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Sample records for chemically modified rna

  1. Efficient ex vivo delivery of chemically modified messenger RNA using lipofection and magnetofection.

    Science.gov (United States)

    Badieyan, Zohreh Sadat; Pasewald, Tamara; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2017-01-22

    Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. siRNAmod: A database of experimentally validated chemically modified siRNAs.

    Science.gov (United States)

    Dar, Showkat Ahmad; Thakur, Anamika; Qureshi, Abid; Kumar, Manoj

    2016-01-28

    Small interfering RNA (siRNA) technology has vast potential for functional genomics and development of therapeutics. However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. Chemical modifications in siRNAs provide means to overcome these shortcomings and improve their stability and potency. Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. Therefore, we have developed siRNAmod, a specialized databank for chemically modified siRNAs. Currently, our repository contains a total of 4894 chemically modified-siRNA sequences, comprising 128 unique chemical modifications on different positions with various permutations and combinations. It incorporates important information on siRNA sequence, chemical modification, their number and respective position, structure, simplified molecular input line entry system canonical (SMILES), efficacy of modified siRNA, target gene, cell line, experimental methods, reference etc. It is developed and hosted using Linux Apache MySQL PHP (LAMP) software bundle. Standard user-friendly browse, search facility and analysis tools are also integrated. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. siRNAmod is freely available at: http://crdd.osdd.net/servers/sirnamod.

  3. Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA

    Directory of Open Access Journals (Sweden)

    Eva Schrom

    2017-06-01

    Full Text Available Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2 protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models.

  4. Enzymatic synthesis of tRNA-peptide conjugates and spectroscopic studies of fluorine-modified RNA

    International Nuclear Information System (INIS)

    Graber, D.

    2010-01-01

    The research presented in this thesis concerns the enzymatic synthesis of artificially modified tRNA, in particular the preparation of non-hydrolysable tRNA-peptide conjugates. Another focus is on NMR-spectroscopic investigations of fluorine-modified RNA. In both projects, chemical methods were developed to address specific RNA-biological research questions. In the first part of this thesis the preparation of tRNA-peptide conjugates with a non-hydrolysable 3'-amide linkage is presented. These molecules are of high relevance for the characterization of ribosomal processes that occur in the peptidyl transferase center (such as peptide bond formation, peptide release, or translocation) using X-ray crystallography and biochemical methods. First, a novel concept to prepare chemically modified ('labeled') tRNA was elaborated based on the combination of solid-phase synthesis and enzymatic ligation. Thereby, a variety of differently labeled tRNAs was achieved. Moreover, the most successful high-yield ligation sites were identified to be situated within the TΨ C-loop. Optimization of the synthesis and the corresponding HPLC-purification of the conjugates were initially conducted with puromycin derivatized tRNA. In the course of this project, also two tRNAs with a ribose 3'-amino group at the terminal adenosine A76 were synthesized. For that purpose a protection group pattern had to be developed to obtain a functionalized solid-support bound to 3'-amino-3'-deoxyadenosine which was appropriate for RNA solid-phase synthesis. The successful preparation of tRNA-peptide conjugates was accomplished in cooperation with Holger Moroder and Jessica Steger (Micura group) who contributed short synthetic RNA-peptide conjugates. These fragments represented the tRNA 3'-termini that were required for exploring the new ligation strategies for non-hydrolisable tRNA - a main aim of this thesis. If the 5'-fragments are synthesized by solid-phase synthesis or in vitro transcription they do not

  5. Covalent Chemical 5'-Functionalization of RNA with Diazo Reagents.

    Science.gov (United States)

    Gampe, Christian M; Hollis-Symynkywicz, Micah; Zécri, Frédéric

    2016-08-22

    Functionalization of RNA at the 5'-terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5'-nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5'-phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5'-end of RNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries

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    Yuuya Kasahara

    2012-01-01

    Full Text Available Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.

  7. Artificial specific binders directly recovered from chemically modified nucleic acid libraries.

    Science.gov (United States)

    Kasahara, Yuuya; Kuwahara, Masayasu

    2012-01-01

    Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.

  8. Chemically modified oligonucleotides with efficient RNase H response

    DEFF Research Database (Denmark)

    Vester, Birte; Boel, Anne Marie; Lobedanz, Sune

    2008-01-01

    Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly...... in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage....

  9. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies

    KAUST Repository

    Chawla, Mohit

    2015-06-27

    Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.

  10. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies

    KAUST Repository

    Chawla, Mohit; Oliva, R.; Bujnicki, J. M.; Cavallo, Luigi

    2015-01-01

    Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.

  11. Treating respiratory viral diseases with chemically modified, second generation intranasal siRNAs.

    Science.gov (United States)

    Barik, Sailen

    2009-01-01

    Chemically synthesized short interfering RNA (siRNA) of pre-determined sequence has ushered a new era in the application of RNA interference (RNAi) against viral genes. We have paid particular attention to respiratory viruses that wreak heavy morbidity and mortality worldwide. The clinically significant ones include respiratory syncytial virus (RSV), parainfluenza virus (PIV) and influenza virus. As the infection by these viruses is clinically restricted to the respiratory tissues, mainly the lungs, the logical route for the application of the siRNA was also the same, i.e., via the nasal route. Following the initial success of intranasal siRNA against RSV, second-generation siRNAs were made against the viral polymerase large subunit (L) that were chemically modified and screened for improved stability, activity and pharmacokinetics. 2'-O-methyl (2'-O-Me) and 2'-deoxy-2'-fluoro (2'-F) substitutions in the ribose ring were incorporated in different positions of the sense and antisense strands and the resultant siRNAs were tested with various transfection reagents intranasally against RSV. Based on these results, we propose the following consensus for designing intranasal antiviral siRNAs: (i) modified 19-27 nt long double-stranded siRNAs are functional in the lung, (ii) excessive 2'-OMe and 2'-F modifications in either or both strands of these siRNAs reduce efficacy, and (iii) limited modifications in the sense strand are beneficial, although their precise efficacy may be position-dependent.

  12. Genetic and chemical modifiers of a CUG toxicity model in Drosophila.

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    Amparo Garcia-Lopez

    2008-02-01

    Full Text Available Non-coding CUG repeat expansions interfere with the activity of human Muscleblind-like (MBNL proteins contributing to myotonic dystrophy 1 (DM1. To understand this toxic RNA gain-of-function mechanism we developed a Drosophila model expressing 60 pure and 480 interrupted CUG repeats in the context of a non-translatable RNA. These flies reproduced aspects of the DM1 pathology, most notably nuclear accumulation of CUG transcripts, muscle degeneration, splicing misregulation, and diminished Muscleblind function in vivo. Reduced Muscleblind activity was evident from the sensitivity of CUG-induced phenotypes to a decrease in muscleblind genetic dosage and rescue by MBNL1 expression, and further supported by the co-localization of Muscleblind and CUG repeat RNA in ribonuclear foci. Targeted expression of CUG repeats to the developing eye and brain mushroom bodies was toxic leading to rough eyes and semilethality, respectively. These phenotypes were utilized to identify genetic and chemical modifiers of the CUG-induced toxicity. 15 genetic modifiers of the rough eye phenotype were isolated. These genes identify putative cellular processes unknown to be altered by CUG repeat RNA, and they include mRNA export factor Aly, apoptosis inhibitor Thread, chromatin remodelling factor Nurf-38, and extracellular matrix structural component Viking. Ten chemical compounds suppressed the semilethal phenotype. These compounds significantly improved viability of CUG expressing flies and included non-steroidal anti-inflammatory agents (ketoprofen, muscarinic, cholinergic and histamine receptor inhibitors (orphenadrine, and drugs that can affect sodium and calcium metabolism such as clenbuterol and spironolactone. These findings provide new insights into the DM1 phenotype, and suggest novel candidates for DM1 treatments.

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

    Science.gov (United States)

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

    2017-10-01

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

  14. Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

    Science.gov (United States)

    Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

    2016-08-26

    The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

    Science.gov (United States)

    Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

    2018-03-05

    Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

  16. Chemical fidelity of an RNA polymerase ribozyme

    DEFF Research Database (Denmark)

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

    2013-01-01

    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......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...

  17. Screening of Modified RNA duplexes

    DEFF Research Database (Denmark)

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

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

  18. LNA-modified oligonucleotides mediate specific inhibition of microRNA function

    DEFF Research Database (Denmark)

    Ørom, Ulf Andersson; Kauppinen, Sakari; Lund, Anders H

    2006-01-01

    microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been...... observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high...... specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus...

  19. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.

    Science.gov (United States)

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

    2016-04-20

    DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Mechanical properties of chemically modified portuguese pinewood

    OpenAIRE

    Lopes, Duarte B; Mai, Carsten; Militz, Holger

    2014-01-01

    To turn wood into a construction material with enhanced properties, many methods of chemical modification have been developed in the last few decades. In this work, mechanical properties of pine wood were chemically modified, compared and evaluated. Maritime pine wood (Pinus pinaster) was modified with four chemical processes: 1,3-dimethylol-4,5- dihydroxyethyleneurea, N-methylol melamine formaldehyde, tetra-alkoxysilane and wax. The following mechanical properties were assessed experiment...

  1. Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

    Science.gov (United States)

    Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

    2016-01-01

    In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

  2. A novel method of modifying immune responses by vaccination with lipiodol-siRNA mixtures

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    Yijian Li

    2006-01-01

    Full Text Available Abstract The dendritic cell (DC possesses the ability to stimulate both T helper 1 (Th1 and Th2 responses depending on activation stimuli. Although it is known that chemically or genetically modified DC can be used therapeutically to steer immune responses towards either Th1 or Th2, cellular therapy with ex vivo manipulated DC is clinically difficult. Here we demonstrate a novel method of switching immune responses from Th1 to Th2 through in vivo immune modulation by administration of siRNA. We demonstrate that siRNA targeting of the IL-12p35 gene leads to a Th2 bias in vitro through an IL-10 dependent mechanism. In vivo administration of siRNA admixed with the oil-based contrast agent lipiodol in the presence of antigen and adjuvant induced a deviation in recall response to reduced production of IFN-γ and augmented IL-4 response using either KLH or ovalbumin. This simple method of in vivo modification of immune response possesses therapeutic potential in Th1-mediated diseases such as multiple sclerosis and autoimmune diabetes.

  3. The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

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    Negin Saffarzadeh

    2014-10-01

    Full Text Available Objective(s: Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG, DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P

  4. Epoxy modified bitumen : Chemical hardening and its interpretation

    NARCIS (Netherlands)

    Apostolidis, P.; Pipintakos, G.; van de Ven, M.F.C.; Liu, X.; Erkens, Sandra; Scarpas, Athanasios

    2018-01-01

    Epoxy modified bitumen (EMB) is a promising technology for long lasting paving materials ensuring higher resistance to rutting, oxygen- and moisture-induced damage. In this paper, an analysis of the chemical reactions that take place during the chemical hardening process (curing) of epoxy modified

  5. The effects of environmental chemical carcinogens on the microRNA machinery.

    Science.gov (United States)

    Izzotti, A; Pulliero, A

    2014-07-01

    The first evidence that microRNA expression is early altered by exposure to environmental chemical carcinogens in still healthy organisms was obtained for cigarette smoke. To date, the cumulative experimental data indicate that similar effects are caused by a variety of environmental carcinogens, including polycyclic aromatic hydrocarbons, nitropyrenes, endocrine disruptors, airborne mixtures, carcinogens in food and water, and carcinogenic drugs. Accordingly, the alteration of miRNA expression is a general mechanism that plays an important pathogenic role in linking exposure to environmental toxic agents with their pathological consequences, mainly including cancer development. This review summarizes the existing experimental evidence concerning the effects of chemical carcinogens on the microRNA machinery. For each carcinogen, the specific microRNA alteration signature, as detected in experimental studies, is reported. These data are useful for applying microRNA alterations as early biomarkers of biological effects in healthy organisms exposed to environmental carcinogens. However, microRNA alteration results in carcinogenesis only if accompanied by other molecular damages. As an example, microRNAs altered by chemical carcinogens often inhibits the expression of mutated oncogenes. The long-term exposure to chemical carcinogens causes irreversible suppression of microRNA expression thus allowing the transduction into proteins of mutated oncogenes. This review also analyzes the existing knowledge regarding the mechanisms by which environmental carcinogens alter microRNA expression. The underlying molecular mechanism involves p53-microRNA interconnection, microRNA adduct formation, and alterations of Dicer function. On the whole, reported findings provide evidence that microRNA analysis is a molecular toxicology tool that can elucidate the pathogenic mechanisms activated by environmental carcinogens. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing

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    Elisabeth H. Vollmann

    2017-06-01

    Full Text Available Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.

