H.; Narisawa, K. [Tohoku Univ. School of Medicine, Sendai (Japan)

    1994-09-01

    Deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive trait and clinically characterized by metabolic ketoacidosis. Previous studies of Caucasian and African American patients identified seven MCM mutations, and we also detected four missense substitutions (Ala197Thr, Val368Asp, Arg369His and Val669Glu). However, mutations with decreased level of MCM mRNA, which accounts for at least 25% of mutations among Caucasian patients, have not been reported. Our study on eight Japanese patients indicated that 13 of 16 mutant alleles (81%) showed decreased level of MCM mRNA, suggesting that these {open_quotes}low message{close_quotes} alleles are likely to be common contributors to MCM deficiency. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of these mutant mRNAs was less than 1% controls. We were able to amplify such mutant mRNAs by nested PCR and directly determine the primary structure. Sequence analysis revealed three novel mutations: a G-to-T substitution at nucleotide position 425, a 2 bp deletion at nt 769 and 770, and a G-to-T substitution at nt 326. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The analysis of 17 Japanese patients revealed the presence of G425T in 7 alleles (21%), suggesting a relatively high incidence of the mutation among Japanese patients. This observation is in sharp contrast to previous reports describing diverse heterogeneity of MCM mutations among Caucasians. Our report is the first to identify MCM mutations that decrease the stability of MCM mRNA. Amplification of trace amount of mRNA followed by sequencing analysis may provide useful tool for identifying such mutations.

  15. Binding properties of ruthenium(II) complexes [Ru(bpy)2(ppn)](2+) and [Ru(phen)2(ppn)](2+) with triplex RNA: As molecular "light switches" and stabilizers for poly(U)·poly(A)*poly(U) triplex.

    Science.gov (United States)

    Li, Jia; Sun, Yanmei; Zhu, Zhiyuan; Zhao, Hong; Tan, Lifeng

    2016-08-01

    Stable RNA triplexes play key roles in many biological processes, while triplexes are thermodynamically less stable than the corresponding duplexes due to the Hoogsteen base pairing. To understand the factors affecting the stabilization of RNA triplexes by octahedral ruthenium(II) complexes, the binding of [Ru(bpy)2(ppn)](2+) (1, bpy=2,2'-bipyridine, ppn=2,4-diaminopyrimido[5,6-b]dipyrido[2,3-f:2',3'-h]quinoxaline) and [Ru(phen)2(ppn)](2+) (2, phen=1,10-phenanthroline) to poly(U)·poly(A)*poly(U) (· denotes the Watson-Crick base pairing and * denotes the Hoogsteen base pairing) has been investigated. The main results obtained here suggest that complexes 1 and 2 can serve as molecular "light switches" and stabilizers for poly(U)·poly(A)*poly(U), while the effectiveness of complex 2 are more marked, suggesting that the hydrophobicity of ancillary ligands has a significant effect on the two Ru(II) complexes binding to poly(U)·poly(A)*poly(U). This study further advances our knowledge on the binding of RNA triplexes with metal complexes, particularly with octahedral ruthenium polypyridyl complexes. Copyright © 2016. Published by Elsevier Inc.

  16. MetalionRNA: computational predictor of metal-binding sites in RNA structures.

    Science.gov (United States)

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Boniecki, Michal; Rother, Kristian; Bujnicki, Janusz M

    2012-01-15

    Metal ions are essential for the folding of RNA molecules into stable tertiary structures and are often involved in the catalytic activity of ribozymes. However, the positions of metal ions in RNA 3D structures are difficult to determine experimentally. This motivated us to develop a computational predictor of metal ion sites for RNA structures. We developed a statistical potential for predicting positions of metal ions (magnesium, sodium and potassium), based on the analysis of binding sites in experimentally solved RNA structures. The MetalionRNA program is available as a web server that predicts metal ions for RNA structures submitted by the user. The MetalionRNA web server is accessible at http://metalionrna.genesilico.pl/.

  17. Docking and Molecular Dynamics Calculations of Some Previously Studied and newly Designed Ligands to Catalytic Core Domain of HIV-1 Integrase and an Investigation to Effects of Conformational Changes of Protein on Docking Results

    Directory of Open Access Journals (Sweden)

    Selami Ercan

    2016-10-01

    Full Text Available Nowadays, AIDS still remains as a worldwide pandemic and continues to cause many death which arise from HIV-1 virus. For nearly 35 years, drugs that target various steps of virus life cycle have been developed. HIV-1 integrase is the one of these steps which is essential for virus life cycle. Computer aided drug design is being used in many drug design studies as also used in development of the first HIV-1 integrase inhibitor Raltegravir. In this study 3 ligands which are used as HIV-1 integrase inhibitors and 4 newly designed ligands were docked to catalytic core domain of HIV-1 integrase. Each of ligands docked to three different conformations of protein. Prepared complexes (21 item were carried out by 50 ns MD simulations and results were analyzed. Finally, the binding free energies of ligands were calculated. Hereunder, it was determined that designed ligands L01 and L03 gave favorable results. The questions about the ligands which have low docking scores in a conformation of protein could give better scores in another conformation of protein and if the MD simulations carry the different oriented and different localized ligands in same position at the end of simulation were answered.

  18. Machine Learning Approaches Toward Building Predictive Models for Small Molecule Modulators of miRNA and Its Utility in Virtual Screening of Molecular Databases.

    Science.gov (United States)

    Periwal, Vinita; Scaria, Vinod

    2017-01-01

    The ubiquitous role of microRNAs (miRNAs) in a number of pathological processes has suggested that they could act as potential drug targets. RNA-binding small molecules offer an attractive means for modulating miRNA function. The availability of bioassay data sets for a variety of biological assays and molecules in public domain provides a new opportunity toward utilizing them to create models and further utilize them for in silico virtual screening approaches to prioritize or assign potential functions for small molecules. Here, we describe a computational strategy based on machine learning for creation of predictive models from high-throughput biological screens for virtual screening of small molecules with the potential to inhibit microRNAs. Such models could be potentially used for computational prioritization of small molecules before performing high-throughput biological assay.

  19. Crystal structure of the RNA component of bacterial ribonuclease P

    Energy Technology Data Exchange (ETDEWEB)

    Torres-Larios, Alfredo; Swinger, Kerren K.; Krasilnikov, Andrey S.; Pan, Tao; Mondragon, Alfonso (NWU); (UC)

    2010-03-08

    Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 {angstrom} resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.

  20. Genetics and Molecular Biology of Epstein-Barr Virus-Encoded BART MicroRNA: A Paradigm for Viral Modulation of Host Immune Response Genes and Genome Stability

    Directory of Open Access Journals (Sweden)

    David H. Dreyfus

    2017-01-01

    Full Text Available Epstein-Barr virus, a ubiquitous human herpesvirus, is associated through epidemiologic evidence with common autoimmune syndromes and cancers. However, specific genetic mechanisms of pathogenesis have been difficult to identify. In this review, the author summarizes evidence that recently discovered noncoding RNAs termed microRNA encoded by Epstein-Barr virus BARF (BamHI A right frame termed BART (BamHI A right transcripts are modulators of human immune response genes and genome stability in infected and bystander cells. BART expression is apparently regulated by complex feedback loops with the host immune response regulatory NF-κB transcription factors. EBV-encoded BZLF-1 (ZEBRA protein could also regulate BART since ZEBRA contains a terminal region similar to ankyrin proteins such as IκBα that regulate host NF-κB. BALF-2 (BamHI A left frame transcript, a viral homologue of the immunoglobulin and T cell receptor gene recombinase RAG-1 (recombination-activating gene-1, may also be coregulated with BART since BALF-2 regulatory sequences are located near the BART locus. Viral-encoded microRNA and viral mRNA transferred to bystander cells through vesicles, defective viral particles, or other mechanisms suggest a new paradigm in which bystander or hit-and-run mechanisms enable the virus to transiently or chronically alter human immune response genes as well as the stability of the human genome.

  1. Dipeptide catalysed prebiotic polymerization of RNA

    DEFF Research Database (Denmark)

    Wieczorek, Rafal; Luisi, Pier Luigi; Monnard, Pierre-Alain

    2011-01-01

    toward more peptide synthesis. In the present work we describe a prebiotically plausible system in which the SerHis dipeptide acts as catalyst for the formation of RNA oligomers from imidazole derivatives of mononucleotides. The thermodynamic shift towards condensation was achieved using water......-concentrated in the remaining liquid microinclusions, thus creating an environment with low water activity in which condensation reactions can occur. Successful oligomerization of RNA monomers catalysed by the SerHis dipeptide was observed in a broad range of pH, and with all four natural nucleobases. The isomeric dipeptide...... HisSer did not exhibit any catalytic properties thus indicating that the specific, spatial arrangement of amino acid residues in the SerHis structure is responsible for its catalytic activity. Establishing novel synthetic pathways to RNA polymerization is important, as to date no convincing prebiotic...

  2. Catalytic bioreactors and methods of using same

    Science.gov (United States)

    Worden, Robert Mark; Liu, Yangmu Chloe

    2017-07-25

    Various embodiments provide a bioreactor for producing a bioproduct comprising one or more catalytically active zones located in a housing and adapted to keep two incompatible gaseous reactants separated when in a gas phase, wherein each of the one or more catalytically active zones may comprise a catalytic component retainer and a catalytic component retained within and/or thereon. Each of the catalytically active zones may additionally or alternatively comprise a liquid medium located on either side of the catalytic component retainer. Catalytic component may include a microbial cell culture located within and/or on the catalytic component retainer, a suspended catalytic component suspended in the liquid medium, or a combination thereof. Methods of using various embodiments of the bioreactor to produce a bioproduct, such as isobutanol, are also provided.

  3. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    RNA modification has attracted increasing interest as it is realized that epitranscriptomics is important in disease development. In type 2 diabetes we have suggested that high urinary excretion of 8-oxo-2'-Guanosine (8oxoGuo), as a measure of global RNA oxidation, is associated with poor survival.......9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...... diabetes. In agreement with our previous finding, DNA oxidation did not show any prognostic value. RNA oxidation represents oxidative stress intracellularly, presumably predominantly in the cytosol. The mechanism of RNA oxidation is not clear, but hypothesized to result from mitochondrial dysfunction...

  4. Molecular mechanism of action of plant DRM de novo DNA methyltransferases.

    Science.gov (United States)

    Zhong, Xuehua; Du, Jiamu; Hale, Christopher J; Gallego-Bartolome, Javier; Feng, Suhua; Vashisht, Ajay A; Chory, Joanne; Wohlschlegel, James A; Patel, Dinshaw J; Jacobsen, Steven E

    2014-05-22

    DNA methylation is a conserved epigenetic gene-regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homodimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the small interfering RNA (siRNA) effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V-mediated noncoding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Science.gov (United States)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  6. Prokaryotic Argonautes – variations on the RNA interference theme

    Directory of Open Access Journals (Sweden)

    John van der Oost

    2014-04-01

    Full Text Available The discovery of RNA interference (RNAi has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago, an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes.

  7. Prokaryotic Argonautes - variations on the RNA interference theme

    Science.gov (United States)

    van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

    2014-01-01

    The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

  8. Flowthrough Reductive Catalytic Fractionation of Biomass

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Eric M.; Stone, Michael L.; Katahira, Rui; Reed, Michelle; Beckham, Gregg T.; Román-Leshkov, Yuriy

    2017-11-01

    Reductive catalytic fractionation (RCF) has emerged as a leading biomass fractionation and lignin valorization strategy. Here, flowthrough reactors were used to investigate RCF of poplar. Most RCF studies to date have been conducted in batch, but a flow-based process enables the acquisition of intrinsic kinetic and mechanistic data essential to accelerate the design, optimization, and scale-up of RCF processes. Time-resolved product distributions and yields obtained from experiments with different catalyst loadings were used to identify and deconvolute events during solvolysis and hydrogenolysis. Multi-bed RCF experiments provided unique insights into catalyst deactivation, showing that leaching, sintering, and surface poisoning are causes for decreased catalyst performance. The onset of catalyst deactivation resulted in higher concentrations of unsaturated lignin intermediates and increased occurrence of repolymerization reactions, producing high-molecular-weight species. Overall, this study demonstrates the concept of flowthrough RCF, which will be vital for realistic scale-up of this promising approach.