  7. An RNA-seq transcriptome analysis of histone modifiers and RNA silencing genes in soybean during floral initiation process.

    Directory of Open Access Journals (Sweden)

    Lim Chee Liew

    Full Text Available Epigenetics has been recognised to play vital roles in many plant developmental processes, including floral initiation through the epigenetic regulation of gene expression. The histone modifying proteins that mediate these modifications involve the SET domain-containing histone methyltransferases, JmjC domain-containing demethylase, acetylases and deacetylases. In addition, RNA interference (RNAi-associated genes are also involved in epigenetic regulation via RNA-directed DNA methylation and post-transcriptional gene silencing. Soybean, a major crop legume, requires a short day to induce flowering. How histone modifications regulate the plant response to external cues that initiate flowering is still largely unknown. Here, we used RNA-seq to address the dynamics of transcripts that are potentially involved in the epigenetic programming and RNAi mediated gene silencing during the floral initiation of soybean. Soybean is a paleopolyploid that has been subjected to at least two rounds of whole genome duplication events. We report that the expanded genomic repertoire of histone modifiers and RNA silencing genes in soybean includes 14 histone acetyltransferases, 24 histone deacetylases, 47 histone methyltransferases, 15 protein arginine methyltransferases, 24 JmjC domain-containing demethylases and 47 RNAi-associated genes. To investigate the role of these histone modifiers and RNA silencing genes during floral initiation, we compared the transcriptional dynamics of the leaf and shoot apical meristem at different time points after a short-day treatment. Our data reveal that the extensive activation of genes that are usually involved in the epigenetic programming and RNAi gene silencing in the soybean shoot apical meristem are reprogrammed for floral development following an exposure to inductive conditions.

  8. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    . This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)+ RNA accumulation within......In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues...

  9. Modified beacon probe assisted dual signal amplification for visual detection of microRNA.

    Science.gov (United States)

    Sun, Xiuwei; Ying, Na; Ju, Chuanjing; Li, Zhongyi; Xu, Na; Qu, Guijuan; Liu, Wensen; Wan, Jiayu

    2018-04-21

    In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Chemical composition of silica-based biocidal modifier

    Directory of Open Access Journals (Sweden)

    Grishina Anna Nikolaevna

    2016-11-01

    Full Text Available Increase of the amount of fungi spores and micotixines causes the increase in the number of different diseases. Because of this, ensuring the biological safety in buildings is becoming more and more important today. The preferred way to guarantee the biological safety of a building is to employ modern building materials that prevent the settlement of the fungi colonies on the inner surfaces of walls. Such building materials can be produced using novel biocidal modifiers that allow controlling the number of microorganisms on the surface and in the bulk of a composite construction. The precipitation product of zinc hydrosilicates and sodium sulfate is one of the mentioned modifiers. Till now, the exact chemical composition of such precipitation product is controversial; it is obvious, though, that the efficacy of the biocidal modifier is mostly determined by the type of the copper compounds. In the present work an integrated approach is used for the investigation of the chemical composition of the biocidal modifier. Such an approach consists in the examination of the modifier’s composition by means of different, yet complementary, research methods: X-ray diffraction, infrared spectroscopy and DTA. It is shown that the chemical composition of the modifier mainly depends on the amount of precipitant. X-ray diffraction reveals that the major part of the modifier is represented by amorphous phase. Along with the increase of the precipitant’s amount the crystalline phase Zn4SO4(OH6•xH2O formation takes place. Such a crystalline phase is not appropriate as a component of the biocidal modifier. Another two methods - DTA and IR spectroscopy - reveal that the amorphous phase consists essentially of zinc hydrosilicates.

  11. Biosorption of uranium by chemically modified Rhodotorula glutinis

    International Nuclear Information System (INIS)

    Bai Jing; Yao Huijun; Fan Fangli; Lin Maosheng; Zhang Lina; Ding Huajie; Lei Fuan; Wu Xiaolei; Li, Xiaofei; Guo Junsheng; Qin Zhi

    2010-01-01

    The present paper reports the biosorption of uranium onto chemically modified yeast cells, Rhodotorula glutinis, in order to study the role played by various functional groups in the cell wall. Esterification of the carboxyl groups and methylation of the amino groups present in the cells were carried out by methanol and formaldehyde treatment, respectively. The uranium sorption capacity increased 31% for the methanol-treated biomass and 11% for the formaldehyde-treated biomass at an initial uranium concentration of 140 mg/L. The enhancement of uranium sorption capacity was investigated by Fourier transform infrared (FTIR) spectroscopy analysis, with amino and carboxyl groups were determined to be the important functional groups involved in uranium binding. The biosorption isotherms of uranium onto the raw and chemically modified biomass were also investigated with varying uranium concentrations. Langmuir and Freundlich models were well able to explain the sorption equilibrium data with satisfactory correlation coefficients higher than 0.9. -- Research highlights: → Uranium biosorption on to chemically modified yeast cells → Cells before and after uranium sorption were investigate by FTIR spectroscopy → Amino and carboxyl groups were important functional groups involved in uranium binding → The sorption equilibrium date of raw and chemically modified biomass fitted well with Langmuir and Freundlich models

  12. Biosorption of uranium by chemically modified Rhodotorula glutinis

    Energy Technology Data Exchange (ETDEWEB)

    Bai Jing, E-mail: baijing@impcas.ac.c [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Yao Huijun [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Fan Fangli; Lin Maosheng [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang Lina; Ding Huajie; Lei Fuan; Wu Xiaolei [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Li, Xiaofei [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Guo Junsheng; Qin Zhi [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China)

    2010-11-15

    The present paper reports the biosorption of uranium onto chemically modified yeast cells, Rhodotorula glutinis, in order to study the role played by various functional groups in the cell wall. Esterification of the carboxyl groups and methylation of the amino groups present in the cells were carried out by methanol and formaldehyde treatment, respectively. The uranium sorption capacity increased 31% for the methanol-treated biomass and 11% for the formaldehyde-treated biomass at an initial uranium concentration of 140 mg/L. The enhancement of uranium sorption capacity was investigated by Fourier transform infrared (FTIR) spectroscopy analysis, with amino and carboxyl groups were determined to be the important functional groups involved in uranium binding. The biosorption isotherms of uranium onto the raw and chemically modified biomass were also investigated with varying uranium concentrations. Langmuir and Freundlich models were well able to explain the sorption equilibrium data with satisfactory correlation coefficients higher than 0.9. -- Research highlights: {yields} Uranium biosorption on to chemically modified yeast cells {yields} Cells before and after uranium sorption were investigate by FTIR spectroscopy {yields} Amino and carboxyl groups were important functional groups involved in uranium binding {yields} The sorption equilibrium date of raw and chemically modified biomass fitted well with Langmuir and Freundlich models

  13. The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

    OpenAIRE

    Negin Saffarzadeh; Seyed Mehdi Kalantar; Ali Jebali; Seyed Hossein Hekmatimoghaddam; Mohammad Hassan Sheikhha; Ehsan Farashahi

    2014-01-01

    Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE)-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium...

  14. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases....

  15. Steric restrictions of RISC in RNA interference identified with size-expanded RNA nucleobases.

    Science.gov (United States)

    Hernández, Armando R; Peterson, Larryn W; Kool, Eric T

    2012-08-17

    Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

  16. Synthesis and Structural Characterization of 2'-Fluoro-α-L-RNA-Modified Oligonucleotides

    DEFF Research Database (Denmark)

    Bundgaard Jensen, Troels; Pasternak, Anna; Stahl Madsen, Andreas

    2011-01-01

    with the smallest destabilization towards RNA. Thermodynamic data show that the duplex formation with 2'-fluoro-α-L-RNA nucleotides is enthalpically disfavored but entropically favored. 2'-Fluoro-α-L-RNA nucleotides exhibit very good base pairing specificity following Watson-Crick rules. The 2'-fluoro......-α-L-RNA monomer was designed as a monocyclic mimic of the bicyclic α-L-LNA, and molecular modeling showed that this indeed is the case as the 2'-fluoro monomer adopts a C3'-endo/C2'-exo sugar pucker. Molecular modeling of modified duplexes show that the 2'-fluoro-α-L-RNA nucleotides partake in Watson-Crick base......We describe the synthesis and binding properties of oligonucleotides that contain one or more 2'-fluoro-α-L-RNA thymine monomer(s). Incorporation of 2'-fluoro-α-L-RNA thymine into oligodeoxynucleotides decreased thermal binding stability slightly upon hybridization with complementary DNA and RNA...

  17. Chemically-modified cellulose paper as a microstructured catalytic reactor.

    Science.gov (United States)

    Koga, Hirotaka; Kitaoka, Takuya; Isogai, Akira

    2015-01-15

    We discuss the successful use of chemically-modified cellulose paper as a microstructured catalytic reactor for the production of useful chemicals. The chemical modification of cellulose paper was achieved using a silane-coupling technique. Amine-modified paper was directly used as a base catalyst for the Knoevenagel condensation reaction. Methacrylate-modified paper was used for the immobilization of lipase and then in nonaqueous transesterification processes. These catalytic paper materials offer high reaction efficiencies and have excellent practical properties. We suggest that the paper-specific interconnected microstructure with pulp fiber networks provides fast mixing of the reactants and efficient transport of the reactants to the catalytically-active sites. This concept is expected to be a promising route to green and sustainable chemistry.

  18. Chemically-Modified Cellulose Paper as a Microstructured Catalytic Reactor

    Directory of Open Access Journals (Sweden)

    Hirotaka Koga

    2015-01-01

    Full Text Available We discuss the successful use of chemically-modified cellulose paper as a microstructured catalytic reactor for the production of useful chemicals. The chemical modification of cellulose paper was achieved using a silane-coupling technique. Amine-modified paper was directly used as a base catalyst for the Knoevenagel condensation reaction. Methacrylate-modified paper was used for the immobilization of lipase and then in nonaqueous transesterification processes. These catalytic paper materials offer high reaction efficiencies and have excellent practical properties. We suggest that the paper-specific interconnected microstructure with pulp fiber networks provides fast mixing of the reactants and efficient transport of the reactants to the catalytically-active sites. This concept is expected to be a promising route to green and sustainable chemistry.

  19. Recognition of RNA by amide modified backbone nucleic acids: molecular dynamics simulations of DNA-RNA hybrids in aqueous solution.

    Science.gov (United States)

    Nina, Mafalda; Fonné-Pfister, Raymonde; Beaudegnies, Renaud; Chekatt, Habiba; Jung, Pierre M J; Murphy-Kessabi, Fiona; De Mesmaeker, Alain; Wendeborn, Sebastian

    2005-04-27

    Thermodynamic and structural properties of a chemically modified DNA-RNA hybrid in which a phosphodiester linkage is replaced by a neutral amide-3 linkage (3'-CH(2)-CONH-5') were investigated using UV melting experiments, molecular dynamics simulations in explicit water, and continuum solvent models. van't Hoff analysis of the experimental UV melting curves suggests that the significant increase of the thermodynamic stability of a 15-mer DNA-RNA with seven alternated amide-3 modifications (+11 degrees C) is mainly due to an increased binding enthalpy. To further evaluate the origin in the observed affinities differences, the electrostatic contribution to the binding free energy was calculated by solving the Poisson-Boltzmann equation numerically. The nonelectrostatic contribution was estimated as the product of a hydrophobic surface tension coefficient and the surface area that is buried upon double strand formation. Structures were taken from 10 ns molecular dynamics simulations computed in a consistent fashion using explicit solvent, counterions, and the particle-mesh Ewald procedure. The present preliminary thermodynamic study suggests that the favorable binding free energy of the amide-3 DNA single strand to the complementary RNA is equally driven by electrostatic and nonpolar contributions to the binding compared to their natural analogues. In addition, molecular dynamics simulations in explicit water were performed on an amide-3 DNA single strand and the corresponding natural DNA. Results from the conformations cluster analysis of the simulated amide-3 DNA single strand ensembles suggest that the 25% of the population sampled within 10 ns has a pre-organized conformation where the sugar C3' endo pucker is favored at the 3'-flanking nucleotides. These structural and thermodynamic features contribute to the understanding of the observed increased affinities of the amide-3 DNA-RNA hybrids at the microscopic level.