  9. A ribosome without RNA

    Directory of Open Access Journals (Sweden)

    Harold S Bernhardt

    2015-11-01

    Full Text Available It was Francis Crick who first asked why the ribosome contains so much RNA, and discussed the implications of this for the direct flow of genetic information from DNA to protein. Remarkable advances in our understanding of the ribosome and protein synthesis, including the recent publication of two mammalian mitochondrial ribosome structures, have shed new light on this intriguing aspect of evolution in molecular biology. We examine here whether RNA is indispensable for coded protein synthesis, or whether an all-protein ‘ribosome’ (or ‘synthosome’ might be possible, with a protein enzyme catalyzing peptide synthesis, and release factor-like protein adaptors able to read a message composed of deoxyribonucleotides. We also compare the RNA world hypothesis with the alternative ‘proteins first’ hypothesis in terms of their different understandings of the evolution of the ribosome, and whether this might have been preceded by an ancestral form of nonribosomal peptide synthesis catalyzed by protein enzymes.

  10. Catalytic Decoupling of Quantum Information

    DEFF Research Database (Denmark)

    Majenz, Christian; Berta, Mario; Dupuis, Frédéric

    2017-01-01

    The decoupling technique is a fundamental tool in quantum information theory with applications ranging from quantum thermodynamics to quantum many body physics to the study of black hole radiation. In this work we introduce the notion of catalytic decoupling, that is, decoupling in the presence...... of an uncorrelated ancilla system. This removes a restriction on the standard notion of decoupling, which becomes important for structureless resources, and yields a tight characterization in terms of the max-mutual information. Catalytic decoupling naturally unifies various tasks like the erasure of correlations...

  11. Catalytic activity of Au nanoparticles

    DEFF Research Database (Denmark)

    Larsen, Britt Hvolbæk; Janssens, Ton V.W.; Clausen, Bjerne

    2007-01-01

    Au is usually viewed as an inert metal, but surprisingly it has been found that Au nanoparticles less than 3–5 nm in diameter are catalytically active for several chemical reactions. We discuss the origin of this effect, focusing on the way in which the chemical activity of Au may change with par......Au is usually viewed as an inert metal, but surprisingly it has been found that Au nanoparticles less than 3–5 nm in diameter are catalytically active for several chemical reactions. We discuss the origin of this effect, focusing on the way in which the chemical activity of Au may change...

  12. Molecular cloning and RNA interference-mediated functional characterization of a Halloween gene spook in the white-backed planthopper Sogatella furcifera.

    Science.gov (United States)

    Jia, Shuang; Wan, Pin-Jun; Zhou, Li-Tao; Mu, Li-Li; Li, Guo-Qing

    2013-09-04

    Ecdysteroid hormones ecdysone and 20-hydroxyecdysone play fundamental roles in insect postembryonic development and reproduction. Five cytochrome P450 monooxygenases (CYPs), encoded by Halloween genes, have been documented to be involved in the ecdysteroidogenesis in insect species of diverse orders such as Diptera, Lepidoptera and Orthoptera. Up to now, however, the involvement of the Halloween genes in ecdysteroid synthesis has not been confirmed in hemipteran insect species. In the present paper, a Halloween gene spook (Sfspo, Sfcyp307a1) was cloned in the hemipteran Sogatella furcifera. SfSPO has three insect conserved P450 motifs, i.e., Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfspo were evaluated by qPCR. Sfspo showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. On day 3 of the fourth-instar nymphs, Sfspo clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA (dsRNA) of Sfspo into the second instars successfully knocked down the target gene, and greatly reduced expression level of ecdysone receptor (EcR) gene. Moreover, knockdown of Sfspo caused lethality and delayed development during nymphal stages. Furthermore, application of 20-hydroxyecdysone on Sfspo-dsRNA-exposed nymphs did not increase Sfspo expression, but could almost completely rescue SfEcR expression, and relieved the negative effects on nymphal survival and development. In S. furcifera, Sfspo was cloned and the conservation of SfSPO is valid. Thus, SfSPO is probably also involved in ecdysteroidogenesis for hemiptera.

  13. Molecular Characterization of the 16S rRNA Gene of Phytoplasmas Detected in Two Leafhopper Species Associated with Alfalfa Plants Infected with Witches' Broom in Oman

    Directory of Open Access Journals (Sweden)

    A.J. Khan

    2003-12-01

    Full Text Available Two leafhopper species, Austroagallia avicula and Empoasca sp., were consistently found in alfalfa fields infected with witches’ broom phytoplasma (OmanAlfWB in the Al-Batinah, Dakhliya, North and South Sharqiya, Muscat, and Al-Bureimi regions of the Sultanate of Oman. Phytoplasmas from both leafhoppers were detected by specific polymerase chain reaction (PCR amplification of the 16S rRNA gene and the spacer region in direct PCR using P1/P7 primer pairs. Comparative RFLP profiles of the amplified rRNA gene and the spacer region from leafhopper phytoplasmas and from 20 phytoplasma controls yielded patterns referable to phytoplasmas belonging to the peanut witches’ broom group (16SrII group. In particular, extensive RFLP analyses with the endonucleases HpaII, Tru9I, Tsp509I, and RsaI indicated that the phytoplasmas in A. avicula and Empoasca sp. were identical but showed some differences from OmanAlfWB; however, RFLP patterns obtained with TaqI showed the OmanAlfWB and the phytoplasmas from the two leafhoppers to be identical. Direct PCR products amplified from phytoplasma leafhopper DNA using the P1/P7 primer pair were cloned and sequenced yielding 1758 bp and 1755 bp products from A. avicula and Empoasca sp. respectively; the homology of these sequences with OmanAlfWB and papaya yellow crinkle phytoplasmas was more than 98%. A phylogenetic tree based on the 16S rRNA gene and spacer region sequences from 44 phytoplasmas revealed that the phytoplasmas from the leafhoppers clustered with OmanAlfWB, papaya yellow crinkle, and gerbera phyllody phytoplasmas, all belonging to 16SrII group, but were distinct from lime witches’ broom phytoplasma, the most commonly found phytoplasma in the Sultanate of Oman.

  14. Molecular characterisation of Mycoplasma hyorhinis isolated from pigs using pulsed-field gel electrophoresis and 16S rRNA sequencing

    OpenAIRE

    Yamaguti, Maur?cio; Oliveira, Ros?ngela C; Marques, Lucas M; Buzinhani, Melissa; Buim, Marcos R; Neto, Renata L; Guimar?es, Ana M?rcia S; Timenetsky, Jorge

    2015-01-01

    Economic loss in pig breeding is common due to respiratory disorders, and Mycoplasma hyopneumoniae and Mycoplasma hyorhinis, namely, are the most common infectious agents. The aim of this study is to recover these mollicutes and detect their genotypic variations by pulsed-field gel electrophoresis (PFGE) and sequencing the 16?s rRNA gene. One hundred and twenty-six swabs from tonsil and nasal mucus of pigs with respiratory disorders were analysed. A total of 78 lungs were sampled, as well as ...

  15. Molecular evidence that the opportunistic fungal pathogen Trichosporon asahii is part of the normal fungal microbiota of the human gut based on rRNA genotyping

    Directory of Open Access Journals (Sweden)

    Otomi Cho

    2015-10-01

    Full Text Available The fungus Trichosporon asahii is distributed widely in the environment and is frequently isolated from clinical specimens. It can cause life-threatening disseminated infections, including breakthrough infection after the administration of candin antifungal drugs. However, the definitive infection route is still unclear. In this study, 43 T. asahii genotypes were detected in 72 fecal samples from healthy subjects. The rRNA genotypes of the species were determined using a culture-independent method. The genotypes of T. asahii were almost identical to those of reported clinical isolates. Therefore, T. asahii colonizing the gastrointestinal tract might be associated with the development of trichosporonosis, and not environmental isolates.

  16. Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc- and manganese-superoxide dismutases in disk abalone, Haliotis discus discus.

    Science.gov (United States)

    Kim, Keun-Yong; Lee, Sang Yoon; Cho, Young Sun; Bang, In Chul; Kim, Ki Hong; Kim, Dong Soo; Nam, Yoon Kwon

    2007-11-01

    Complementary DNAs encoding copper/zinc superoxide dismutase (Cu/Zn-SOD; SOD1) and manganese superoxide dismutase (Mn-SOD; SOD2) were isolated from disk abalone, Haliotis discus discus. The open reading frame sequences of Cu/Zn- and Mn-SODs encoded 154 and 226 amino acids, respectively. Multiple sequence alignments using the deduced amino acid sequences revealed that both abalone SODs showed considerable sequence similarities with their orthologues from diverse aerobic organisms, in which the amino acid residues forming metal ligands were highly conserved. All phylogenetic trees for both SOD genes inferred from maximum likelihood and Bayesian inference analyses presented the monophyletic status of Teleostei and Aves/Tetrapoda clades, and recovered relatively close genetic affiliation of H. discus discus with some molluscan species. Expression of both SODs at mRNA levels were highly modulated in various tissues (gill, muscle and hepatopancreas from juveniles, and haemocytes from adults) by experimental exposures to heavy metals (copper, zinc and cadmium) and also by thermal treatments (elevation of temperature). The mRNA levels of both SODs were increased in general during the metal or thermal treatments; however, the transcriptional responses of SOD genes were quite variable depending upon isoforms and tissues based on semi-quantitative and/or real-time RT-PCR assays.

  17. [Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE)].

    Science.gov (United States)

    Master, Adam; Nauman, Alicja

    2014-01-01

    Translation initiation is a key rate-limiting step in cellular protein synthesis. A cap-dependent initiation is the most effective mechanism of the translation. However, some physiological (mitosis) and pathological (oxidative stress) processes may switch the classic mechanism to an alternative one that is regulated by an mRNA element such as IRES, uORF, IRE, CPE, DICE, AURE or CITE. A recently discovered mechanism of RNA hypoxia response element (rHRE)-dependent translation initiation, may change the view of oxygen-regulated translation and give a new insight into unexplained biochemical processes. Hypoxia is one of the better-known factors that may trigger an alternative mechanism of the translation initiation. Temporal events of oxygen deficiency within tissues and organs may activate processes such as angiogenesis, myogenesis, regeneration, wound healing, and may promote an adaptive response in cardiovascular and neurodegenerative diseases. On the other hand, growth of solid tumors may be accompanied by cyclic hypoxia, allowing for synthesis of proteins required for further progression of cancer cells. This paper provides a review of current knowledge on translational control in the context of alternative models of translation initiation.