  20. Posttranscriptional RNA Modifications: playing metabolic games in a cell's chemical Legoland.

    Science.gov (United States)

    Helm, Mark; Alfonzo, Juan D

    2014-02-20

    Nature combines existing biochemical building blocks, at times with subtlety of purpose. RNA modifications are a prime example of this, where standard RNA nucleosides are decorated with chemical groups and building blocks that we recall from our basic biochemistry lectures. The result: a wealth of chemical diversity whose full biological relevance has remained elusive despite being public knowledge for some time. Here, we highlight several modifications that, because of their chemical intricacy, rely on seemingly unrelated pathways to provide cofactors for their synthesis. Besides their immediate role in affecting RNA function, modifications may act as sensors and transducers of information that connect a cell's metabolic state to its translational output, carefully orchestrating a delicate balance between metabolic rate and protein synthesis at a system's level. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

    Directory of Open Access Journals (Sweden)

    Bing Bai

    2013-01-01

    Full Text Available The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering.

  2. Calculation of Multiphase Chemical Equilibrium by the Modified RAND Method

    DEFF Research Database (Denmark)

    Tsanas, Christos; Stenby, Erling Halfdan; Yan, Wei

    2017-01-01

    method. The modified RAND extends the classical RAND method from single-phase chemical reaction equilibrium of ideal systems to multiphase chemical equilibrium of nonideal systems. All components in all phases are treated in the same manner and the system Gibbs energy can be used to monitor convergence....... This is the first time that modified RAND was applied to multiphase chemical equilibrium systems. The combined algorithm was tested using nine examples covering vapor–liquid (VLE) and vapor–liquid–liquid equilibria (VLLE) of ideal and nonideal reaction systems. Successive substitution provided good initial......A robust and efficient algorithm for simultaneous chemical and phase equilibrium calculations is proposed. It combines two individual nonstoichiometric solving procedures: a nested-loop method with successive substitution for the first steps and final convergence with the second-order modified RAND...

  3. Database proton NMR chemical shifts for RNA signal assignment and validation

    Energy Technology Data Exchange (ETDEWEB)

    Barton, Shawn; Heng Xiao [University of Maryland, Baltimore County, Howard Hughes Medical Institute (United States); Johnson, Bruce A., E-mail: bruce@onemoonscientific.com [University of Maryland, Baltimore County, Department of Chemistry and Biochemistry (United States); Summers, Michael F., E-mail: summers@hhmi.umbc.edu [University of Maryland, Baltimore County, Howard Hughes Medical Institute (United States)

    2013-01-15

    The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the {sup 1}H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson-Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 4{sup 3} possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA {sup 1}H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.

  4. The use of simultaneous chemical precipitation in modified activated ...

    African Journals Online (AJOL)

    The use of simultaneous chemical precipitation in modified activated sludge systems exhibiting biological excess phosphate removal: Part 6: Modelling of simultaneous chemical-biological P removal - review of existing models.

  5. Chemically modified graphene based supercapacitors for flexible and miniature devices

    Science.gov (United States)

    Ghosh, Debasis; Kim, Sang Ouk

    2015-09-01

    Rapid progress in the portable and flexible electronic devises has stimulated supercapacitor research towards the design and fabrication of high performance flexible devices. Recent research efforts for flexible supercapacitor electrode materials are highly focusing on graphene and chemically modified graphene owing to the unique properties, including large surface area, high electrical and thermal conductivity, excellent mechanical flexibility, and outstanding chemical stability. This invited review article highlights current status of the flexible electrode material research based on chemically modified graphene for supercapacitor application. A variety of electrode architectures prepared from chemically modified graphene are summarized in terms of their structural dimensions. Novel prototypes for the supercapacitor aiming at flexible miniature devices, i.e. microsupercapacitor with high energy and power density are highlighted. Future challenges relevant to graphene-based flexible supercapacitors are also suggested. [Figure not available: see fulltext.

  6. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

    Directory of Open Access Journals (Sweden)

    Eikan Mishima

    Full Text Available The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A, N6-methyladenosine (m6A, pseudouridine, and 5-methylcytidine (m5C showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

  7. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

    Science.gov (United States)

    Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

    2015-01-01

    The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

  8. Development of antibody-modified chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier as a strategy for inhibiting HIV replication in astrocytes.

    Science.gov (United States)

    Gu, Jijin; Al-Bayati, Karam; Ho, Emmanuel A

    2017-08-01

    RNA interference (RNAi)-mediated gene silencing offers a novel treatment and prevention strategy for human immunodeficiency virus (HIV) infection. HIV was found to infect and replicate in human brain cells and can cause neuroinfections and neurological deterioration. We designed dual-antibody-modified chitosan/small interfering RNA (siRNA) nanoparticles to deliver siRNA across the blood-brain barrier (BBB) targeting HIV-infected brain astrocytes as a strategy for inhibiting HIV replication. We hypothesized that transferrin antibody and bradykinin B2 antibody could specifically bind to the transferrin receptor (TfR) and bradykinin B2 receptor (B2R), respectively, and deliver siRNA across the BBB into astrocytes as potential targeting ligands. In this study, chitosan nanoparticles (CS-NPs) were prepared by a complex coacervation method in the presence of siRNA, and antibody was chemically conjugated to the nanoparticles. The antibody-modified chitosan nanoparticles (Ab-CS-NPs) were spherical in shape, with an average particle size of 235.7 ± 10.2 nm and a zeta potential of 22.88 ± 1.78 mV. The therapeutic potential of the nanoparticles was evaluated based on their cellular uptake and gene silencing efficiency. Cellular accumulation and gene silencing efficiency of Ab-CS-NPs in astrocytes were significantly improved compared to non-modified CS-NPs and single-antibody-modified CS-NPs. These results suggest that the combination of anti-Tf antibody and anti-B2 antibody significantly increased the knockdown effect of siRNA-loaded nanoparticles. Thus, antibody-mediated dual-targeting nanoparticles are an efficient and promising delivery strategy for inhibiting HIV replication in astrocytes. Graphical abstract Graphic representation of dual-antibody-conjugated chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier (BBB) for inhibiting HIV replication in astrocytes. a Nanoparticle delivery to the BBB and penetration. b Tf

  9. Testing insecticidal activity of novel chemically synthesized siRNA against Plutella xylostella under laboratory and field conditions.

    Directory of Open Access Journals (Sweden)

    Liang Gong

    Full Text Available BACKGROUND: Over the last 60 years, synthetic chemical pesticides have served as a main tactic in the field of crop protection, but their availability is now declining as a result of the development of insect resistance. Therefore, alternative pest management agents are needed. However, the demonstration of RNAi gene silencing in insects and its successful usage in disrupting the expression of vital genes opened a door to the development of a variety of novel, environmentally sound approaches for insect pest management. METHODOLOGY/PRINCIPAL FINDINGS: Six small interfering RNAs (siRNAs were chemically synthesized and modified according to the cDNA sequence of P. xylostella acetylcholine esterase genes AChE1 and AChE2. All of them were formulated and used in insecticide activity screening against P. xylostella. Bioassay data suggested that Si-ace1_003 and Si-ace2_001 at a concentration of 3 µg cm(-2 displayed the best insecticidal activity with 73.7% and 89.0%, mortality, respectively. Additional bioassays were used to obtain the acute lethal concentrations of LC50 and LC90 for Si-ace2_001, which were 53.66 µg/ml and 759.71 µg/ml, respectively. Quantitative Real-time PCR was used to confirm silencing and detected that the transcript levels of P. xylostella AChE2 (PxAChE2 were reduced by 5.7-fold compared to the control group. Consequently, AChE activity was also reduced by 1.7-fold. Finally, effects of the siRNAs on treated plants of Brassica oleracea and Brassica alboglabra were investigated with different siRNA doses. Our results showed that Si-ace2_001 had no negative effects on plant morphology, color and growth of vein under our experimental conditions. CONCLUSIONS: The most important finding of this study is the discovery that chemically synthesized and modified siRNA corresponding to P. xylostella AChE genes cause significant mortality of the insect both under laboratory and field conditions, which provides a novel strategy to control P

  10. A novel tyrosine-modified low molecular weight polyethylenimine (P10Y) for efficient siRNA delivery in vitro and in vivo.

    Science.gov (United States)

    Ewe, Alexander; Przybylski, Susanne; Burkhardt, Jana; Janke, Andreas; Appelhans, Dietmar; Aigner, Achim

    2016-05-28

    The delivery of nucleic acids, particularly of small RNA molecules like siRNAs for the induction of RNA interference (RNAi), still represents a major hurdle with regard to their application in vivo. Possible therapeutic applications thus rely on the development of efficient non-viral gene delivery vectors. While low molecular weight polyethylenimines (PEIs) have been successfully explored, the introduction of chemical modifications offers an avenue towards the development of more efficient vectors. In this paper, we describe the synthesis of a novel tyrosine-modified low-molecular weight polyethylenimine (P10Y) for efficient siRNA complexation and delivery. The comparison with the respective parent PEI reveals that knockdown efficacies are considerably enhanced by the tyrosine modification, as determined in different reporter cell lines, without appreciable cytotoxicity. We furthermore identify optimal conditions for complex preparation as well as for storing or lyophilization of the complexes without loss of biological activity. Beyond reporter cell lines, P10Y/siRNA complexes mediate the efficient knockdown of endogenous target genes and, upon knockdown of the anti-apoptotic oncogene survivin, tumor cell inhibitory effects in different carcinoma cell lines. Pushing the system further towards its therapeutic in vivo application, we demonstrate in mice the delivery of intact siRNAs and distinct biodistribution profiles upon systemic (intravenous or intraperitoneal) injection. No adverse effects (hepatotoxicity, immunostimulation/alterations in immunophenotype, weight loss) are observed. More importantly, profound tumor-inhibitory effects in a melanoma xenograft mouse model are observed upon systemic application of P10Y/siRNA complexes for survivin knockdown, indicating the therapeutic efficacy of P10Y/siRNA complexes. Taken together, we (i) establish tyrosine-modified PEI (P10Y) as efficient platform for siRNA delivery in vitro and in vivo, (ii) identify optimal

  11. Dynamics of prebiotic RNA reproduction illuminated by chemical game theory.

    Science.gov (United States)

    Yeates, Jessica A M; Hilbe, Christian; Zwick, Martin; Nowak, Martin A; Lehman, Niles

    2016-05-03

    Many origins-of-life scenarios depict a situation in which there are common and potentially scarce resources needed by molecules that compete for survival and reproduction. The dynamics of RNA assembly in a complex mixture of sequences is a frequency-dependent process and mimics such scenarios. By synthesizing Azoarcus ribozyme genotypes that differ in their single-nucleotide interactions with other genotypes, we can create molecules that interact among each other to reproduce. Pairwise interplays between RNAs involve both cooperation and selfishness, quantifiable in a 2 × 2 payoff matrix. We show that a simple model of differential equations based on chemical kinetics accurately predicts the outcomes of these molecular competitions using simple rate inputs into these matrices. In some cases, we find that mixtures of different RNAs reproduce much better than each RNA type alone, reflecting a molecular form of reciprocal cooperation. We also demonstrate that three RNA genotypes can stably coexist in a rock-paper-scissors analog. Our experiments suggest a new type of evolutionary game dynamics, called prelife game dynamics or chemical game dynamics. These operate without template-directed replication, illustrating how small networks of RNAs could have developed and evolved in an RNA world.

  12. Dynamics of prebiotic RNA reproduction illuminated by chemical game theory

    Science.gov (United States)

    Yeates, Jessica A. M.; Hilbe, Christian; Zwick, Martin; Nowak, Martin A.; Lehman, Niles

    2016-01-01

    Many origins-of-life scenarios depict a situation in which there are common and potentially scarce resources needed by molecules that compete for survival and reproduction. The dynamics of RNA assembly in a complex mixture of sequences is a frequency-dependent process and mimics such scenarios. By synthesizing Azoarcus ribozyme genotypes that differ in their single-nucleotide interactions with other genotypes, we can create molecules that interact among each other to reproduce. Pairwise interplays between RNAs involve both cooperation and selfishness, quantifiable in a 2 × 2 payoff matrix. We show that a simple model of differential equations based on chemical kinetics accurately predicts the outcomes of these molecular competitions using simple rate inputs into these matrices. In some cases, we find that mixtures of different RNAs reproduce much better than each RNA type alone, reflecting a molecular form of reciprocal cooperation. We also demonstrate that three RNA genotypes can stably coexist in a rock–paper–scissors analog. Our experiments suggest a new type of evolutionary game dynamics, called prelife game dynamics or chemical game dynamics. These operate without template-directed replication, illustrating how small networks of RNAs could have developed and evolved in an RNA world. PMID:27091972

  13. ZnS nanoflakes deposition by modified chemical method

    International Nuclear Information System (INIS)

    Desai, Mangesh A.; Sartale, S. D.

    2014-01-01

    We report deposition of zinc sulfide nanoflakes on glass substrates by modified chemical method. The modified chemical method involves adsorption of zinc–thiourea complex on the substrate and its dissociation in presence of hydroxide ions to release sulfur ions from thiourea which react with zinc ions present in the complex to form zinc sulfide nanoflakes at room temperature. Influence of zinc salt and thiourea concentrations ratios on the morphology of the films was investigated by scanning electron microscope (SEM). The ratio of zinc and thiourea in the zinc–thiourea complex significantly affect the size of the zinc sulfide nanoflakes, especially width and density of the nanoflakes. The X-ray diffraction analysis exhibits polycrystalline nature of the zinc sulfide nanoflakes with hexagonal phase

  14. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

    Science.gov (United States)

    Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

    2015-03-11

    A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Highly stable aptamers selected from a 2'-fully modified fGmH RNA library for targeting biomaterials.

    Science.gov (United States)

    Friedman, Adam D; Kim, Dongwook; Liu, Rihe

    2015-01-01

    When developed as targeting ligands for the in vivo delivery of biomaterials to biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2' modification. This study aims to develop a novel class of highly stable, 2'-fully modified RNA aptamers that are ideal for the targeted delivery of biomaterials. We demonstrated the facile transcription of a fGmH (2'-F-dG, 2'-OMe-dA/dC/dU) RNA library with unexpected hydrophobicity, the direct selection of aptamers from a fGmH RNA library that bind Staphylococcus aureus Protein A (SpA) as a model target, and the superior nuclease and serum stability of these aptamers compared to 2'-partially modified RNA variants. Characterizations of fGmH RNA aptamers binding to purified SpA and to endogenous SpA present on the surface of S. aureus cells demonstrate fGmH RNA aptamer selectivity and stability. Significantly, fGmH RNA aptamers were able to functionalize, stabilize, and specifically deliver aggregation-prone silver nanoparticles (AgNPs) to S. aureus with SpA-dependent antimicrobial effects. This study describes a novel aptamer class with considerable potential to improve the in vivo applicability of nucleic acid-based affinity molecules to biomaterials.

  16. Encoded libraries of chemically modified peptides.

    Science.gov (United States)

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries. Copyright © 2015. Published by Elsevier Ltd.

  17. In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts

    NARCIS (Netherlands)

    Mook, O. R. F.; Vreijling, Jeroen; Wengel, Suzy L.; Wengel, Jesper; Zhou, Chuanzheng; Chattopadhyaya, Jyoti; Baas, Frank; Fluiter, Kees

    2010-01-01

    The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and

  18. Biological resistance of polyethylene composites made with chemically modified fiber or flour

    Science.gov (United States)

    Rebecca E. Ibach; Craig M. Clemons

    2002-01-01

    The role of moisture in the biological decay of wood-plastic composites was investigated. Southern pine wood fiber and ponderosa pine wood flour were chemically modified using either acetic anhydride (AA), butylene oxide (BO), or propylene oxide (PO). A 50:50 mixture of high density polyethylene and either chemically modified fiber or flour, or untreated fiber or flour...

  19. Chemical sensors based on molecularly modified metallic nanoparticles

    International Nuclear Information System (INIS)

    Haick, Hossam

    2007-01-01

    This paper presents a concise, although admittedly non-exhaustive, didactic review of some of the main concepts and approaches related to the use of molecularly modified metal nanoparticles in or as chemical sensors. This paper attempts to pull together different views and terminologies used in sensors based on molecularly modified metal nanoparticles, including those established upon electrochemical, optical, surface Plasmon resonance, piezoelectric and electrical transduction approaches. Finally, this paper discusses briefly the main advantages and disadvantages of each of the presented class of sensors. (review article)

  20. MicroRNA silencing in primates: towards development of novel therapeutics

    DEFF Research Database (Denmark)

    Petri, Andreas; Lindow, Morten; Kauppinen, Sakari

    2009-01-01

    MicroRNAs (miRNA) comprise an abundant class of small noncoding RNAs that act as important posttranscriptional regulators of gene expression. Accumulating evidence showing that aberrantly expressed miRNAs play important roles in human cancers underscores them as potential targets for therapeutic ...... intervention. Recent reports on efficient miRNA silencing in rodents and nonhuman primates using high-affinity targeting by chemically modified antisense oligonucleotides highlight the utility of such compounds in the development of miRNA-based cancer therapeutics....

  1. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    Science.gov (United States)

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  2. Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy

    Directory of Open Access Journals (Sweden)

    Hong-Hao Yu

    2016-10-01

    Full Text Available Dystrophinopathy, including Duchenne muscle dystrophy (DMD and Becker muscle dystrophy (BMD is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

  3. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Julia F Alterman

    2015-01-01

    Full Text Available Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

  4. tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Mayumi Okamoto

    2014-09-01

    Full Text Available Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD. The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

  5. Highly Stable Aptamers Selected from a 2′-Fully Modified fGmH RNA Library for Targeting Biomaterials

    Science.gov (United States)

    Friedman, Adam D.; Kim, Dongwook; Liu, Rihe

    2014-01-01

    When developed as targeting ligands for the in vivo delivery of biomaterials to biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2′ modification. This study aims to develop a novel class of highly stable, 2′-fully modified RNA aptamers that are ideal for the targeted delivery of biomaterials. We demonstrated the facile transcription of a fGmH (2′-F-dG, 2′-OMe-dA/dC/dU) RNA library with unexpected hydrophobicity, the direct selection of aptamers from a fGmH RNA library that bind Staphylococcus aureus Protein A (SpA) as a model target, and the superior nuclease and serum stability of these aptamers compared to 2′-partially modified RNA variants. Characterizations of fGmH RNA aptamers binding to purified SpA and to endogenous SpA present on the surface of S. aureus cells demonstrate fGmH RNA aptamer selectivity and stability. Significantly, fGmH RNA aptamers were able to functionalize, stabilize, and further deliver aggregation-prone silver nanoparticles (AgNPs) to S. aureus with SpA-dependent antimicrobial effects. This study describes a novel aptamer class with considerable potential to improve the in vivo applicability of nucleic acid-based affinity molecules to biomaterials. PMID:25443790

  6. 3-(3-amino-3-carboxypropyl)-5,6-Dihydrouridine is one of two novel post-transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Krog, Jesper Schak; Español, Yaiza; Giessing, Anders M B

    2011-01-01

    tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar. Furtherm......tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar...... of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher-order tandem MS. Phylogenetic comparison with modifications in tRNA(Lys) from other organisms was used through......: a minor fraction with the previously described 2-methylthio-N(6) -threonylcarbamoyl-modification, and a major fraction with A37 being modified by a 294.0-Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin....

  7. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang

    2016-02-04

    Chromatin is the universal template of genetic information in all eukaryotic organisms. Chemical modifications of the DNA-packaging histone proteins and the DNA bases are crucial signaling events in directing the use and readout of eukaryotic genomes. The enzymes that install and remove these chromatin modifications as well as the proteins that bind these marks govern information that goes beyond the sequence of DNA. Therefore, these so-called epigenetic regulators are intensively studied and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity, and target sites of chromatin modifying enzymes and reader proteins.

  8. Chemically modified carbon fibers and their applications

    International Nuclear Information System (INIS)

    Ermolenko, I.N.; Lyubliner, I.P.; Gulko, N.V.

    1990-01-01

    This book gives a comprehensive review about chemically modified carbon fibers (e.g. by incorporation of other elements) and is structured as follows: 1. Types of carbon fibers, 2. Structure of carbon fibers, 3. Properties of carbon fibers, 4. The cellulose carbonization process, 5. Formation of element-carbon fiber materials, 6. Surface modification of carbon fibers, and 7. Applications of carbon fibers (e.g. adsorbents, catalysts, constituents of composites). (MM)

  9. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

    Science.gov (United States)

    Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

    2015-01-01

    Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

  10. Modified method for combined DNA and RNA isolation from peanut and other oil seeds.

    Science.gov (United States)

    Dang, Phat M; Chen, Charles Y

    2013-02-01

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.

  11. Supersensitive fingerprinting of explosives by chemically modified nanosensors arrays

    Science.gov (United States)

    Lichtenstein, Amir; Havivi, Ehud; Shacham, Ronen; Hahamy, Ehud; Leibovich, Ronit; Pevzner, Alexander; Krivitsky, Vadim; Davivi, Guy; Presman, Igor; Elnathan, Roey; Engel, Yoni; Flaxer, Eli; Patolsky, Fernando

    2014-06-01

    The capability to detect traces of explosives sensitively, selectively and rapidly could be of great benefit for applications relating to civilian national security and military needs. Here, we show that, when chemically modified in a multiplexed mode, nanoelectrical devices arrays enable the supersensitive discriminative detection of explosive species. The fingerprinting of explosives is achieved by pattern recognizing the inherent kinetics, and thermodynamics, of interaction between the chemically modified nanosensors array and the molecular analytes under test. This platform allows for the rapid detection of explosives, from air collected samples, down to the parts-per-quadrillion concentration range, and represents the first nanotechnology-inspired demonstration on the selective supersensitive detection of explosives, including the nitro- and peroxide-derivatives, on a single electronic platform. Furthermore, the ultrahigh sensitivity displayed by our platform may allow the remote detection of various explosives, a task unachieved by existing detection technologies.

  12. 2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity.

    Science.gov (United States)

    Wu, Sherry Y; Yang, Xianbin; Gharpure, Kshipra M; Hatakeyama, Hiroto; Egli, Martin; McGuire, Michael H; Nagaraja, Archana S; Miyake, Takahito M; Rupaimoole, Rajesha; Pecot, Chad V; Taylor, Morgan; Pradeep, Sunila; Sierant, Malgorzata; Rodriguez-Aguayo, Cristian; Choi, Hyun J; Previs, Rebecca A; Armaiz-Pena, Guillermo N; Huang, Li; Martinez, Carlos; Hassell, Tom; Ivan, Cristina; Sehgal, Vasudha; Singhania, Richa; Han, Hee-Dong; Su, Chang; Kim, Ji Hoon; Dalton, Heather J; Kovvali, Chandra; Keyomarsi, Khandan; McMillan, Nigel A J; Overwijk, Willem W; Liu, Jinsong; Lee, Ju-Seog; Baggerly, Keith A; Lopez-Berestein, Gabriel; Ram, Prahlad T; Nawrot, Barbara; Sood, Anil K

    2014-03-12

    Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2'-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.