  18. Molecular characterization of a new monopartite dsRNA mycovirus from mycorrhizal Thelephora terrestris (Ehrh.) and its detection in soil oribatid mites (Acari: Oribatida)

    Energy Technology Data Exchange (ETDEWEB)

    Petrzik, Karel, E-mail: petrzik@umbr.cas.cz [Department of Plant Virology, Institute of Plant Molecular Biology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice (Czech Republic); Sarkisova, Tatiana [Department of Plant Virology, Institute of Plant Molecular Biology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice (Czech Republic); Starý, Josef [Institute of Soil Biology, Biology Centre of the Czech Academy of Sciences, Na Sádkách 7, 370 05 České Budějovice (Czech Republic); Koloniuk, Igor [Department of Plant Virology, Institute of Plant Molecular Biology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice (Czech Republic); and others

    2016-02-15

    A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris (Ehrh.) and sequenced. This virus, named Thelephora terrestris virus 1 (TtV1), contains two reading frames in different frames but with the possibility that ORF2 could be translated as a fusion polyprotein after ribosomal -1 frameshifting. Picornavirus 2A-like motif, nudix hydrolase, phytoreovirus S7, and RdRp domains were found in a unique arrangement on the polyprotein. A new genus named Phlegivirus and containing TtV1, PgLV1, RfV1 and LeV is therefore proposed. Twenty species of oribatid mites were identified in soil material in the vicinity of T. terrestris. TtV1 was detected in large amounts in Steganacarus (Tropacarus) carinatus (C.L. Koch, 1841) and in much smaller amounts in Nothrus silvestris (Nicolet). This is the first description of mycovirus presence in oribatid mites. - Highlights: • A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris. • A new virus genus Phlegivirus is proposed. • The mycovirus was firstly detected in oribatid mites.

  19. Molecular characterization of a new monopartite dsRNA mycovirus from mycorrhizal Thelephora terrestris (Ehrh.) and its detection in soil oribatid mites (Acari: Oribatida)

    International Nuclear Information System (INIS)

    Petrzik, Karel; Sarkisova, Tatiana; Starý, Josef; Koloniuk, Igor

    2016-01-01

    A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris (Ehrh.) and sequenced. This virus, named Thelephora terrestris virus 1 (TtV1), contains two reading frames in different frames but with the possibility that ORF2 could be translated as a fusion polyprotein after ribosomal -1 frameshifting. Picornavirus 2A-like motif, nudix hydrolase, phytoreovirus S7, and RdRp domains were found in a unique arrangement on the polyprotein. A new genus named Phlegivirus and containing TtV1, PgLV1, RfV1 and LeV is therefore proposed. Twenty species of oribatid mites were identified in soil material in the vicinity of T. terrestris. TtV1 was detected in large amounts in Steganacarus (Tropacarus) carinatus (C.L. Koch, 1841) and in much smaller amounts in Nothrus silvestris (Nicolet). This is the first description of mycovirus presence in oribatid mites. - Highlights: • A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris. • A new virus genus Phlegivirus is proposed. • The mycovirus was firstly detected in oribatid mites.

  20. Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.

    Science.gov (United States)

    Murayama, Mayumi; Kakinuma, Yuki; Maeda, Yasunori; Rao, Juluri R; Matsuda, Motoo; Xu, Jiru; Moore, Peter J A; Millar, B Cherie; Rooney, Paul J; Goldsmith, Colin E; Loughrey, Anne; McMahon, M Ann S; McDowell, David A; Moore, John E

    2010-03-01

    Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals. (c) 2009 Elsevier Inc. All rights reserved.

  1. The lethal giant larvae Gene in Tribolium castaneum: Molecular Properties and Roles in Larval and Pupal Development as Revealed by RNA Interference

    Directory of Open Access Journals (Sweden)

    Da Xiao

    2014-04-01

    Full Text Available We identified and characterized the TcLgl gene putatively encoding lethal giant larvae (Lgl protein from the red flour beetle (Tribolium castaneum. Analyses of developmental stage and tissue-specific expression patterns revealed that TcLgl was constitutively expressed. To examine the role of TcLgl in insect development, RNA interference was performed in early (1-day larvae, late (20-day larvae, and early (1-day pupae. The early larvae injected with double-stranded RNA of TcLgl (dsTcLgl at 100, 200, and 400 ng/larva failed to pupate, and 100% mortality was achieved within 20 days after the injection or before the pupation. The late larvae injected with dsTcLgl at these doses reduced the pupation rates to only 50.3%, 36.0%, and 18.2%, respectively. The un-pupated larvae gradually died after one week, and visually unaffected pupae failed to emerge into adults and died during the pupal stage. Similarly, when early pupae were injected with dsTcLgl at these doses, the normal eclosion rates were reduced to only 22.5%, 18.0%, and 11.2%, respectively, on day 7 after the injection, and all the adults with abnormal eclosion died in two days after the eclosion. These results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.

  2. Engineering reactors for catalytic reactions

    Indian Academy of Sciences (India)

    ... improvements in reactor performance. In this article, application of recent (and not so recent) developments in engineering reactors for catalytic reactions is discussed. Some examples where performance enhancement was realized by catalyst design, appropriate choice of reactor, better injection and dispersion strategies ...

  3. Catalytic properties of niobium compounds

    International Nuclear Information System (INIS)

    Tanabe, K.; Iizuka, T.

    1983-04-01

    The catalytic activity and selectivity of niobium compounds including oxides, salts, organometallic compounds and others are outlined. The application of these compounds as catalysts to diversified reactions is reported. The nature and action of niobium catalysts are characteristic and sometimes anomalous, suggesting the necessity of basic research and the potential use as catalysts for important processes in the chemical industry. (Author) [pt

  4. Catalytic carboxyester hydrolysis by diaminodiphenols

    Indian Academy of Sciences (India)

    Administrator

    Two diaminodiphenols, 1 and 2, have been examined as catalysts for the hydrolysis of 4- nitrophenyl acetate (NA) and 4-nitrophenylphosphate (NP) in aqueous-acetonitrile (25% acetonitrile v/v) media at 35ºC, I = 1·0 mol dm–3. The compound 1 enhances the hydrolysis rate of NA more than 105 times. Its catalytic efficiency ...

  5. Engineering reactors for catalytic reactions

    Indian Academy of Sciences (India)

    on selectivity can make substantial impact on process viability and economics. Extensive studies have been conducted to establish sound basis for design and engineering of reactors for practising such catalytic reactions and for realizing improvements in reactor performance. In this article, application of recent (and not so ...

  6. Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM.

    Science.gov (United States)

    Petkovic, Sonja; Badelt, Stefan; Block, Stephan; Flamm, Christoph; Delcea, Mihaela; Hofacker, Ivo; Müller, Sabine

    2015-07-01

    Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and self-concatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions. © 2015 Petkovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. Molecular evidence that the opportunistic fungal pathogen Trichosporon asahii is part of the normal fungal microbiota of the human gut based on rRNA genotyping.

    Science.gov (United States)

    Cho, Otomi; Matsukura, Manami; Sugita, Takashi

    2015-10-01

    The fungus Trichosporon asahii is distributed widely in the environment and is frequently isolated from clinical specimens. It can cause life-threatening disseminated infections, including breakthrough infection after the administration of candin antifungal drugs. However, the definitive infection route is still unclear. In this study, 43 T. asahii genotypes were detected in 72 fecal samples from healthy subjects. The rRNA genotypes of the species were determined using a culture-independent method. The genotypes of T. asahii were almost identical to those of reported clinical isolates. Therefore, T. asahii colonizing the gastrointestinal tract might be associated with the development of trichosporonosis, and not environmental isolates. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Immersion calorimetry as a tool to evaluate the catalytic performance of titanosilicate materials in the epoxidation of cyclohexene.

    Science.gov (United States)

    Vernimmen, Jarian; Guidotti, Matteo; Silvestre-Albero, Joaquin; Jardim, Erika O; Mertens, Myrjam; Lebedev, Oleg I; Van Tendeloo, Gustaaf; Psaro, Rinaldo; Rodríguez-Reinoso, Francisco; Meynen, Vera; Cool, Pegie

    2011-04-05

    Different types of titanosilicates are synthesized, structurally characterized, and subsequently catalytically tested in the liquid-phase epoxidation of cyclohexene. The performance of three types of combined zeolitic/mesoporous materials is compared with that of widely studied Ti-grafted-MCM-41 molecular sieve and the TS-1 microporous titanosilicate. The catalytic test results are correlated with the structural characteristics of the different catalysts. Moreover, for the first time, immersion calorimetry with the same substrate molecule as in the catalytic test reaction is applied as an extra means to interpret the catalytic results. A good correlation between catalytic performance and immersion calorimetry results is found. This work points out that the combination of catalytic testing and immersion calorimetry can lead to important insights into the influence of the materials structural characteristics on catalysis. Moreover, the potential of using immersion calorimetry as a screening tool for catalysts in epoxidation reactions is shown.

  9. Temperature requirements for initiation of RNA-dependent RNA polymerization

    International Nuclear Information System (INIS)

    Yang Hongyan; Gottlieb, Paul; Wei Hui; Bamford, Dennis H.; Makeyev, Eugene V.

    2003-01-01

    To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages (Cystoviridae), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive (ts) point mutant of the polymerase subunit (Pol) from bacteriophage phi12. Interestingly, initiation by both wt and the ts phi12 Pol was notably more sensitive to increased temperatures than the elongation step, the absolute value of the nonpermissive temperature being lower for the ts enzyme. Experiments with the Pol subunit of related cystovirus phi6 revealed a similar differential sensitivity of the initiation and elongation steps. This is consistent with the previous result showing that de novo initiation by RdRp from dengue virus is inhibited at elevated temperatures, whereas the elongation phase is relatively thermostable. Overall, these data suggest that de novo RNA-dependent RNA synthesis in many viral systems includes a specialized thermolabile state of the RdRp initiation complex

  10. Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation

    OpenAIRE

    Saramago, Margarida; Peregrina, Alexandra; Robledo, Marta; Matos, Rute G.; Hilker, Rolf; Serrania, Javier; Becker, Anke; Arraiano, Cecilia M.; Jim?nez-Zurdo, Jos? I.

    2016-01-01

    Abstract Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously...

  11. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  12. Enhanced catalytic behavior of Ni alloys in steam methane reforming

    Science.gov (United States)

    Yoon, Yeongpil; Kim, Hanmi; Lee, Jaichan

    2017-08-01

    The dissociation process of methane on Ni and Ni alloys are investigated by density functional theory (DFT) in terms of catalytic efficiency and carbon deposition. Examining the dissociation to CH3, CH2, CH, C, and H is not sufficient to properly predict the catalytic efficiency and carbon deposition, and further investigation of the CO gas-evolving reaction is required to completely understand methane dissociation in steam. The location of alloying element in Ni alloy needed be addressed from the results of ab-inito molecular dynamics (MD). The reaction pathway of methane dissociation associated with CO gas evolution is traced by performing first-principles calculations of the adsorption and activation energies of each dissociation step. During the dissociation process, two alternative reaction steps producing adsorbed C and H or adsorbed CO are critically important in determining coking inhibition as well as H2 gas evolution (i.e., the catalytic efficiency). The theoretical calculations presented here suggest that alloying Ni with Ru is an effective way to reduce carbon deposition and enhance the catalytic efficiency of H2 fueling in solid oxide fuel cells (SOFCs).