  13. DNA methyltransferase 1-targeting miRNA-148aof dairymilk: apotential bioactive modifier of thehumanepigenome

    Directory of Open Access Journals (Sweden)

    Bodo C. Melnik

    2017-09-01

    Full Text Available Background: The perception of milk has changed from a “simple food” to a more sophisticated bioactive functional signaling system that promotes mTORC1-driven postnatal anabolism, growth, and development of the newborn infant. Accumulating evidence supports the view that milk´s miRNAs significantly contribute to these processes. The most abundant miRNA of milk found in milk fat and milk exosomes is miRNA-148a, which targets DNA methyltransferase 1 (DNMT1, a pivotal epigenetic regulator that suppresses transcription. Furthermore, milk-derived miRNA-125b, miRNA-30d, and miRNA-25 target TP53, the guardian of the genome that interacts with DNMT1 and regulates metabolism, cell kinetics, and apoptosis. Thus, the question arose whether cow´s milk-derived miRNAs may modify epigenetic regulation of the human milk consumer. Methods: To understand the potential impact of dairy milk consumption on human epigenetics, we have analyzed all relevant research-based bioinformatics data related to milk, milk miRNAs, epigenetic regulation, and lactation performance with special attention to bovine miRNAs that modify gene expression of DNA methyltransferase 1 (DNMT1 and p53 (TP53, the two guardians of the mammalian genome. By means of translational research and comparative functional genomics, we investigated the potential impact of cow´s milk miRNAs on epigenetic regulation of human DNMT1, TP53, FOXP3, and FTO, which are critically involved in immunologic and metabolic programming respectively. miRNA sequences have been obtained from mirbase.org. miRNA-target site prediction has been performed using TargetScan release 7.0. Results: The most abundant miRNA of cow´s milk is miRNA-148a, which represents more than 10% of all miRNAs of cow´s milk, survives pasteurization and refrigerated storage. The seed sequence of human and bovine miRNA-148a-3p is identical. Furthermore, human and bovine DNMT1 mRNA share 88% identity. The miRNA-148a 7mer seed is conserved in

  14. Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA.

    Science.gov (United States)

    Jensen, Ditte Krohn; Jensen, Linda Boye; Koocheki, Saeid; Bengtson, Lasse; Cun, Dongmei; Nielsen, Hanne Mørck; Foged, Camilla

    2012-01-10

    Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local

  15. Simple methods for the 3' biotinylation of RNA.

    Science.gov (United States)

    Moritz, Bodo; Wahle, Elmar

    2014-03-01

    Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3' ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3'-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3' ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of 29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme's proofreading activity, can cope with more extended 3' heterogeneities.

  16. 2’f-OMe-phosphorodithioate modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity

    Science.gov (United States)

    Wu, Sherry Y.; Yang, Xianbin; Gharpure, Kshipra M.; Hatakeyama, Hiroto; Egli, Martin; McGuire, Michael H.; Nagaraja, Archana S.; Miyake, Takahito M.; Rupaimoole, Rajesha; Pecot, Chad V.; Taylor, Morgan; Pradeep, Sunila; Sierant, Malgorzata; Rodriguez-Aguayo, Cristian; Choi, Hyun J.; Previs, Rebecca A.; Armaiz-Pena, Guillermo N.; Huang, Li; Martinez, Carlos; Hassell, Tom; Ivan, Cristina; Sehgal, Vasudha; Singhania, Richa; Han, Hee-Dong; Su, Chang; Kim, Ji Hoon; Dalton, Heather J.; Kowali, Chandra; Keyomarsi, Khandan; McMillan, Nigel A.J.; Overwijk, Willem W.; Liu, Jinsong; Lee, Ju-Seog; Baggerly, Keith A.; Lopez-Berestein, Gabriel; Ram, Prahlad T.; Nawrot, Barbara; Sood, Anil K.

    2014-01-01

    Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2’-O-Methyl (2’-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2’-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM Domain Containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumors following MePS2-modified siRNA treatment, leading to a synergistic anti-tumor effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types. PMID:24619206

  17. Modified NASA-Lewis chemical equilibrium code for MHD applications

    Science.gov (United States)

    Sacks, R. A.; Geyer, H. K.; Grammel, S. J.; Doss, E. D.

    1979-01-01

    A substantially modified version of the NASA-Lewis Chemical Equilibrium Code was recently developed. The modifications were designed to extend the power and convenience of the Code as a tool for performing combustor analysis for MHD systems studies. The effect of the programming details is described from a user point of view.

  18. Reactive chemically modified piezoelectric crystal detectors: A new class of high-selectivity sensors

    International Nuclear Information System (INIS)

    Fadeev, A.Yu.; Filatov, A.L.; Lisichkin, G.V.

    1994-01-01

    A great number of works have focused on the study of properties of modified piezoelectric quartz crystal detectors (PQCDs) coated with sorbing substrates and on applying sensors based on them for the analysis of diluted gas mixtures and solutions. This work offers a new class of gravemetric sensors characterized by a reversible chemical reaction that occurs on their surface. Silica films are proposed as a sorbing coating of quartz detectors, and a chemical modification of a surface is suggested for covalent fixation of the necessary compounds. PQCDs were chemically modified with reactive diene derivatives that can also act as dienophiles. Hexachlorocyclopentadiene (HCCPD, resonater I) and cyclopentadiene (CPD, resonator II) were fixed on a PQCD surface in several stages. After treatment with the resonaters, the PQCD in a CPD gas phase exhibited time dependent frequency shifts from 20-100 Hz. The results suggest that there is a reversible chemical reaction on the electrode surface of resonators I and II when they interact with CPD vapors. Therefore, PQCDs modified with reactive dienes were prepared for the first time and may be employed as selective sensors for CPD

  19. RNA Nanoparticles Derived from Three-Way Junction of Phi29 Motor pRNA Are Resistant to I-125 and Cs-131 Radiation

    Science.gov (United States)

    Li, Hui; Rychahou, Piotr G.; Cui, Zheng; Pi, Fengmei; Evers, B. Mark; Shu, Dan

    2015-01-01

    Radiation reagents that specifically target tumors are in high demand for the treatment of cancer. The emerging field of RNA nanotechnology might provide new opportunities for targeted radiation therapy. This study investigates whether chemically modified RNA nanoparticles derived from the packaging RNA (pRNA) three-way junction (3WJ) of phi29 DNA-packaging motor are resistant to potent I-125 and Cs-131 radiation, which is a prerequisite for utilizing these RNA nanoparticles as carriers for targeted radiation therapy. pRNA 3WJ nanoparticles were constructed and characterized, and the stability of these nanoparticles under I-125 and Cs-131 irradiation with clinically relevant doses was examined. RNA nanoparticles derived from the pRNA 3WJ targeted tumors specifically and they were stable under irradiation of I-125 and Cs-131 with clinically relevant doses ranging from 1 to 90 Gy over a significantly long time up to 20 days, while control plasmid DNA was damaged at 20 Gy or higher. PMID:26017686

  20. Carbon nanotube-polyamidoamine dendrimer hybrid-modified electrodes for highly sensitive electrochemical detection of microRNA24.

    Science.gov (United States)

    Li, Fengye; Peng, Jing; Zheng, Qiong; Guo, Xiang; Tang, Hao; Yao, Shouzhuo

    2015-01-01

    A simple and ultrasensitive microRNA (miRNA) electrochemical biosensor employing multiwalled carbon nanotube (MWCNT)-polyamidoamine (PAMAM) dendrimer and methylene blue (MB) redox indicator is reported in this work. The assay utilizes a glass carbon (GC) electrode modified with MWCNT-PAMAM, on which the oligonucleotide capture probes are immobilized. The electrochemical detection of miRNAs is completed by measuring the reduction signal change of MB before and after the probe hybridization with target miRNA (miRNA24 is used as a model case). The MWCNT-PAMAM/GC electrode shows greatly enhanced signal to MB reduction in contrast to bare GC electrode. The functionalization of MWCNT with PAMAM maintains the electrochemical property of MWCNT to MB reduction but minimizes the undesired adsorption of MB on the MWCNT surface. The effect of experimental variables on the miRNA detection is investigated and optimized. A detection limit of 0.5 fM and a linear peak current density-concentration relationship up to 100 nM are obtained following 60 min hybridization. The proposed assay is successfully used to detect miRNA24 in total RNA sample extracted from HeLa cells.

  1. X-Ray Photoelectron Spectroscopic Characterization of Chemically Modified Electrodes Used as Chemical Sensors and Biosensors: A Review

    Directory of Open Access Journals (Sweden)

    Elio Desimoni

    2015-04-01

    Full Text Available The characterization of chemically modified sensors and biosensors is commonly performed by cyclic voltammetry and electron microscopies, which allow verifying electrode mechanisms and surface morphologies. Among other techniques, X-ray photoelectron spectroscopy (XPS plays a unique role in giving access to qualitative, quantitative/semi-quantitative and speciation information concerning the sensor surface. Nevertheless, XPS remains rather underused in this field. The aim of this paper is to review selected articles which evidence the useful performances of XPS in characterizing the top surface layers of chemically modified sensors and biosensors. A concise introduction to X-ray Photoelectron Spectroscopy gives to the reader the essential background. The application of XPS for characterizing sensors suitable for food and environmental analysis is highlighted.

  2. Surface-modified polymeric pads for enhanced performance during chemical mechanical planarization

    International Nuclear Information System (INIS)

    Deshpande, S.; Dakshinamurthy, S.; Kuiry, S.C.; Vaidyanathan, R.; Obeng, Y.S.; Seal, S.

    2005-01-01

    The chemical mechanical planarization (CMP) process occurs at an atomic level at the slurry/wafer interface and hence slurries and polishing pads play a critical role in their successful implementation. Polyurethane is a commonly used polymer in the manufacturing of CMP pads. These pads are incompatible with some chemicals present in the CMP slurries, such as hydrogen peroxide. To overcome these problems, Psiloquest has developed new Application Specific Pads (ASP). Surface of such pads has been modified by depositing a thin film of tetraethyl orthosilicate using plasma-enhanced chemical vapor deposition (PECVD) process. In the present study, mechanical properties of such coated pads have been investigated using nanoindentation. The surface morphology and the chemistry of the ASP were studied using scanning electron microcopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy techniques. It was observed that mechanical and chemical properties of the pad top surface are a function of the PECVD coating time. Such PECVD-treated pads are found to be hydrophilic and do not require storage in aqueous media during the not-in-use period. The metal removal rate using such surface-modified polishing pads was found to increase linearly with the PECVD coating time

  3. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

    OpenAIRE

    Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

    2015-01-01

    Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we sh...

  4. Osteogenesis ability of biomimetic modified 3Y-TZP ceramic using chemical treatment

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Shih-Kuang [Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taichung 40601, Taiwan, ROC (China); Chang, Pai-Ling [Taoyuan General Hospital, Taoyuan 33004, Taiwan, ROC (China); Ho, Wen-Fu [Department of Chemical and Materials Engineering, National University of Kaohsiung, Kaohsiung 81148, Taiwan, ROC (China); Hsu, Hsueh-Chuan; Liao, Huei-Jyuan [Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taichung 40601, Taiwan, ROC (China); Wu, Shih-Ching, E-mail: scwu@ctust.edu.tw [Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taichung 40601, Taiwan, ROC (China)

    2015-12-01

    In this study, RGD peptide derived from extracellular matrix proteins was employed to modify the surface of yttria-tetragonal zirconia polycrystal (3Y-TZP) to promote cell adhesion. The surface of 3Y-TZP ceramic specimens was first modified using chemical treatment with aqueous solutions of H{sub 3}PO{sub 4}, CH{sub 3}COOH, and NaOH, for the formation of Zr–OH surface functional groups. Then, the RGD peptide was immobilized on the surface of the 3Y-TZP through silanization method, with covalent bonding via the Zr–OH surface functional groups. From this study, the RGD peptide can successfully be grafted onto the chemical modified 3Y-TZP surface. The –OH functional groups formed on the surface of 3Y-TZP after acid/alkaline chemical treatment contribute to the grafting reaction of RGD peptides. The use of phosphoric acid solution in 3Y-TZP surface treatment before RGD peptide grafting for biomimetic modification can significantly enhance cell adhesion, proliferation, and differentiation. - Highlights: • This study successfully immobilized the peptides onto the surface of zirconia. • Acid/alkaline chemical treatment promotes the formation of − OH functional groups. • The use of phosphoric acid solution produced the formation of most − OH. • Peptides can significantly enhance cell adhesion, proliferation, and differentiation.

  5. Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA

    OpenAIRE

    Warren, Luigi; Manos, Philip D.; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj; Smith, Zachary D.; Meissner, Alexander; Daley, George Q.; Brack, Andrew S.; Collins, James J.; Cowan, Chad; Schlaeger, Thorsten M.

    2010-01-01

    Clinical application of induced pluripotent stem (iPS) cells is limited by the low efficiency of iPS derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-integrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral re...

  6. Small Molecule Modifiers of the microRNA and RNA Interference Pathway

    OpenAIRE

    Deiters, Alexander

    2009-01-01

    Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, whic...

  7. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

    Science.gov (United States)

    Warren, Luigi; Manos, Philip D; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj K; Smith, Zachary D; Meissner, Alexander; Daley, George Q; Brack, Andrew S; Collins, James J; Cowan, Chad; Schlaeger, Thorsten M; Rossi, Derrick J

    2010-11-05

    Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. A procedure to validate and correct the {sup 13}C chemical shift calibration of RNA datasets

    Energy Technology Data Exchange (ETDEWEB)

    Aeschbacher, Thomas; Schubert, Mario, E-mail: schubert@mol.biol.ethz.ch; Allain, Frederic H.-T., E-mail: allain@mol.biol.ethz.ch [ETH Zuerich, Institute for Molecular Biology and Biophysics (Switzerland)

    2012-02-15

    Chemical shifts reflect the structural environment of a certain nucleus and can be used to extract structural and dynamic information. Proper calibration is indispensable to extract such information from chemical shifts. Whereas a variety of procedures exist to verify the chemical shift calibration for proteins, no such procedure is available for RNAs to date. We present here a procedure to analyze and correct the calibration of {sup 13}C NMR data of RNAs. Our procedure uses five {sup 13}C chemical shifts as a reference, each of them found in a narrow shift range in most datasets deposited in the Biological Magnetic Resonance Bank. In 49 datasets we could evaluate the {sup 13}C calibration and detect errors or inconsistencies in RNA {sup 13}C chemical shifts based on these chemical shift reference values. More than half of the datasets (27 out of those 49) were found to be improperly referenced or contained inconsistencies. This large inconsistency rate possibly explains that no clear structure-{sup 13}C chemical shift relationship has emerged for RNA so far. We were able to recalibrate or correct 17 datasets resulting in 39 usable {sup 13}C datasets. 6 new datasets from our lab were used to verify our method increasing the database to 45 usable datasets. We can now search for structure-chemical shift relationships with this improved list of {sup 13}C chemical shift data. This is demonstrated by a clear relationship between ribose {sup 13}C shifts and the sugar pucker, which can be used to predict a C2 Prime - or C3 Prime -endo conformation of the ribose with high accuracy. The improved quality of the chemical shift data allows statistical analysis with the potential to facilitate assignment procedures, and the extraction of restraints for structure calculations of RNA.

  9. Construction of an Aptamer-SiRNA Chimera-Modified Tissue-Engineered Blood Vessel for Cell-Type-Specific Capture and Delivery.

    Science.gov (United States)

    Chen, Wen; Zeng, Wen; Sun, Jiansen; Yang, Mingcan; Li, Li; Zhou, Jingting; Wu, Yangxiao; Sun, Jun; Liu, Ge; Tang, Rui; Tan, Ju; Zhu, Chuhong

    2015-06-23

    The application of tissue-engineered blood vessels (TEBVs) is the main developmental direction of vascular replacement therapy. Due to few and/or dysfunctional endothelial progenitor cells (EPCs), it is difficult to successfully construct EPC capture TEBVs in diabetes. RNA has a potential application in cell protection and diabetes treatment, but poor specificity and low efficiency of RNA transfection in vivo limit the application of RNA. On the basis of an acellular vascular matrix, we propose an aptamer-siRNA chimera-modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consists of two parts, CD133-adenosine kinase (ADK) chimeras and a TEBV scaffold. Our results showed that CD133-ADK chimeras could selectively capture the CD133-positive cells in vivo, and then captured cells can internalize the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK siRNA by a dicer, resulting in depletion of ADK. An ADK-deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce nonspecific RNA transfection, we increased the proportion of HAuCl4 during the material preparation, through which the transfection capacity of polyethylenimine (PEI)/polyethylene glycol (PEG)-capped gold nanoparticles (PEI/PEG-AuNPs) was significantly decreased and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2'-O-methyl modification was used to enhance the in vivo stability of RNA chimeras. At day 30 postgrafting, the patency rate of CD133-ADK chimera-modified TEBVs reached 90% in diabetic rats and good endothelialization was observed.

  10. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

  11. Characterisation of chemically-modified proteins by electrospray ionisation mass spectrometry

    International Nuclear Information System (INIS)

    Bennett, K.L.

    1996-09-01

    Electrospray mass spectrometry (ESI-MS) has been used to examine a range of intact monoclonal antibodies (MAbs), antibody fragments such as F(ab') 2 , F ab and F c , chemically-modified fragments and a range of other chemically-modified peptides and proteins as part of a broader study aimed at establishing ESI-MS as a method for the characterisation of radioimmunoconjugates (radiolabelled monoclonal antibodies). For example, the addition of up to 10 biotin molecules to the 'papain-sensitive' 50 kDa F ab fragment can be easily detected in ESI mass spectra. For intact MAbs, however, it is only possible to detect average shifts in the mass of intact antibodies following modification. Successful ESI-MS analysis of complexes formed between chelators and other small molecules conjugated to synthetic peptides, hen egg-white Iysozyme (HEL) (M r 14 306) and horse heart myoglobin (M r 16 951) has been demonstrated. ESI-MS offers considerable advantages compared with existing methods for the characterisation of chemically-conjugated proteins including speed and sensitivity of analysis and the capability for obtaining specific structural information. The conditions for ESI-MS of intact MAbs and MAb fragments have been examined in detail and it was found that 150 kDa MAbs generally required lower sample concentration and higher skimmer potentials compared with the 50 kDa F ab fragment and other lower molecular weight proteins. In addition, the m/z range over which ions from MAbs were observed was higher (m/z ∼2000-4500) than for smaller proteins. ESI-MS was also found to be useful for probing the action of the protease papain, that is used to generate MAb fragments (F(ab) '2, F ab and F c ). Further, different sensitivities to papain for different MAb preparations was demonstrated. Finally, the tandem mass spectra of a range of peptides modified by iodine and biotin were examined. In the case of biotinylated peptides, a characteristic fragment ion was identified that could

  12. Integrating chemical footprinting data into RNA secondary structure prediction.

    Directory of Open Access Journals (Sweden)

    Kourosh Zarringhalam

    Full Text Available Chemical and enzymatic footprinting experiments, such as shape (selective 2'-hydroxyl acylation analyzed by primer extension, yield important information about RNA secondary structure. Indeed, since the [Formula: see text]-hydroxyl is reactive at flexible (loop regions, but unreactive at base-paired regions, shape yields quantitative data about which RNA nucleotides are base-paired. Recently, low error rates in secondary structure prediction have been reported for three RNAs of moderate size, by including base stacking pseudo-energy terms derived from shape data into the computation of minimum free energy secondary structure. Here, we describe a novel method, RNAsc (RNA soft constraints, which includes pseudo-energy terms for each nucleotide position, rather than only for base stacking positions. We prove that RNAsc is self-consistent, in the sense that the nucleotide-specific probabilities of being unpaired in the low energy Boltzmann ensemble always become more closely correlated with the input shape data after application of RNAsc. From this mathematical perspective, the secondary structure predicted by RNAsc should be 'correct', in as much as the shape data is 'correct'. We benchmark RNAsc against the previously mentioned method for eight RNAs, for which both shape data and native structures are known, to find the same accuracy in 7 out of 8 cases, and an improvement of 25% in one case. Furthermore, we present what appears to be the first direct comparison of shape data and in-line probing data, by comparing yeast asp-tRNA shape data from the literature with data from in-line probing experiments we have recently performed. With respect to several criteria, we find that shape data appear to be more robust than in-line probing data, at least in the case of asp-tRNA.

  13. Mechano-sorptive creep of Portuguese pinewood chemically modified

    Directory of Open Access Journals (Sweden)

    Barroso Lopes Duarte

    2014-03-01

    Full Text Available The effect of chemical modification on mechano-sorptive creep in bending was studied by experimental work. Stakes with 20 × 20 × 400 mm RTL of Portuguese wood species (Pinus pinaster Aiton modified with 1,3-dimethylol-4,5- dihydroxyethyleneurea (DMDHEU, m-methylated melamine resin (MMF, tetraethoxysilane (TEOS and amid wax (WA were measured under asymmetric moistening conditions over a period of 42 days (app. 1000 hours with stress level (SL of 12 MPa, according to ENV 1156.

  14. Sedimentation behaviour and colloidal properties of porous, chemically modified silicas in non-aqueous solvents

    NARCIS (Netherlands)

    Vissers, J.P.C.; Laven, J.; Claessens, H.A.; Cramers, C.A.M.G.; Agterof, W.G.M.

    1997-01-01

    The sedimentation behaviour and colloidal properties of porous, chemically modified silicas dispersed in non-aqueous solvents have been studied. The free settling behaviour of non-aggregated silica suspensions could effectively be described with a modified Stokes equation that takes into account the

  15. Chemically Modified Polyvinyl Chloride for Removal of Thionine Dye (Lauth’s Violet

    Directory of Open Access Journals (Sweden)

    Helena Ma A. M. M. S. Ali

    2017-11-01

    Full Text Available The chemical modification of hydrophobic polymer matrices is an alternative way to elchange their surface properties. The introduction of sulfonic groups in the polymer changes the surface properties such as adhesion, wettability, catalytic ability, and adsorption capacity. This work describes the production and application of chemically modified polyvinyl chloride (PVC as adsorbent for dyes removal. Chemical modification of PVC was evaluated by infrared spectroscopy and elemental analysis, which indicated the presence of sulfonic groups on PVC. The chemically modified PVC (PVCDS showed an ion exchange capacity of 1.03 mmol−1, and efficiently removed the thionine dye (Lauth’s violet from aqueous solutions, reaching equilibrium in 30 min. The adsorption kinetics was better adjusted for a pseudo second order model. This result indicates that the adsorption of thionine onto PVCDS occurs by chemisorption. Among the models for the state of equilibrium, SIPS and Langmuir exhibited the best fit to the experimental results and PVCDS showed high adsorption capacities (370 mg−1. Thus, it is assumed that the system presents homogeneous characteristics to the distribution of active sites. The modification promoted the formation of surface characteristics favorable to the dye adsorption by the polymer.

  16. Dehydration and hydration behavior of metal-salt-modified materials for chemical heat pumps

    International Nuclear Information System (INIS)

    Ishitobi, Hirokazu; Uruma, Keirei; Takeuchi, Masato; Ryu, Junichi; Kato, Yukitaka

    2013-01-01

    Lithium chloride (LiCl) modified magnesium hydroxide (Mg(OH) 2 ) is a potential new material for chemical heat pumps. However, there is insufficient information concerning its dehydration and hydration behavior. In this study, the dehydration and hydration reactions, corresponding to the heat storage and the heat output operations, respectively, of authentic Mg(OH) 2 and LiCl-modified Mg(OH) 2 were investigated by thermogravimetric methods and near infrared spectroscopy. The dehydration of authentic Mg(OH) 2 proceeded as a one-step reaction. In contrast, the dehydration of LiCl-modified Mg(OH) 2 occurred in two steps. The dehydration reaction rates were increased by LiCl modification of the Mg(OH) 2 surface, while the activation energy for the first-order dehydration reaction was lowered. The mechanism for the hydration reaction of magnesium oxide (MgO) was different to that for the hydration of LiCl-modified MgO. This difference was explained by the effect of the LiCl on the MgO particle surface. - Highlights: ► LiCl-modified Mg(OH) 2 is a candidate material for chemical heat pumps. ► The dehydration reaction of LiCl-modified Mg(OH) 2 is a two-step reaction. ► The dehydration reaction of Mg(OH) 2 was enhanced by LiCl modification. ► The hydration mechanisms of authentic MgO and LiCl-modified MgO were different.

  17. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  18. Modified clay minerals efficiency against chemical and biological warfare agents for civil human protection.