  13. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    Science.gov (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4-fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  14. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    Science.gov (United States)

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  15. Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

    International Nuclear Information System (INIS)

    Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

    2003-01-01

    The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

  16. A WHEP Domain Regulates the Dynamic Structure and Activity of Caenorhabditis elegans Glycyl-tRNA Synthetase.

    Science.gov (United States)

    Chang, Chih-Yao; Chien, Chin-I; Chang, Chia-Pei; Lin, Bo-Chun; Wang, Chien-Chia

    2016-08-05

    WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases and play roles in tRNA or protein binding. We present evidence herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. The cytoplasmic form possessed an N-terminal WHEP domain, whereas its mitochondrial isoform possessed an extra N-terminal sequence consisting of an mitochondrial targeting signal and an appended domain. Cross-species complementation assays showed that CeGRS1 effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 knock-out strain. Although both forms of CeGlyRS efficiently charged the cytoplasmic tRNAs(Gly) of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNA(Gly) isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA binding activity, deletion of this domain reduced the catalytic efficiency of the enzyme. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests a role for the WHEP domain as a regulator of the dynamic structure and activity of the enzyme. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Theoretical study of binding of hydrated Zn(II) and Mg(II) cations to 5'-guanosine monophosphate. Toward polarizable molecular mechanics for DNA and RNA

    Czech Academy of Sciences Publication Activity Database

    Gresh, N.; Šponer, Judit E.; Špačková, Naďa; Leszczynski, J.; Šponer, Jiří

    2003-01-01

    Roč. 107, č. 33 (2003), s. 8669-8681 ISSN 1520-6106 R&D Projects: GA MŠk LN00A016 Grant - others:National Science Foundation(US) CREST 9805465; National Institutes of Health(US) RCMI G1 2RR13459-21; Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : polarizable molecular mechanics * quantum-chemical computations * hydrated cations Subject RIV: BO - Biophysics Impact factor: 3.679, year: 2003

  18. Morphological analysis of the reproductive system and molecular identification by mitochondrial markers COI and 16S rRNA of Megalobulimus oblongus (Mollusca, Strophocheilidae) of Colombia

    OpenAIRE

    Jaramillo Roldán, Erika; 1 Grupo de Microbiología Ambiental, Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia. 3 Programa de Estudio y Control de Enfermedades Tropicales – PECET, Sede de Investigación Universitaria – SIU, Medellín, Colombia.; López Martínez, Jessika; 1 Grupo de Microbiología Ambiental, Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia. 3 Programa de Estudio y Control de Enfermedades Tropicales – PECET, Sede de Investigación Universitaria – SIU, Medellín, Colombia.; Ramírez, Rina; Laboratorio de Sistemática Molecular y Filogeografía, Facultad de Ciencias Biológicas; y Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Av. Arenales 1256, Lima-11, Perú.; Velásquez Trujillo, Luz Elena; 1 Grupo de Microbiología Ambiental, Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia. 3 Programa de Estudio y Control de Enfermedades Tropicales – PECET, Sede de Investigación Universitaria – SIU, Medellín, Colombia.

    2014-01-01

    En este trabajo se hizo el análisis morfológico y molecular a 28 caracoles terrestres de Megalobulimus oblongus, colectados en diferentes departamentos de Colombia, depositados en una colección de referencia. Para la caracterización morfológica, los animales se disecaron al estéreomicroscopio. Se describieron el sistema reproductor y la concha. Del sistema reproductor se tomaron medidas a las estructuras que lo componen. De la concha se describió la forma, el color, el número de espiras y la ...

  19. Conserved and variable domains of RNase MRP RNA.

    Science.gov (United States)

    Dávila López, Marcela; Rosenblad, Magnus Alm; Samuelsson, Tore

    2009-01-01

    Ribonuclease MRP is a eukaryotic ribonucleoprotein complex consisting of one RNA molecule and 7-10 protein subunits. One important function of MRP is to catalyze an endonucleolytic cleavage during processing of rRNA precursors. RNase MRP is evolutionary related to RNase P which is critical for tRNA processing. A large number of MRP RNA sequences that now are available have been used to identify conserved primary and secondary structure features of the molecule. MRP RNA has structural features in common with P RNA such as a conserved catalytic core, but it also has unique features and is characterized by a domain highly variable between species. Information regarding primary and secondary structure features is of interest not only in basic studies of the function of MRP RNA, but also because mutations in the RNA give rise to human genetic diseases such as cartilage-hair hypoplasia.

  20. Ribosome-inactivating proteins: potent poisons and molecular tools.

    Science.gov (United States)

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-11-15

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

  1. RNA Study Using DNA Nanotechnology.

    Science.gov (United States)

    Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

    2016-01-01

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Linear and non-linear dependencies between copy number aberrations and mRNA expression reveal distinct molecular pathways in breast cancer

    Directory of Open Access Journals (Sweden)

    Frigessi Arnoldo

    2011-05-01

    Full Text Available Abstract Background Elucidating the exact relationship between gene copy number and expression would enable identification of regulatory mechanisms of abnormal gene expression and biological pathways of regulation. Most current approaches either depend on linear correlation or on nonparametric tests of association that are insensitive to the exact shape of the relationship. Based on knowledge of enzyme kinetics and gene regulation, we would expect the functional shape of the relationship to be gene dependent and to be related to the gene regulatory mechanisms involved. Here, we propose a statistical approach to investigate and distinguish between linear and nonlinear dependences between DNA copy number alteration and mRNA expression. Results We applied the proposed method to DNA copy numbers derived from Illumina 109 K SNP-CGH arrays (using the log R values and expression data from Agilent 44 K mRNA arrays, focusing on commonly aberrated genomic loci in a collection of 102 breast tumors. Regression analysis was used to identify the type of relationship (linear or nonlinear, and subsequent pathway analysis revealed that genes displaying a linear relationship were overall associated with substantially different biological processes than genes displaying a nonlinear relationship. In the group of genes with a linear relationship, we found significant association to canonical pathways, including purine and pyrimidine metabolism (for both deletions and amplifications as well as estrogen metabolism (linear amplification and BRCA-related response to damage (linear deletion. In the group of genes displaying a nonlinear relationship, the top canonical pathways were specific pathways like PTEN and PI13K/AKT (nonlinear amplification and Wnt(B and IL-2 signalling (nonlinear deletion. Both amplifications and deletions pointed to the same affected pathways and identified cancer as the top significant disease and cell cycle, cell signaling and cellular

  3. Molecular heterogeneous catalysts derived from bipyridine-based organosilica nanotubes for C–H bond activation† †Electronic supplementary information (ESI) available: Experimental details, material characterization data, catalytic measurement details. See DOI: 10.1039/c7sc00713b Click here for additional data file.

    Science.gov (United States)

    Zhang, Shengbo; Wang, Hua; Li, Mei; Han, Jinyu

    2017-01-01

    Heterogeneous metal complex catalysts for direct C–H activation with high activity and durability have always been desired for transforming raw materials into feedstock chemicals. This study described the design and synthesis of one-dimensional organosilica nanotubes containing 2,2′-bipyridine (bpy) ligands in the framework (BPy-NT) and their post-synthetic metalation to provide highly active and robust molecular heterogeneous catalysts. By adjusting the ratios of organosilane precursors, very short BPy-NT with ∼50 nm length could be controllably obtained. The post-synthetic metalation of bipyridine-functionalized nanotubes with [IrCp*Cl(μ-Cl)]2 (Cp* = η5-pentamethylcyclopentadienyl) and [Ir(cod)(OMe)]2 (cod = 1,5-cyclooctadiene) afforded solid catalysts, IrCp*-BPy-NT and Ir(cod)-BPy-NT, which were utilized for C–H oxidation of heterocycles and cycloalkanes as well as C–H borylation of arenes. The cut-short nanotube catalysts displayed enhanced activities and durability as compared to the analogous homogeneous catalysts and other conventional heterogeneous catalysts, benefiting from the isolated active sites as well as the fast transport of substrates and products. After the reactions, a detailed characterization of Ir-immobilized BPy-NT via TEM, SEM, nitrogen adsorption, UV/vis, XPS, and 13C CP MAS NMR indicated the molecular nature of the active species as well as stable structures of nanotube scaffolds. This study demonstrates the potential of BPy-NT with a short length as an integration platform for the construction of efficient heterogeneous catalytic systems for organic transformations. PMID:28970878

  4. Vapor Phase Catalytic Ammonia Reduction

    Science.gov (United States)

    Flynn, Michael T.; Harper, Lynn D. (Technical Monitor)

    1994-01-01

    This paper discusses the development of a Vapor Phase Catalytic Ammonia Reduction (VPCAR) teststand and the results of an experimental program designed to evaluate the potential of the technology as a water purification process. In the experimental program the technology is evaluated based upon product water purity, water recovery rate, and power consumption. The experimental work demonstrates that the technology produces high purity product water and attains high water recovery rates at a relatively high specific power consumption. The experimental program was conducted in 3 phases. In phase I an Igepon(TM) soap and water mixture was used to evaluate the performance of an innovative Wiped-Film Rotating-Disk evaporator and associated demister. In phase II a phenol-water solution was used to evaluate the performance of the high temperature catalytic oxidation reactor. In phase III a urine analog was used to evaluate the performance of the combined distillation/oxidation functions of the processor.

  5. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

    Science.gov (United States)

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-01-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  6. Inorganic membranes and catalytic reactors

    OpenAIRE

    Rangel, Maria do Carmo

    1997-01-01

    Membrane reactors are reviewed with emphasis in their applications in catalysis field. The basic principles of these systems are presented as well as a historical development. The several kinds of catalytic membranes and their preparations are discussed including the problems, needs and challenges to be solved in order to use these reactors in commercial processes. Some applications of inorganic membrane reactors are also shown. It was concluded that these systems have a great potential for i...

  7. Molecular characterization of a new monopartite dsRNA mycovirus from mycorrhizal Thelephora terrestris (Ehrh.) and its detection in soil oribatid mites (Acari: Oribatida).

    Science.gov (United States)

    Petrzik, Karel; Sarkisova, Tatiana; Starý, Josef; Koloniuk, Igor; Hrabáková, Lenka; Kubešová, Olga

    2016-02-01

    A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris (Ehrh.) and sequenced. This virus, named Thelephora terrestris virus 1 (TtV1), contains two reading frames in different frames but with the possibility that ORF2 could be translated as a fusion polyprotein after ribosomal -1 frameshifting. Picornavirus 2A-like motif, nudix hydrolase, phytoreovirus S7, and RdRp domains were found in a unique arrangement on the polyprotein. A new genus named Phlegivirus and containing TtV1, PgLV1, RfV1 and LeV is therefore proposed. Twenty species of oribatid mites were identified in soil material in the vicinity of T. terrestris. TtV1 was detected in large amounts in Steganacarus (Tropacarus) carinatus (C.L. Koch, 1841) and in much smaller amounts in Nothrus silvestris (Nicolet). This is the first description of mycovirus presence in oribatid mites. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. 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.

  9. Pyrite footprinting of RNA

    Energy Technology Data Exchange (ETDEWEB)

    Schlatterer, Joerg C., E-mail: joerg.schlatterer@einstein.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY (United States); Wieder, Matthew S. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY (United States); Jones, Christopher D.; Pollack, Lois [School of Applied and Engineering Physics, Cornell University, Ithaca, NY (United States); Brenowitz, Michael [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY (United States)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer RNA structure is mapped by pyrite mediated {sup {center_dot}}OH footprinting. Black-Right-Pointing-Pointer Repetitive experiments can be done in a powdered pyrite filled cartridge. Black-Right-Pointing-Pointer High {sup {center_dot}}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 ({sup {center_dot}}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{sub 2}) can produce sufficient {sup {center_dot}}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 {sup {center_dot}}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 {sup {center_dot}}OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

  10. tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

    Science.gov (United States)

    Myers, Christopher A; Kuhla, Birte; Cusack, Stephen; Lambowitz, Alan M

    2002-03-05

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

  11. Oxoperoxomolybdenum(VI complexes of catalytic and biomedical relevance: Synthesis, characterization, antibacterial activity and 3D-molecular modeling of some oxoperoxomolybdenum(VI chelates in mixed (O,O coordination environment involving maltol and β-diketoenolates

    Directory of Open Access Journals (Sweden)

    R.C. Maurya

    2016-09-01

    Full Text Available A new series of four mixed-ligand complexes of oxoperoxomolybdenum(VI of the general composition [MoO(O2(ma(L]·H2O, where maH = 3-hydroxy-2-methyl-4-pyrone and LH = o-acetoacetanisidide (o-aansH, o-acetoacetotoluidide (o-aatdH, acetylacetone (acacH or methylactoacetate (macacH, has been synthesized by the interaction of [MoO(O2]2+ (obtained in situ during the interaction of MoO3 and 30% H2O2 on continuous stirring at 50 °C for 24 h and the said ligands in aqueous-ethanol medium. The complexes so obtained have been characterized by elemental analyses, molybdenum determination, molar conductance, decomposition temperature and magnetic measurements, thermogravimetric studies, 1H NMR, IR, mass and electronic spectral studies. Antibacterial studies of one of the ligands maltol and the two representative metal complexes were also carried out and it has been observed that the complexes are more potent bactericides than the ligand. The 3D-molecular modeling and analysis for bond lengths and bond angles have also been carried out for one of the representative compound (1 to substantiate the proposed structure. Pseudopentagonal bipyramidal structures have been proposed for these complexes.