    Science.gov (United States)

    Plachá, Daniela; Rosenbergová, Kateřina; Slabotínský, Jiří; Kutláková, Kateřina Mamulová; Studentová, Soňa; Martynková, Gražyna Simha

    2014-04-30

    Sorption efficiencies of modified montmorillonite and vermiculite of their mono ionic Na and organic HDTMA and HDP forms were studied against chemical and biological warfare agents such as yperite and selected bacterial strains. Yperite interactions with modified clay minerals were observed through its capture in low-density polyethylene foil-modified clay composites by measuring yperite gas permeation with using chemical indication and gas chromatography methods. The antibacterial activities of synthetized organoclays were tested against selected Gram-positive and Gram-negative bacterial species in minimum inhibitory concentration tests. The obtained results showed a positive influence of modified clay minerals on the significant yperite breakthrough-time increase. The most effective material was the polyethylene-Na form montmorillonite, while the polyethylene-Na form vermiculite showed the lowest efficiency. With increasing organic cations loading in the interlayer space the montmorillonite efficiency decreased, and in the case of vermiculite an opposite effect was observed. Generally the modified montmorillonites were more effective than modified vermiculites. The HDP cations seem to be more effective compare to the HDTMA. The antibacterial activity tests confirmed efficiency of all organically modified clay minerals against Gram-positive bacteria. The confirmation of antibacterial activity against Y. pestis, plague bacteria, is the most interesting result of this part of the study. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Photoelectrocatalytic reduction of CO2 into chemicals using Pt-modified reduced graphene oxide combined with Pt-modified TiO2 nanotubes.

    Science.gov (United States)

    Cheng, Jun; Zhang, Meng; Wu, Gai; Wang, Xin; Zhou, Junhu; Cen, Kefa

    2014-06-17

    The photoelectrocatalytic (PEC) reduction of CO2 into high-value chemicals is beneficial in alleviating global warming and advancing a low-carbon economy. In this work, Pt-modified reduced graphene oxide (Pt-RGO) and Pt-modified TiO2 nanotubes (Pt-TNT) were combined as cathode and photoanode catalysts, respectively, to form a PEC reactor for converting CO2 into valuable chemicals. XRD, XPS, TEM, AFM, and SEM were employed to characterize the microstructures of the Pt-RGO and Pt-TNT catalysts. Reduction products, such as C2H5OH and CH3COOH, were obtained from CO2 under band gap illumination and biased voltage. A combined liquid product generation rate (CH3OH, C2H5OH, HCOOH, and CH3COOH) of approximately 600 nmol/(h·cm(2)) was observed. Carbon atom conversion rate reached 1,130 nmol/(h·cm(2)), which were much higher than those achieved using Pt-modified carbon nanotubes and platinum carbon as cathode catalysts.

  20. mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic.

    Science.gov (United States)

    Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

    2013-02-01

    Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and "naked mRNA" for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.

  1. Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicus

    International Nuclear Information System (INIS)

    Pleshe, Elizabeth; Truesdell, John; Batey, Robert T.

    2005-01-01

    The crystal structure of Aquifex aeolicus TrmH, a member of the a/b-knot superfamily responsible for O methylation of G18 of tRNAs, was determined to 1.85 Å resolution using the molecular-replacement method. Biological RNAs contain a variety of post-transcriptional modifications that facilitate their efficient function in the cellular environment. One of the two most common forms of modification is methylation of the 2′-hydroxyl group of the ribose sugar, which is performed by a number of S-adenosylmethionine (SAM) dependent methyltransferases. In bacteria, many of these modifications in tRNA and rRNA are carried out by the α/β-knot superfamily of enzymes, whose SAM-binding pocket is created by a characteristic deep trefoil knot. TrmH, an enzyme found throughout all three kingdoms of life, modifies the universally conserved guanosine 18 position of tRNA. The crystal structure of TrmH from the thermophilic bacterium Aquifex aeolicus has been determined at 1.85 Å resolution using data collected from a synchrotron-radiation source. The protein reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the α/β-knot superfamily, with α-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding

  2. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

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

  3. Sensory and rheological properties of transgenically and chemically modified starch ingredients as evaluated in a food product model

    DEFF Research Database (Denmark)

    Ahmt, T.; Wischmann, Bente; Blennow, A.

    2004-01-01

    gels with a higher tendency to retrograde and a low freeze/thaw stability as compared to starches with shorter amylopectin chains and lower phosphorous content. The textural properties of the food product model prepared from genetically and chemically modified starches were characterised by sensory......Starches derived from five genetically modified potato lines, two chemically modified potato starches and two native starches from potato and maize were subjected to physical and chemical analyses and their functionality evaluated in a milk-based food product model. The transgenic starches were...... and rheological analyses. To clearly visualise the effects of the modifications, data was evaluated by radar plots and multiple regression analysis (chemometrics). Genetically modified potato starches with longer amylopectin chains and increased phosphorous content gave a more gelled and a shorter texture...

  4. Chemical treatment of the intra-canal dentin surface: a new approach to modify dentin hydrophobicity

    Directory of Open Access Journals (Sweden)

    Cesar GAITAN-FONSECA

    2013-01-01

    Full Text Available Objective This study evaluated the hydrophobicity of dentin surfaces that were modified through chemical silanization with octadecyltrichlorosilane (OTS. Material and Methods An in vitro experimental study was performed using 40 human permanent incisors that were divided into the following two groups: non-silanized and silanized. The specimens were pretreated and chemically modified with OTS. After the chemical modification, the dentin hydrophobicity was examined using a water contact angle measurement (WCA. The effectiveness of the modification of hydrophobicity was verified by the fluid permeability test (FPT. Results and Conclusions Statistically significant differences were found in the values of WCA and FPT between the two groups. After silanization, the hydrophobic intraradicular dentin surface exhibited in vitro properties that limit fluid penetration into the sealed root canal. This chemical treatment is a new approach for improving the sealing of the root canal system.

  5. Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

    Science.gov (United States)

    Jobert, Laure; Nilsen, Hilde

    2014-07-01

    The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

  6. Modified mesenchymal stem cells using miRNA transduction alter lung injury in a bleomycin model.

    Science.gov (United States)

    Huleihel, Luai; Sellares, Jacobo; Cardenes, Nayra; Álvarez, Diana; Faner, Rosa; Sakamoto, Koji; Yu, Guoying; Kapetanaki, Maria G; Kaminski, Naftali; Rojas, Mauricio

    2017-07-01

    Although different preclinical models have demonstrated a favorable role for bone marrow-derived mesenchymal stem cells (B-MSC) in preventing fibrosis, this protective effect is not observed with late administration of these cells, when fibrotic changes are consolidated. We sought to investigate whether the late administration of B-MSCs overexpressing microRNAs (miRNAs) let-7d (antifibrotic) or miR-154 (profibrotic) could alter lung fibrosis in a murine bleomycin model. Using lentiviral vectors, we transduced miRNAs (let-7d or miR-154) or a control sequence into human B-MSCs. Overexpression of let-7d or miR-154 was associated with changes in the mesenchymal properties of B-MSCs and in their cytokine expression. Modified B-MSCs were intravenously administered to mice at day 7 after bleomycin instillation, and the mice were euthanized at day 14 Bleomycin-injured animals that were treated with let-7d cells were found to recover quicker from the initial weight loss compared with the other treatment groups. Interestingly, animals treated with miR-154 cells had the lowest survival rate. Although a slight reduction in collagen mRNA levels was observed in lung tissue from let-7d mice, no significant differences were observed in Ashcroft score and OH-proline. However, the distinctive expression in cytokines and CD45-positive cells in the lung suggests that the differential effects observed in both miRNA mice groups were related to an effect on the immunomodulation function. Our results establish the use of miRNA-modified mesenchymal stem cells as a potential future research in lung fibrosis. Copyright © 2017 the American Physiological Society.

  7. Chemically modified cellulose paper as a thin film microextraction phase.

    Science.gov (United States)

    Saraji, Mohammad; Farajmand, Bahman

    2013-11-01

    In this paper, chemically modified cellulose paper was introduced as a novel extracting phase for thin film microextraction (TFME). Different reagents (Octadecyltrichlorosilane, diphenyldichlorosilane, cyclohexyl isocyanate and phenyl isocyanate) were used to modify the cellulose papers. The modified papers were evaluated as a sorbent for the extraction of some synthetic and natural estrogenic hormones (17α-ethynylestradiol, estriol and estradiol) from aqueous samples. Liquid chromatography-fluorescence detection was used for the quantification of the extracted compounds. The cellulose paper modified with phenyl isocyanate showed the best affinity to the target compounds. TEME parameters such as desorption condition, shaking rate, sample ionic strength and extraction time were investigated and optimized. Limit of detections were between 0.05 and 0.23μgL(-1) and relative standard deviations were less than 11.1% under the optimized condition. The calibration curves were obtained in the range of 0.2-100μgL(-1) with a good linearity (r(2)>0.9935). Wastewater, human urine, pool and river water samples were studied as real samples for the evaluation of the method. Relative recoveries were found to be between 75% and 101%. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Chemical functionalization of ceramic tile surfaces by silane coupling agents: polymer modified mortar adhesion mechanism implications

    Directory of Open Access Journals (Sweden)

    Alexandra Ancelmo Piscitelli Mansur

    2008-09-01

    Full Text Available Adhesion between tiles and mortars are crucial to the stability of ceramic tile systems. From the chemical point of view, weak forces such as van der Waals forces and hydrophilic interactions are expected to be developed preferably at the tiles and polymer modified Portland cement mortar interface. The main goal of this paper was to use organosilanes as primers to modify ceramic tile hydrophilic properties to improve adhesion between ceramic tiles and polymer modified mortars. Glass tile surfaces were treated with several silane derivatives bearing specific functionalities. Contact angle measurements and Fourier Transform Infrared Spectroscopy (FTIR were used for evaluating the chemical changes on the tile surface. In addition, pull-off tests were conducted to assess the effect on adhesion properties between tile and poly(ethylene-co-vinyl acetate, EVA, modified mortar. The bond strength results have clearly shown the improvement of adherence at the tile-polymer modified mortar interface, reflecting the overall balance of silane, cement and polymer interactions.

  9. Chemically modified tetracyclines stimulate matrix metalloproteinase-s production by periodontal ligament cells

    NARCIS (Netherlands)

    Bildt, M.M.; Snoek-van Beurden, A.M.P.; Groot, J. de; El, B. van; Kuijpers-Jagtman, A.M.; Hoff, J.W. van den

    2006-01-01

    Background and Objective: The purpose of this study was to investigate the effects of chemically modified tetracyclines (CMTs) on the production of gelatinases [matrix metalloproteinase (MMP)-2 and -9] by human periodontal ligament (PDL) cells, and on the activity of recombinant gelatinases.

  10. Comparative Profiling of microRNA Expression in Soybean Seeds from Genetically Modified Plants and their Near-Isogenic Parental Lines

    Science.gov (United States)

    Wang, Yong; Lan, Qingkuo; Zhao, Xin; Xu, Wentao; Li, Feiwu; Wang, Qinying; Chen, Rui

    2016-01-01

    MicroRNAs (miRNAs) have been widely demonstrated to play fundamental roles in gene regulation in most eukaryotes. To date, there has been no study describing the miRNA composition in genetically modified organisms (GMOs). In this study, small RNAs from dry seeds of two GM soybean lines and their parental cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, several differentially expressed gma-miRNAs were found between the GM and non-GM soybeans. Meanwhile, more differentially expressed gma-miRNAs were identified between distantly relatednon-GM soybeans, indicating that the miRNA components of soybean seeds varied among different soybean lines, including the GM and non-GM soybeans, and the extent of difference might be related to their genetic relationship. Additionally, fourteen novel gma-miRNA candidates were predicted in soybean seeds including a potential bidirectionally transcribed miRNA family with two genomic loci (gma-miR-N1). Our findings firstly provided useful data for miRNA composition in edible GM crops and also provided valuable information for soybean miRNA research. PMID:27214227

  11. Comparative Profiling of microRNA Expression in Soybean Seeds from Genetically Modified Plants and their Near-Isogenic Parental Lines.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available MicroRNAs (miRNAs have been widely demonstrated to play fundamental roles in gene regulation in most eukaryotes. To date, there has been no study describing the miRNA composition in genetically modified organisms (GMOs. In this study, small RNAs from dry seeds of two GM soybean lines and their parental cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, several differentially expressed gma-miRNAs were found between the GM and non-GM soybeans. Meanwhile, more differentially expressed gma-miRNAs were identified between distantly relatednon-GM soybeans, indicating that the miRNA components of soybean seeds varied among different soybean lines, including the GM and non-GM soybeans, and the extent of difference might be related to their genetic relationship. Additionally, fourteen novel gma-miRNA candidates were predicted in soybean seeds including a potential bidirectionally transcribed miRNA family with two genomic loci (gma-miR-N1. Our findings firstly provided useful data for miRNA composition in edible GM crops and also provided valuable information for soybean miRNA research.