  12. Ultrasensitive Label-Free Sensing of IL-6 Based on PASE Functionalized Carbon Nanotube Micro-Arrays with RNA-Aptamers as Molecular Recognition Elements.

    Science.gov (United States)

    Khosravi, Farhad; Loeian, Seyed Masoud; Panchapakesan, Balaji

    2017-04-17

    This study demonstrates the rapid and label-free detection of Interleukin-6 (IL-6) using carbon nanotube micro-arrays with aptamer as the molecular recognition element. Single wall carbon nanotubes micro-arrays biosensors were manufactured using photo-lithography, metal deposition, and etching techniques. Nanotube biosensors were functionalized with 1-Pyrenebutanoic Acid Succinimidyl Ester (PASE) conjugated IL-6 aptamers. Real time response of the sensor conductance was monitored with increasing concentration of IL-6 (1 pg/mL to 10 ng/mL), exposure to the sensing surface in buffer solution, and clinically relevant spiked blood samples. Non-specific Bovine Serum Albumin (BSA), PBS samples, and anti-IgG functionalized devices gave similar signatures in the real time conductance versus time experiments with no significant change in sensor signal. Exposure of the aptamer functionalized nanotube surface to IL-6 decreased the conductance with increasing concentration of IL-6. Experiments based on field effect transistor arrays suggested shift in drain current versus gate voltage for 1 pg and 1 ng of IL-6 exposure. Non-specific BSA did not produce any appreciable shift in the I ds versus V g suggesting specific interactions of IL-6 on PASE conjugated aptamer surface gave rise to the change in electrical signal. Both Z axis and phase image in an Atomic Force Microscope (AFM) suggested unambiguous molecular interaction of the IL-6 on the nanotube-aptamer surface at 1 pg/mL concentration. The concentration of 1 pg falls below the diagnostic gray zone for cancer (2.3 pg-4 ng/mL), which is an indicator of early stage cancer. Thus, nanotube micro-arrays could potentially be developed for creating multiplexed assays involving cancer biomarker proteins and possibly circulating tumor cells all in a single assay using PASE functionalization protocol.

  13. Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP.

    Science.gov (United States)

    Perederina, Anna; Khanova, Elena; Quan, Chao; Berezin, Igor; Esakova, Olga; Krasilnikov, Andrey S

    2011-10-01

    Ribonuclease (RNase) MRP is a multicomponent ribonucleoprotein complex closely related to RNase P. RNase MRP and eukaryotic RNase P share most of their protein components, as well as multiple features of their catalytic RNA moieties, but have distinct substrate specificities. While RNase P is practically universally found in all three domains of life, RNase MRP is essential in eukaryotes. The structural organizations of eukaryotic RNase P and RNase MRP are poorly understood. Here, we show that Pop5 and Rpp1, protein components found in both RNase P and RNase MRP, form a heterodimer that binds directly to the conserved area of the putative catalytic domain of RNase MRP RNA. The Pop5/Rpp1 binding site corresponds to the protein binding site in bacterial RNase P RNA. Structural and evolutionary roles of the Pop5/Rpp1 heterodimer in RNases P and MRP are discussed.

  14. The Effect of Formaldehyde Fixation on RNA

    Science.gov (United States)

    Evers, David L.; Fowler, Carol B.; Cunningham, Brady R.; Mason, Jeffrey T.; O'Leary, Timothy J.

    2011-01-01

    Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems. We studied the reversal of formaldehyde fixation of RNA using a 50 base RNA oligonucleotide and total cellular RNA. Formaldehyde-adducted, native, and hydrolyzed RNA species were identified by their bioanalyzer electrophoretic migration patterns and RT–quantitative PCR. Demodification conditions included temperature, time, buffer, and pH. The reversal of formaldehyde-fixed RNA to native species without apparent RNA hydrolysis was most successfully performed in dilute Tris, phosphate, or similar buffers (pH 8) at 70°C for 30 minutes. Amines were not required for efficient formaldehyde demodification. Formaldehyde-fixed RNA was more labile than native RNA to treatment with heat and buffer, suggesting that antigen retrieval methods for proteins may impede RNA hybridization or RNA extraction. Taken together, the data indicate that reliable conditions may be used to remove formaldehyde adducts from RNA to improve the quality of RNA available for molecular studies. PMID:21497290

  15. Molecular characterization, RNA interference and recombinant protein approach to study the function of the putative Molt Inhibiting Hormone (FmMIH1) gene from the shrimp Fenneropenaeus merguiensis.

    Science.gov (United States)

    Liang, Huafang; Liu, Yan; Zhou, Ting Ting; Li, Xiaoyuan; Li, Bin; Chan, Siuming F

    2017-12-14

    The Molt Inhibiting Hormone gene and cDNA of the banana shrimp Fenneropenaeus merguiensis (FmMIH1) has been cloned and characterized. FmMIH1 possesses most of the characteristics of the eyestalk CHH/MIH/GIH family subtype-II neuropeptides. FmMIH1 open reading frame consists of 315 bp encoding for 105 amino acid residues. The mature peptide of FmMIH1 consists of 76 amino acid residues, a glycine residue at position 11 of the mature peptide and 6 cysteine residues located in the conserved position. In addition to eyestalk, high levels of FmMIH1 transcript could also be detected in the intestine. FmMIH1 transcript level is low throughout the post-molt, early to mid-intermolt and premolt. However, a sharp increase could be observed in late intermolt (C3 stage). Both alignment and phylogenetic analysis reveal that FmMIH1 is most similar to the MIH1 of other shrimps. For functional assay, RNA interference results show that a significant 2.3 days (P < 0.05) reduction in molt cycle duration could be observed in shrimp receiving dsFmMIH1 injection. Surprisingly, injection of recombinant FmMIH1 could also cause a significant reduction of the molt cycle (average 1.9 days, P < 0.05). We hypothesize that the recombinant protein is biological inactive but it competes with the endogenous MIH for carrier protein binding and consequently reduces the amount of biological MIH that could reach the targets. In conclusion, the result of this study will provide us new insight in molting/growth control in crustacean. Copyright © 2017. Published by Elsevier Inc.

  16. New Catalytic DNA Biosensors for Radionuclides and Metal ions

    International Nuclear Information System (INIS)

    Lu, Yi

    2003-01-01

    The goals of the project are to develop new catalytic DNA biosensors for simultaneous detection and quantification of bioavailable radionuclides and metal ions, and apply the sensors for on-site, real-time assessment of concentration, speciation and stability of the individual contaminants during and after bioremediation. A negative selection strategy was tested and validated. In vitro selection was shown to yield highly active and specific transition metal ion-dependent catalytic DNA/RNA. A fluorescence resonance energy transfer (FRET) study of in vitro selected DNA demonstrated that the trifluorophore labeled system is a simple and powerful tool in studying complex biomolecules structure and dynamics, and is capable of revealing new sophisticated structural changes. New fluorophore/quenchers in a single fluorosensor yielded improved signal to noise ratio in detection, identification and quantification of metal contaminants. Catalytic DNA fluorescent and colorimetric sensors were shown useful in sensing lead in lake water and in leaded paint. Project results were described in two papers and two patents, and won an international prize

  17. Structure and function of echinoderm telomerase RNA

    OpenAIRE

    Podlevsky, Joshua D.; Li, Yang; Chen, Julian J.-L.

    2016-01-01

    Telomerase is a ribonucleoprotein (RNP) enzyme that requires an integral telomerase RNA (TR) subunit, in addition to the catalytic telomerase reverse transcriptase (TERT), for enzymatic function. The secondary structures of TRs from the three major groups of species, ciliates, fungi, and vertebrates, have been studied extensively and demonstrate dramatic diversity. Herein, we report the first comprehensive secondary structure of TR from echinoderms—marine invertebrates closely related to vert...

  18. Nuclear Export of Messenger RNA

    Directory of Open Access Journals (Sweden)

    Jun Katahira

    2015-03-01

    Full Text Available Transport of messenger RNA (mRNA from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.

  19. Nuclear Export of Messenger RNA

    Science.gov (United States)

    Katahira, Jun

    2015-01-01

    Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

  20. RNA viruses in the sea.

    Science.gov (United States)

    Lang, Andrew S; Rise, Matthew L; Culley, Alexander I; Steward, Grieg F

    2009-03-01

    Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

  1. Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel.

    Directory of Open Access Journals (Sweden)

    Guo-Rui Yuan

    Full Text Available Ryanodine receptors (RyRs are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel, a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97% to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.

  2. Dimerization interface of 3-hydroxyacyl-CoA dehydrogenase tunes the formation of its catalytic intermediate.

    Directory of Open Access Journals (Sweden)

    Yingzhi Xu

    Full Text Available 3-Hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.35 is a homodimeric enzyme localized in the mitochondrial matrix, which catalyzes the third step in fatty acid β-oxidation. The crystal structures of human HAD and subsequent complexes with cofactor/substrate enabled better understanding of HAD catalytic mechanism. However, numerous human diseases were found related to mutations at HAD dimerization interface that is away from the catalytic pocket. The role of HAD dimerization in its catalytic activity needs to be elucidated. Here, we solved the crystal structure of Caenorhabditis elegans HAD (cHAD that is highly conserved to human HAD. Even though the cHAD mutants (R204A, Y209A and R204A/Y209A with attenuated interactions on the dimerization interface still maintain a dimerization form, their enzymatic activities significantly decrease compared to that of the wild type. Such reduced activities are in consistency with the reduced ratios of the catalytic intermediate formation. Further molecular dynamics simulations results reveal that the alteration of the dimerization interface will increase the fluctuation of a distal region (a.a. 60-80 that plays an important role in the substrate binding. The increased fluctuation decreases the stability of the catalytic intermediate formation, and therefore the enzymatic activity is attenuated. Our study reveals the molecular mechanism about the essential role of the HAD dimerization interface in its catalytic activity via allosteric effects.

  3. A DNA enzyme that cleaves RNA

    Science.gov (United States)

    Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

    1994-01-01

    BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

  4. Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: A non-conventional hemolysin and a ribosomal RNA methyl transferase

    Directory of Open Access Journals (Sweden)

    Ahmed Neesar

    2010-09-01

    was significantly slower than mock vector transformed E. coli. The S30 extract of E. coli expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [3H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity. Conclusions The Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by Mycobacterium tuberculosis.

  5. Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

    Science.gov (United States)

    Phiriyangkul, Pharima; Utarabhand, Prapaporn

    2006-04-01

    In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp. Copyright 2006 Wiley-Liss, Inc.

  6. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae

    Directory of Open Access Journals (Sweden)

    Kang-Kang Xu

    2013-08-01

    Full Text Available Chitin synthase (CHS, a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2 was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

  7. Performance of a Novel Hydrophobic Mesoporous Material for High Temperature Catalytic Oxidation of Naphthalene

    Directory of Open Access Journals (Sweden)

    Guotao Zhao

    2014-01-01

    Full Text Available A high surface area, hydrophobic mesoporous material, MFS, has been successfully synthesized by a hydrothermal synthesis method using a perfluorinated surfactant, SURFLON S-386, as the single template. N2 adsorption and TEM were employed to characterize the pore structure and morphology of MFS. Static water adsorption test indicates that the hydrophobicity of MFS is significantly higher than that of MCM-41. XPS and Py-GC/MS analysis confirmed the existence of perfluoroalkyl groups in MFS which led to its high hydrophobicity. MFS was used as a support for CuO in experiments of catalytic combustion of naphthalene, where it showed a significant advantage over MCM-41 and ZSM-5. SEM was helpful in understanding why CuO-MFS performed so well in the catalytic combustion of naphthalene. Experimental results indicated that MFS was a suitable support for catalytic combustion of large molecular organic compounds, especially for some high temperature catalytic reactions when water vapor was present.

  8. Understanding the mechanism of catalytic fast pyrolysis by unveiling reactive intermediates in heterogeneous catalysis

    Science.gov (United States)

    Hemberger, Patrick; Custodis, Victoria B. F.; Bodi, Andras; Gerber, Thomas; van Bokhoven, Jeroen A.

    2017-06-01

    Catalytic fast pyrolysis is a promising way to convert lignin into fine chemicals and fuels, but current approaches lack selectivity and yield unsatisfactory conversion. Understanding the pyrolysis reaction mechanism at the molecular level may help to make this sustainable process more economic. Reactive intermediates are responsible for product branching and hold the key to unveiling these mechanisms, but are notoriously difficult to detect isomer-selectively. Here, we investigate the catalytic pyrolysis of guaiacol, a lignin model compound, using photoelectron photoion coincidence spectroscopy with synchrotron radiation, which allows for isomer-selective detection of reactive intermediates. In combination with ambient pressure pyrolysis, we identify fulvenone as the central reactive intermediate, generated by catalytic demethylation to catechol and subsequent dehydration. The fulvenone ketene is responsible for the phenol formation. This technique may open unique opportunities for isomer-resolved probing in catalysis, and holds the potential for achieving a mechanistic understanding of complex, real-life catalytic processes.

  9. Understanding the mechanism of catalytic fast pyrolysis by unveiling reactive intermediates in heterogeneous catalysis

    Science.gov (United States)

    Hemberger, Patrick; Custodis, Victoria B. F.; Bodi, Andras; Gerber, Thomas; van Bokhoven, Jeroen A.

    2017-01-01

    Catalytic fast pyrolysis is a promising way to convert lignin into fine chemicals and fuels, but current approaches lack selectivity and yield unsatisfactory conversion. Understanding the pyrolysis reaction mechanism at the molecular level may help to make this sustainable process more economic. Reactive intermediates are responsible for product branching and hold the key to unveiling these mechanisms, but are notoriously difficult to detect isomer-selectively. Here, we investigate the catalytic pyrolysis of guaiacol, a lignin model compound, using photoelectron photoion coincidence spectroscopy with synchrotron radiation, which allows for isomer-selective detection of reactive intermediates. In combination with ambient pressure pyrolysis, we identify fulvenone as the central reactive intermediate, generated by catalytic demethylation to catechol and subsequent dehydration. The fulvenone ketene is responsible for the phenol formation. This technique may open unique opportunities for isomer-resolved probing in catalysis, and holds the potential for achieving a mechanistic understanding of complex, real-life catalytic processes. PMID:28660882

  10. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    Science.gov (United States)

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-07-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or `invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

  11. Infidelity of translation of encephalomyocarditis viral RNA with tRNA from human malignant trophoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, O.K.; Kuchino, Y.

    1977-09-23

    We have investigated tRNA from the human malignant trophoblastic cells (BeWo cell) and human chorionic tissue for the translation of specific mRNAs, in a tRNA-dependent protein synthesizing system from Ehrlich ascites cells. BeWo cell tRNA and chorionic tRNA supported oviduct mRNA or encephalomyocarditis (EMC) viral RNA directed amino acid incorporation into polypeptides equally effectively. Polypeptides synthesized with oviduct mRNA and tRNA from both sources were identical upon sodium dodecylsulfate polyacrylamide gel electrophoresis. But the EMC RNA directed polypeptides synthesized with BeWo cell tRNA were different from those synthesized with chorionic tRNA. A polypeptide (molecular weight 58,000) was apparently not synthesized and the synthesis of a faster moving component (molecular weight, 14,000) was enhanced when BeWo cell tRNA was used. These results imply a functional difference in tRNA from human malignant cells compared to their normal counterpart.

  12. Catalytic Organometallic Reactions of Ammonia

    Science.gov (United States)

    Klinkenberg, Jessica L.

    2012-01-01

    Until recently, ammonia had rarely succumbed to catalytic transformations with homogeneous catalysts, and the development of such reactions that are selective for the formation of single products under mild conditions has encountered numerous challenges. However, recently developed catalysts have allowed several classes of reactions to create products with nitrogen-containing functional groups from ammonia. These reactions include hydroaminomethylation, reductive amination, alkylation, allylic substitution, hydroamination, and cross-coupling. This Minireview describes examples of these processes and the factors that control catalyst activity and selectivity. PMID:20857466

  13. Studies of Catalytic Model Systems

    DEFF Research Database (Denmark)

    Holse, Christian

    of the Cu/ZnO nanoparticles is highly relevant to industrial methanol synthesis for which the direct interaction of Cu and ZnO nanocrystals synergistically boost the catalytic activity. The dynamical behavior of the nanoparticles under reducing and oxidizing environments were studied by means of ex situ X...... as the nanoparticles are reduced. The Cu/ZnO nanoparticles are tested on a  µ-reactor platform and prove to be active towards methanol synthesis, making it an excellent model system for further investigations into activity depended morphology changes....

  14. Catalytic Combustion of Ethyl Acetate

    OpenAIRE

    ÖZÇELİK, Tuğba GÜRMEN; ATALAY, Süheyda; ALPAY, Erden

    2014-01-01

    The catalytic combustion of ethyl acetate over prepared metal oxide catalysts was investigated. CeO, Co2O3, Mn2O3, Cr2O3, and CeO-Co2O3 catalysts were prepared on monolith supports and they were tested. Before conducting the catalyst experiments, we searched for the homogeneous gas phase combustion reaction of ethyl acetate. According to the homogeneous phase experimental results, 45% of ethyl acetate was converted at the maximum reactor temperature tested (350 °C). All the prepare...

  15. Kinetics of heterogeneous catalytic reactions

    CERN Document Server

    Boudart, Michel

    2014-01-01

    This book is a critical account of the principles of the kinetics of heterogeneous catalytic reactions in the light of recent developments in surface science and catalysis science. Originally published in 1984. The Princeton Legacy Library uses the latest print-on-demand technology to again make available previously out-of-print books from the distinguished backlist of Princeton University Press. These paperback editions preserve the original texts of these important books while presenting them in durable paperback editions. The goal of the Princeton Legacy Library is to vastly increase acc

  16. Heterogeneous Catalytic Oligomerization of Ethylene

    Science.gov (United States)

    Jan, Oliver Dennis

    increased with temperature, with 17 wt.% observed at 190ºC. Higher reaction temperatures led to the formation of odd-numbered oligomers primarily due to acid-catalyzed cracking reactions. In the range of space velocities tested, a moderate WHSV of 2.0 hr-1 resulted in a local maximum of 10.6 wt.% of liquid hydrocarbon yield. A moderate nickel loading of 3.4 wt.% also resulted in the highest liquid yield out of the three loadings tested (10.6 wt.%). The variation in nickel loading revealed the importance of having a synergistic balance of nickel and acid sites on the catalyst to maximize ethylene conversion and maintain high liquid hydrocarbon yield. Lastly, we used supercritical ethylene as both a solvent and as a reactant for ethylene oligomerization over two silica-alumina type catalysts: Ni-Hbeta and Ni-Al-SBA-15. Specifically, the effect of pressure and temperature on the overall conversion and product selectivity were evaluated in the range from 0 to 65 bar and 30 to 120ºC. At subcritical conditions, the ethylene conversion reached a plateau of around 50%. By increasing the pressure past the critical point of ethylene, the conversion drastically increased to 71%. The increased conversion can be attributed to the solubility of certain oligomers, namely butene, in supercritical ethylene that promotes desorption from catalytic active site before further oligomerization. We also tested a mesoporous catalyst, Ni-Al-SBA-15 and observed conversion trends analogous to that of Ni-Hbeta. At supercritical conditions, ethylene oligomerization over Ni-Al-SBA-15 was more selective towards the butene product, with nearly 74 wt.% butenes observed. The catalyst activity increased with temperature from 30ºC to 120ºC. The experiment conducted at 30ºC showed very little activity and ethylene conversion, however it effectively heavy molecular weight species from the catalyst. This condition, albeit being not effective for ethylene oligomerization, could be implemented as an in

  17. HIV-1 as RNA evolution machine

    NARCIS (Netherlands)

    Berkhout, Ben

    2011-01-01

    We have over the years studied several sequence or structural elements within the HIV-1 RNA genome. Molecular mechanisms have been proposed for the role of these RNA motifs in virus replication. We have developed HIV-1 evolution as a powerful research method to study different aspects of the viral

  18. The crystal structure of tRNA

    Indian Academy of Sciences (India)

    Madhu

    of yeast alanine tRNA by Robert Holley's group at Cornell. University (lthaca, NY, USA) in 1965, earned Holley a ... 1964, to a staff position at the MRC LMB's Division of. Molecular Genetics, then co-headed by Francis ... purification methods for tRNAs, especially for the initiator. tRNA. We were becoming less interested in ...

  19. Catalytic enantioselective Reformatsky reaction with ketones

    NARCIS (Netherlands)

    Fernandez-Ibanez, M. Angeles; Macia, Beatriz; Minnaard, Adriaan J.; Feringa, Ben L.

    2008-01-01

    Chiral tertiary alcohols were obtained with good yields and enantioselectivities via a catalytic Reformatsky reaction with ketones, including the challenging diaryl ketones, using chiral BINOL derivatives.

  20. Análisis morfológico del sistema reproductor e identificación molecular a través de los marcadores mitocondriales COI y 16S rRNA de Megalobulimus oblongus (Mollusca, Strophocheilidae de Colombia

    Directory of Open Access Journals (Sweden)

    Erika Jaramillo Roldán

    2014-05-01

    Full Text Available En este trabajo se hizo el análisis morfológico y molecular a 28 caracoles terrestres de Megalobulimus oblongus, colectados en diferentes departamentos de Colombia, depositados en una colección de referencia. Para la caracterización morfológica, los animales se disecaron al estéreomicroscopio. Se describieron el sistema reproductor y la concha. Del sistema reproductor se tomaron medidas a las estructuras que lo componen. De la concha se describió la forma, el color, el número de espiras y la ornamentación e igualmente se tomaron medidas básicas usando un calibrador digital. Para el análisis molecular se usaron dos marcadores mitocondriales, el 16S rRNA y el gen Citocromo Oxidasa C subunidad I (COI. Ambos marcadores confirmaron la presencia de un único haplotipo en los ejemplares, aun para individuos situados en regiones biogeográficas diferentes y distantes. Este estudio sugiere que en Colombia M. oblongus se encuentra en peligro, por lo que urgen investigaciones sobre reproducción, genética poblacional y biogeografía que esclarezcan su situación en el país. Demuestra además que las colecciones de referencia y los bancos de tejidos son fuentes de información de gran valor, ya que permiten conocer aspectos relacionados con el riesgo en que se encuentran las especies y que sirven de insumo para el diseño de acciones de conservación.

  1. Catalytic converters in the fireplace

    International Nuclear Information System (INIS)

    Kouki, J.

    1995-01-01

    In addition to selecting the appropriate means of heating and using dry fuel, the amount of harmful emissions contained by flue gases produced by fireplaces can be reduced by technical means. One such option is to use an oxidising catalytic converter. Tests at TTS Institute's Heating Studies Experimental Station have focused on two such converters (dense and coarse) mounted in light-weight iron heating stoves. The ability of the dense catalytic converter to oxidise carbon monoxide gases proved to be good. The concentration of carbon monoxide in the flue gases was reduced by as much as 90 %. Measurements conducted by VTT (Technical Research Centre of Finland) showed that the conversion of other gases, e.g. of methane, was good. The exhaust resistance caused by the dense converter was so great as to necessitate the mounting of a fluegas evacuation fan in the chimney for the purpose of creating sufficient draught. When relying on natural draught, the dense converter requires a chimney of at least 7 metres and a by-pass connection while the fire is being lit. In addition, the converter will have to be constructed to be less dense and this will mean that it's capability to oxidise non-combusted gases will be reduced. The coarse converter did not impair the draught but it's oxidising property was insufficient. With the tests over, the converter was not observed to have become blocked up by impurities

  2. Electrostatic interactions in catalytic centers of F1-ATPase

    Science.gov (United States)

    Pogrebnaya, Alexandra F.; Romanovsky, Yury M.; Tikhonov, Alexander N.

    2003-10-01

    F1-ATPase is one of the most important enzymes of membrane bioenergetics. F1-ATPase is the constituent complex that provides the ATP formation from ADP and inorganic phosphate (Pi) at the expense of energy of electrochemical gradient of hydrogen ions generated across the energy transducing mitochondrial, chloroplast or bacterial membrane. F1-ATPase is a reversible molecular machine that can work as a proton pump due to energy released in the course of ATP hydrolysis (ATPase reaction). The unusual feature of this enzyme is that it operates as a rotary molecular motor. Recently, using the fluorescence microscopy method for the real time visualization of molecular mobility of individual molecules, it was demonstrated directly that the ATP hydrolysis by F1-ATPase is accompanied by unidirectional rotations of mobile subunits (rotor) of F1F0-ATP synthase. In this work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), products molecules (MgADP and Pi), and charged amino acid residuals of ATPase molecule to the energy changes associated with the substrate binding and their chemical transformations in the catalytic centers located at the interface of α and β subunits of the enzyme (oligomer complex α3β3γ of bovine mitochondria ATPase). A catalytic cycle of ATP hydrolysis considered in our work includes conformational changes of α and β subunits caused by unidirectional rotations of an eccentric γ subunit. The knowledge of energy characteristics and force field in catalytic center of an enzyme in different conformational states may be important for further simulation dynamic properties of ATP synthase complex.

  3. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.

    OpenAIRE

    Richards, O C; Baker, S; Ehrenfeld, E

    1996-01-01

    The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and...

  4. Cofactors in the RNA World

    Science.gov (United States)

    Ditzler, Mark A.

    2014-01-01

    RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

  5. Clinical and molecular analysis of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss associated with the mitochondrial 12S rRNA C1494T mutation

    International Nuclear Information System (INIS)

    Wang Qiuju; Li Qingzhong; Han Dongyi; Zhao Yali; Zhao Lidong; Qian Yaping; Yuan Hu; Li Ronghua; Zhai Suoqiang; Young Wieyen; Guan Minxin

    2006-01-01

    We report here the clinical, genetic, and molecular characterization of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Five of nine matrilineal relatives had aminoglycoside-induced hearing loss. These matrilineal relatives exhibited variable severity and audiometric configuration of hearing impairment, despite sharing some common features: being bilateral and having sensorineural hearing impairment. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified 16 variants and the homoplasmic 12S rRNA C1494T mutation, which was associated with hearing loss in the other large Chinese family. In fact, the occurrence of the C1494T mutation in these genetically unrelated pedigrees affected by hearing impairment strongly indicated that this mutation is involved in the pathogenesis of aminoglycoside-induced and nonsyndromic hearing loss. However, incomplete penetrance of hearing loss indicated that the C1494T mutation itself is not sufficient to produce a clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Those mtDNA variants, showing no evolutional conservation, may not have a potential modifying role in the pathogenesis of the C1494T mutation. However, nuclear background seems to contribute to the phenotypic variability of matrilineal relatives in this family. Furthermore, aminoglycosides modulate the expressivity and penetrance of deafness associated with the C1494T mutation in this family

  6. Molecular Surveillance of Cronobacter spp. Isolated from a Wide Variety of Foods from 44 Different Countries by Sequence Typing of 16S rRNA, rpoB and O-Antigen Genes.

    Science.gov (United States)

    Miranda, Nancy; Banerjee, Pratik; Simpson, Steven; Kerdahi, Khalil; Sulaiman, Irshad M

    2017-05-11

    Cronobacter spp. are emerging infectious bacteria that can cause acute meningitis and necrotizing enterocolitis in neonatal and immunocompromised individuals. Although this opportunistic human-pathogenic microorganism has been isolated from a wide variety of food and environmental samples, it has been primarily linked to foodborne outbreaks associated with powdered infant formula. The U.S. Food and Drug Administration use the presence of these microbes as one of the criteria to assess food adulteration and to implement regulatory actions. In this study, we have examined 195 aliquots of enrichments from the nine major categories of foods (including baby and medical food, dairy products, dried food, frozen food, pet food, produce, ready-to-eat snacks, seafood, and spices) from 44 countries using conventional microbiological and molecular techniques. The typical colonies of Cronobacter were then identified by VITEK2 and real-time PCR. Subsequently, sequence typing was performed on the 51 recovered Cronobacter isolates at the 16S rRNA, rpo B and seven O-antigen loci for species identification in order to accomplish an effective surveillance program for the control and prevention of foodborne illnesses.

  7. Surface X-ray studies of catalytic clean technologies.

    Science.gov (United States)

    Lee, Adam F; Prabhakaran, Vinod; Wilson, Karen

    2010-06-14

    The rational design of new heterogeneous catalysts for clean chemical technologies can be accelerated by molecular level insight into surface chemical processes. In situ methodologies, able to provide time-resolved and/or pressure dependent information on the evolution of reacting adsorbed layers over catalytically relevant surfaces, are therefore of especial interest. Here we discuss recent applications of surface X-ray techniques to surface-catalysed oxidations, (de)hydrogenations, C-C coupling, dehalogenation and associated catalyst restructuring, and explore how these may help to shape future sustainable chemistry.

  8. Catalytic pyrolysis of nylon-6 to recover caprolactam

    Energy Technology Data Exchange (ETDEWEB)

    Czernik, Stefan; Elam, Carolyn C.; Evans, Robert J.; Meglen, Robert R.; Moens, Luc; Tatsumoto, Kuniyasu [National Renewable Energy Laboratory, Golden, CO 80401 (United States)

    1998-06-01

    Catalytic pyrolysis has been proposed as a possible process for the recovery of caprolactam from waste nylon-6. Promising process conditions, i.e. catalyst and temperature were identified using a micro-scale reactor/molecular-beam mass-spectrometer system. At 330-360C, in the presence of {alpha}-alumina supported KOH, the reaction proceeded at a high rate and selectivity. Only a few minutes were needed to complete the nylon-6 depolymerization with a caprolactam yield of 85%. These results were confirmed in a bench-scale fluidized bed reactor system

  9. Dedicated Beamline Facilities for Catalytic Research. Synchrotron Catalysis Consortium (SCC)

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jingguang [Columbia Univ., New York, NY; Frenkel, Anatoly [Yeshiva Univ., New York, NY (United States); Rodriguez, Jose [Brookhaven National Lab. (BNL), Upton, NY (United States); Adzic, Radoslav [Brookhaven National Lab. (BNL), Upton, NY (United States); Bare, Simon R. [UOP LLC, Des Plaines, IL (United States); Hulbert, Steve L. [Brookhaven National Lab. (BNL), Upton, NY (United States); Karim, Ayman [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Mullins, David R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Overbury, Steve [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2015-03-04

    Synchrotron spectroscopies offer unique advantages over conventional techniques, including higher detection sensitivity and molecular specificity, faster detection rate, and more in-depth information regarding the structural, electronic and catalytic properties under in-situ reaction conditions. Despite these advantages, synchrotron techniques are often underutilized or unexplored by the catalysis community due to various perceived and real barriers, which will be addressed in the current proposal. Since its establishment in 2005, the Synchrotron Catalysis Consortium (SCC) has coordinated significant efforts to promote the utilization of cutting-edge catalytic research under in-situ conditions. The purpose of the current renewal proposal is aimed to provide assistance, and to develop new sciences/techniques, for the catalysis community through the following concerted efforts: Coordinating the implementation of a suite of beamlines for catalysis studies at the new NSLS-II synchrotron source; Providing assistance and coordination for catalysis users at an SSRL catalysis beamline during the initial period of NSLS to NSLS II transition; Designing in-situ reactors for a variety of catalytic and electrocatalytic studies; Assisting experimental set-up and data analysis by a dedicated research scientist; Offering training courses and help sessions by the PIs and co-PIs.

  10. FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation.

    Science.gov (United States)

    Popow, Johannes; Alleaume, Anne-Marie; Curk, Tomaz; Schwarzl, Thomas; Sauer, Sven; Hentze, Matthias W

    2015-11-01

    Mitochondrial RNA processing is an essential step for the synthesis of the components of the electron transport chain in all eukaryotic organisms, yet several aspects of mitochondrial RNA biogenesis and regulation are not sufficiently understood. RNA interactome capture identified several disease-relevant RNA-binding proteins (RBPs) with noncanonical RNA-binding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. A mutation within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP. FASTKD2 interacts with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. CRISPR-mediated deletion of FASTKD2 leads to aberrant processing and expression of RNR2 and ND6 mRNA that encodes a subunit of the respiratory complex I. Metabolic phenotyping of FASTKD2-deficient cells reveals impaired cellular respiration with reduced activities of all respiratory complexes. This work identifies key aspects of the molecular network of a previously uncharacterized, disease-relevant RNA-binding protein, FASTKD2, by a combination of genomic, molecular, and metabolic analyses. © 2015 Popow et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  11. Biases in small RNA deep sequencing data.

    Science.gov (United States)

    Raabe, Carsten A; Tang, Thean-Hock; Brosius, Juergen; Rozhdestvensky, Timofey S

    2014-02-01

    High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias.

  12. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

    2012-10-10

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

  13. Heterogeneous catalytic degradation of polyacrylamide solution | Hu ...

    African Journals Online (AJOL)

    Modified with trace metal elements, the catalytic activity of Fe2O3/Al2O3 could be changed greatly. Among various trace metal elements, Fe2O3/Al2O3 catalysts modified with Co and Cu showed great increase on catalytic activity. International Journal of Engineering, Science and Technology, Vol. 2, No. 7, 2010, pp. 110- ...

  14. Catalytic gasification of dry and wet biomass

    NARCIS (Netherlands)

    van Rossum, G.; Potic, B.; Kersten, Sascha R.A.; van Swaaij, Willibrordus Petrus Maria

    2009-01-01

    Catalytic gasification of dry biomass and of wet biomass streams in hot compressed water are reviewed and discussed as potential technologies for the production of synthesis gas, hydrogen- and methane-rich gas. Next to literature data also new experimental results from our laboratory on catalytic

  15. Conserved water-mediated H-bonding dynamics of catalytic Asn ...

    Indian Academy of Sciences (India)

    Prakash

    Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that ...

  16. Direct observation of doping incorporation pathways in self-catalytic GaMnAs nanowires

    DEFF Research Database (Denmark)

    Kasama, Takeshi; Thuvander, M.; Siusys, A.

    2015-01-01

    Doping mechanisms of Mn in GaAs nanowires (NWs) that have been grown self-catalytically at 600 degrees C by molecular beam epitaxy (MBE) are investigated using advanced electron microscopy techniques and atom probe tomography, Mn is found to be incorporated primarily in the form of non...

  17. Conserved water-mediated H-bonding dynamics of catalytic Asn ...

    Indian Academy of Sciences (India)

    Prakash

    [Nandi T K, Bairagya H R, Mukhopadhyay B P, Sekar K, Sukul D and Bera A K 2009 Conserved water-mediated H-bonding dynamics of catalytic. Asn 175 in plant thiol protease; J. Biosci. 34 27–34]. Keywords. Conserved water in molecular recognition; MD simulation; plant cysteine protease. Abbreviations used: CHASA ...

  18. Non-catalytic recuperative reformer

    Science.gov (United States)

    Khinkis, Mark J.; Kozlov, Aleksandr P.; Kurek, Harry

    2015-12-22

    A non-catalytic recuperative reformer has a flue gas flow path for conducting hot flue gas from a thermal process and a reforming mixture flow path for conducting a reforming mixture. At least a portion of the reforming mixture flow path is embedded in the flue gas flow path to permit heat transfer from the hot flue gas to the reforming mixture. The reforming mixture flow path contains substantially no material commonly used as a catalyst for reforming hydrocarbon fuel (e.g., nickel oxide, platinum group elements or rhenium), but instead the reforming mixture is reformed into a higher calorific fuel via reactions due to the heat transfer and residence time. In a preferred embodiment, extended surfaces of metal material such as stainless steel or metal alloy that are high in nickel content are included within at least a portion of the reforming mixture flow path.

  19. Electrochemical promotion of catalytic reactions

    Science.gov (United States)

    Imbihl, R.

    2010-05-01

    The electrochemical promotion of heterogeneously catalyzed reactions (EPOC) became feasible through the use of porous metal electrodes interfaced to a solid electrolyte. With the O 2- conducting yttrium stabilized zirconia (YSZ), the Na + conducting β″-Al 2O 3 (β-alumina), and several other types of solid electrolytes the EPOC effect has been demonstrated for about 100 reaction systems in studies conducted mainly in the mbar range. Surface science investigations showed that the physical basis for the EPOC effect lies in the electrochemically induced spillover of oxygen and alkali metal, respectively, onto the surface of the metal electrodes. For the catalytic promotion effect general concepts and mechanistic schemes were proposed but these concepts and schemes are largely speculative. Applying surface analytical tools to EPOC systems the proposed mechanistic schemes can be verified or invalidated. This report summarizes the progress which has been achieved in the mechanistic understanding of the EPOC effect.

  20. Selective catalytic oxidation of ammonia

    Energy Technology Data Exchange (ETDEWEB)

    Leppaelahti, J.; Koljonen, T. [VTT Energy, Espoo (Finland)

    1996-12-31

    In the combustion of fossil fuels, the principal source of nitrogen oxides is nitrogen bound in the fuel structure. In gasification, a large part of fuel nitrogen forms NH{sub 3}, which may form nitrogen oxides during gas combustion. If NH{sub 3} and other nitrogen species could be removed from hot gas, the NO emission could be considerably reduced. However, relatively little attention has been paid to finding new means of removing nitrogen compounds from the hot gasification gas. The possibility of selectively oxidizing NH{sub 3} to N{sub 2} in the hot gasification has been studied at VTT Energy. The largest NH{sub 3} reductions have been achieved by catalytic oxidation on aluminium oxides. (author) (4 refs.)

  1. The Catalytic Role of Coronene for Molecular Hydrogen Formation

    DEFF Research Database (Denmark)

    Mennella, Vito; Hornekær, Liv; Thrower, John

    2012-01-01

    We present the results of an experimental study on the interaction of atomic deuterium with coronene films. The effects of D atom irradiation have been analyzed with infrared spectroscopy. The spectral changes provide evidence for deuteration of the outer edge coronene C sites via a D addition re...

  2. Diesel engine catalytic combustor system. [aircraft engines

    Science.gov (United States)

    Ream, L. W. (Inventor)

    1984-01-01

    A low compression turbocharged diesel engine is provided in which the turbocharger can be operated independently of the engine to power auxiliary equipment. Fuel and air are burned in a catalytic combustor to drive the turbine wheel of turbine section which is initially caused to rotate by starter motor. By opening a flapper value, compressed air from the blower section is directed to catalytic combustor when it is heated and expanded, serving to drive the turbine wheel and also to heat the catalytic element. To start, engine valve is closed, combustion is terminated in catalytic combustor, and the valve is then opened to utilize air from the blower for the air driven motor. When the engine starts, the constituents in its exhaust gas react in the catalytic element and the heat generated provides additional energy for the turbine section.

  3. Short RNA indicator sequences are not completely degraded by autoclaving

    Science.gov (United States)

    Unnithan, Veena V.; Unc, Adrian; Joe, Valerisa; Smith, Geoffrey B.

    2014-01-01

    Short indicator RNA sequences (autoclaving and are recovered intact by molecular amplification. Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses. If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving. PMID:24518856

  4. Predicting and Modeling RNA Architecture

    Science.gov (United States)

    Westhof, Eric; Masquida, Benoît; Jossinet, Fabrice

    2011-01-01

    SUMMARY A general approach for modeling the architecture of large and structured RNA molecules is described. The method exploits the modularity and the hierarchical folding of RNA architecture that is viewed as the assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. Despite the extensive molecular neutrality observed in RNA structures, specificity in RNA folding is achieved through global constraints like lengths of helices, coaxiality of helical stacks, and structures adopted at the junctions of helices. The Assemble integrated suite of computer tools allows for sequence and structure analysis as well as interactive modeling by homology or ab initio assembly with possibilities for fitting within electronic density maps. The local key role of non-Watson-Crick pairs guides RNA architecture formation and offers metrics for assessing the accuracy of three-dimensional models in a more useful way than usual root mean square deviation (RMSD) values. PMID:20504963

  5. Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles.

    Science.gov (United States)

    Afonin, Kirill A; Viard, Mathias; Kagiampakis, Ioannis; Case, Christopher L; Dobrovolskaia, Marina A; Hofmann, Jen; Vrzak, Ashlee; Kireeva, Maria; Kasprzak, Wojciech K; KewalRamani, Vineet N; Shapiro, Bruce A

    2015-01-27

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.

  6. Analysis of RNA by capillary electrophoresis.

    Science.gov (United States)

    Skeidsvoll, J; Ueland, P M

    1996-09-01

    Analytical parameters known to be important for the separation of DNA by capillary electrophoresis, including gel polymer concentration, electrical field strength and temperature, were investigated and optimized for the analysis of RNA molecules from 100 to 2000 bases. Denaturation, essential to obtain uniform and identifiable peaks, was accomplished by heating the sample in 80% formamide prior to electrophoresis and the presence of 2-8 M urea in the electrophoresis buffer. Efficient separations were obtained over a wide range of electrical field strengths and temperatures using the gel polymer hydroxypropylmethylcellulose (HPMC) as separation matrix. Low HPMC concentrations (RNA (> 1000 bases) whereas higher HPMC concentrations were required for optimal separation of low molecular mass RNA. An optimized system was applicable for the separation of the predominating RNA populations (small RNA of 60-300 bases (as a group of unseparated peaks), 18S and 28S rRNA) in total RNA from a human glioma cell line. This is the first systematic investigation of electrophoresis of higher molecular mass RNA in capillaries, and motivates further studies to transfer electrophoresis of RNA to the capillary format.

  7. Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue.

    Science.gov (United States)

    Chène, Patrick; Rudloff, Joëlle; Schoepfer, Joseph; Furet, Pascal; Meier, Peter; Qian, Zhiyan; Schlaeppi, Jean-Marc; Schmitz, Rita; Radimerski, Thomas

    2009-01-07

    Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1) inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

  8. Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue

    Directory of Open Access Journals (Sweden)

    Schlaeppi Jean-Marc

    2009-01-01

    Full Text Available Abstract Background Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. Results Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1 inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. Conclusion The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

  9. Examination of bacterial inhibition using a catalytic DNA.

    Directory of Open Access Journals (Sweden)

    Long Qu

    Full Text Available Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA, RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC of antibiotics much faster than the conventional optical density (OD method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research.

  10. Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance.

    Directory of Open Access Journals (Sweden)

    You R Chng

    Full Text Available African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp. This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

  11. Fast production of homogeneous recombinant RNA--towards large-scale production of RNA

    NARCIS (Netherlands)

    Nelissen, F.H.; Leunissen, E.H.P.; van de Laar, L.; Tessari, M.; Heus, H.A.; Wijmenga, S.S.

    2012-01-01

    In the past decades, RNA molecules have emerged as important players in numerous cellular processes. To understand these processes at the molecular and atomic level, large amounts of homogeneous RNA are required for structural, biochemical and pharmacological investigations. Such RNAs are generally

  12. Catalytic Wittig and aza-Wittig reactions

    Directory of Open Access Journals (Sweden)

    Zhiqi Lao

    2016-11-01

    Full Text Available This review surveys the literature regarding the development of catalytic versions of the Wittig and aza-Wittig reactions. The first section summarizes how arsenic and tellurium-based catalytic Wittig-type reaction systems were developed first due to the relatively easy reduction of the oxides involved. This is followed by a presentation of the current state of the art regarding phosphine-catalyzed Wittig reactions. The second section covers the field of related catalytic aza-Wittig reactions that are catalyzed by both phosphine oxides and phosphines.

  13. Catalytic Wittig and aza-Wittig reactions.

    Science.gov (United States)

    Lao, Zhiqi; Toy, Patrick H

    2016-01-01

    This review surveys the literature regarding the development of catalytic versions of the Wittig and aza-Wittig reactions. The first section summarizes how arsenic and tellurium-based catalytic Wittig-type reaction systems were developed first due to the relatively easy reduction of the oxides involved. This is followed by a presentation of the current state of the art regarding phosphine-catalyzed Wittig reactions. The second section covers the field of related catalytic aza-Wittig reactions that are catalyzed by both phosphine oxides and phosphines.

  14. Statistical evaluation of mature landfill leachate treatment by homogeneous catalytic ozonation

    Directory of Open Access Journals (Sweden)

    A. L. C. Peixoto

    2010-12-01

    Full Text Available This study presents the results of a mature landfill leachate treated by a homogeneous catalytic ozonation process with ions Fe2+ and Fe3+ at acidic pH. Quality assessments were performed using Taguchi's method (L8 design. Strong synergism was observed statistically between molecular ozone and ferric ions, pointing to their catalytic effect on •OH generation. The achievement of better organic matter depollution rates requires an ozone flow of 5 L h-1 (590 mg h-1 O3 and a ferric ion concentration of 5 mg L-1.

  15. Mesoporous molecular sieve catalysts

    DEFF Research Database (Denmark)

    Højholt, Karen Thrane

    This thesis deals with a very specific class of molecular sieves known as zeolites. Zeolites are a class of crystalline aluminosilicates characterised by pores or cavities of molecular dimensions as part of their crystal structure. In this work zeolites were modified for the use and understanding...... of different catalytic applications. Primarily the zeolites were modified regarding the porosity and the introduction of metals to the framework. The obtained materials were used as solid acid catalysts, as an inert matrix for stabilising metal nanoparticles and as an anchoring material for molecular metal....... Furthermore, preliminary work was done using mesoporous ZSM-5 zeolites as support material for anchoring molecular CoMo6 species for the application as potential bi-functional catalyst in simultaneous hydrodesulfurisation (HDS) and hydrocracking. HDS activity tests revealed that the anchoring improved...

  16. Catalytic activity of nuclease P1: Experiment and theory

    International Nuclear Information System (INIS)

    Miller, J.H.; Falcone, J.M.; Shibata, M.; Box, H.C.

    1994-10-01

    Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates

  17. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Directory of Open Access Journals (Sweden)

    Dong Zhao

    2017-01-01

    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  18. Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

    Science.gov (United States)

    Alshehri, Abdullah; Grabowska, Anna; Stolnik, Snow

    2018-02-28

    Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement - a stage between initial cellular internalization and final gene silencing of siRNA delivery systems.

  19. Rice Cellulose SynthaseA8 P