  12. Comparative Profiling of microRNA Expression in Soybean Seeds from Genetically Modified Plants and their Near-Isogenic Parental Lines.

    Science.gov (United States)

    Wang, Yong; Lan, Qingkuo; Zhao, Xin; Xu, Wentao; Li, Feiwu; Wang, Qinying; Chen, Rui

    2016-01-01

    MicroRNAs (miRNAs) have been widely demonstrated to play fundamental roles in gene regulation in most eukaryotes. To date, there has been no study describing the miRNA composition in genetically modified organisms (GMOs). In this study, small RNAs from dry seeds of two GM soybean lines and their parental cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, several differentially expressed gma-miRNAs were found between the GM and non-GM soybeans. Meanwhile, more differentially expressed gma-miRNAs were identified between distantly relatednon-GM soybeans, indicating that the miRNA components of soybean seeds varied among different soybean lines, including the GM and non-GM soybeans, and the extent of difference might be related to their genetic relationship. Additionally, fourteen novel gma-miRNA candidates were predicted in soybean seeds including a potential bidirectionally transcribed miRNA family with two genomic loci (gma-miR-N1). Our findings firstly provided useful data for miRNA composition in edible GM crops and also provided valuable information for soybean miRNA research.

  13. Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice

    OpenAIRE

    Judge, Adam D.; Robbins, Marjorie; Tavakoli, Iran; Levi, Jasna; Hu, Lina; Fronda, Anna; Ambegia, Ellen; McClintock, Kevin; MacLachlan, Ian

    2009-01-01

    siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in b...

  14. Adhesion mapping of chemically modified and poly(ethylene oxide)-grafted glass surfaces

    OpenAIRE

    Jogikalmath, G.; Stuart, J.K.; Pungor, A.; Hlady, V.

    1999-01-01

    Two-dimensional mapping of the adhesion pull-off forces was used to study the origin of surface heterogeneity in the grafted poly(ethylene oxide) (PEO) layer. The variance of the pull-off forces measured over the μm-sized regions after each chemical step of modifying glass surfaces was taken to be a measure of the surface chemical heterogeneity. The attachment of γ-glycidoxypropyltrimethoxy silane (GPS) to glass decreased the pull-off forces relative to the clean glass and made the surface mo...

  15. Dependence of radioprotective effect of chemical modifying agents on their intracellular concentrations

    International Nuclear Information System (INIS)

    Eidus, L.K.; Korystov, Y.N.; Kublik, L.N.; Vexler, A.M.

    1982-01-01

    Regularities of the radioprotective effect of chemical modifying agents cysteamine, caffeine benzoate, thioglycolic acid, and caffeine, all weak electrolytes, have been studied in cultured Chinese hamster cells. Efficiency of protection is shown to be dependent on pH and concentrations of the drug inside the cells and in the medium. Based on the theory of the dissociation of weak electrolytes and their distribution between the cells and the medium a strong correlation between the efficiency of modification of the radiation response and intracellular concentration of a modifying agent is shown. (author)

  16. Prediction of hydrogen and carbon chemical shifts from RNA using database mining and support vector regression

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Joshua D.; Summers, Michael F. [University of Maryland Baltimore County, Howard Hughes Medical Institute (United States); Johnson, Bruce A., E-mail: bruce.johnson@asrc.cuny.edu [University of Maryland Baltimore County, Department of Chemistry and Biochemistry (United States)

    2015-09-15

    The Biological Magnetic Resonance Data Bank (BMRB) contains NMR chemical shift depositions for over 200 RNAs and RNA-containing complexes. We have analyzed the {sup 1}H NMR and {sup 13}C chemical shifts reported for non-exchangeable protons of 187 of these RNAs. Software was developed that downloads BMRB datasets and corresponding PDB structure files, and then generates residue-specific attributes based on the calculated secondary structure. Attributes represent properties present in each sequential stretch of five adjacent residues and include variables such as nucleotide type, base-pair presence and type, and tetraloop types. Attributes and {sup 1}H and {sup 13}C NMR chemical shifts of the central nucleotide are then used as input to train a predictive model using support vector regression. These models can then be used to predict shifts for new sequences. The new software tools, available as stand-alone scripts or integrated into the NMR visualization and analysis program NMRViewJ, should facilitate NMR assignment and/or validation of RNA {sup 1}H and {sup 13}C chemical shifts. In addition, our findings enabled the re-calibration a ring-current shift model using published NMR chemical shifts and high-resolution X-ray structural data as guides.

  17. Recovery of microbial diversity and activity during bioremediation following chemical oxidation of diesel contaminated soils.

    Science.gov (United States)

    Sutton, Nora B; Langenhoff, Alette A M; Lasso, Daniel Hidalgo; van der Zaan, Bas; van Gaans, Pauline; Maphosa, Farai; Smidt, Hauke; Grotenhuis, Tim; Rijnaarts, Huub H M

    2014-03-01

    To improve the coupling of in situ chemical oxidation and in situ bioremediation, a systematic analysis was performed of the effect of chemical oxidation with Fenton's reagent, modified Fenton's reagent, permanganate, or persulfate, on microbial diversity and activity during 8 weeks of incubation in two diesel-contaminated soils (peat and fill). Chemical oxidant and soil type affected the microbial community diversity and biodegradation activity; however, this was only observed following treatment with Fenton's reagent and modified Fenton's reagent, and in the biotic control without oxidation. Differences in the highest overall removal efficiencies of 69 % for peat (biotic control) and 59 % for fill (Fenton's reagent) were partially explained by changes in contaminant soil properties upon oxidation. Molecular analysis of 16S rRNA and alkane monooxygenase (alkB) gene abundances indicated that oxidation with Fenton's reagent and modified Fenton's reagent negatively affected microbial abundance. However, regeneration occurred, and final relative alkB abundances were 1-2 orders of magnitude higher in chemically treated microcosms than in the biotic control. 16S rRNA gene fragment fingerprinting with DGGE and prominent band sequencing illuminated microbial community composition and diversity differences between treatments and identified a variety of phylotypes within Alpha-, Beta-, and Gammaproteobacteria. Understanding microbial community dynamics during coupled chemical oxidation and bioremediation is integral to improved biphasic field application.

  18. The prospects for the research on chemical modifiers of cancer treatment

    International Nuclear Information System (INIS)

    Jin Yizun

    2002-01-01

    The current clinical statue of chemical modifiers of cancer treatment is described. It should enable greater enhancement ratios to be attained clinically. To search for less toxic and more potent sensitizers for radiotherapy and chemotherapy will be improved the cancer treatment. Phase I, II and III clinical trials with TPZ, one of the bioreductive drugs, has shown the benefit to patients with cancer

  19. The ROS-sensitive microRNA-9/9* controls the expression of mitochondrial tRNA-modifying enzymes and is involved in the molecular mechanism of MELAS syndrome.

    Science.gov (United States)

    Meseguer, Salvador; Martínez-Zamora, Ana; García-Arumí, Elena; Andreu, Antonio L; Armengod, M-Eugenia

    2015-01-01

    Mitochondrial dysfunction activates mitochondria-to-nucleus signaling pathways whose components are mostly unknown. Identification of these components is important to understand the molecular mechanisms underlying mitochondrial diseases and to discover putative therapeutic targets. MELAS syndrome is a rare neurodegenerative disease caused by mutations in mitochondrial (mt) DNA affecting mt-tRNA(Leu(UUR)). Patient and cybrid cells exhibit elevated oxidative stress. Moreover, mutant mt-tRNAs(Leu(UUR)) lack the taurine-containing modification normally present at the wobble uridine (U34) of wild-type mt-tRNA(Leu(UUR)), which is considered an etiology of MELAS. However, the molecular mechanism is still unclear. We found that MELAS cybrids exhibit a significant decrease in the steady-state levels of several mt-tRNA-modification enzymes, which is not due to transcriptional regulation. We demonstrated that oxidative stress mediates an NFkB-dependent induction of microRNA-9/9*, which acts as a post-transcriptional negative regulator of the mt-tRNA-modification enzymes GTPBP3, MTO1 and TRMU. Down-regulation of these enzymes by microRNA-9/9* affects the U34 modification status of non-mutant tRNAs and contributes to the MELAS phenotype. Anti-microRNA-9 treatments of MELAS cybrids reverse the phenotype, whereas miR-9 transfection of wild-type cells mimics the effects of siRNA-mediated down-regulation of GTPBP3, MTO1 and TRMU. Our data represent the first evidence that an mt-DNA disease can directly affect microRNA expression. Moreover, we demonstrate that the modification status of mt-tRNAs is dynamic and that cells respond to stress by modulating the expression of mt-tRNA-modifying enzymes. microRNA-9/9* is a crucial player in mitochondria-to-nucleus signaling as it regulates expression of nuclear genes in response to changes in the functional state of mitochondria. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email

  20. Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Suzan M Hammond

    2014-01-01

    Full Text Available Splice switching oligonucleotides (SSOs induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™” to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

  1. Adsorption study of copper (II) by chemically modified orange peel

    International Nuclear Information System (INIS)

    Feng Ningchuan; Guo Xueyi; Liang Sha

    2009-01-01

    An adsorbent, the chemically modified orange peel, was prepared from hydrolysis of the grafted copolymer, which was synthesized by interaction of methyl acrylate with cross-linking orange peel. The presence of poly (acrylic acid) on the biomass surface was verified by infrared spectroscopy (IR), scanning electron microscopy (SEM) and thermogravimetry (TG). Total negative charge in the biomass surface and the zeta potentials were determined. The modified biomass was found to present high adsorption capacity and fast adsorption rate for Cu (II). From Langmuir isotherm, the adsorption capacity for Cu (II) was 289.0 mg g -1 , which is about 6.5 times higher than that of the unmodified biomass. The kinetics for Cu (II) adsorption followed the pseudo-second-order kinetics. The adsorbent was used to remove Cu (II) from electroplating wastewater and was suitable for repeated use for more than four cycles.

  2. Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay.

    Directory of Open Access Journals (Sweden)

    Jun Yang

    Full Text Available Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC. A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.

  3. Adhesive and morphological characteristics of surface chemically modified polytetrafluoroethylene films

    International Nuclear Information System (INIS)

    Hopp, B.; Kresz, N.; Kokavecz, J.; Smausz, T.; Schieferdecker, H.; Doering, A.; Marti, O.; Bor, Z.

    2004-01-01

    In the present paper, we report an experimental determination of adhesive and topographic characteristics of chemically modified surface of polytetrafluoroethylene (PTFE) films. The surface chemistry was modified by ArF excimer laser irradiation in presence of triethylene-tetramine photoreagent. The applied laser fluence was varied in the range of 0.4-9 mJ/cm 2 , and the number of laser pulses incident on the same area was 1500. To detect the changes in the adhesive features of the treated Teflon samples, we measured receding contact angle for distilled water and adhesion strength, respectively. It was found that the receding contact angle decreased from 96 deg. to 30-37 deg. and the adhesion strength of two-component epoxy glue to the treated sample surface increased from 0.03 to 9 MPa in the applied laser fluence range. Additionally, it was demonstrated that the adhesion of human cells to the modified Teflon samples is far better than to the untreated ones. The contact mode and pulsed force mode atomic force microscopic investigations of the treated samples demonstrated that the measured effective contact area of the irradiated films does not differ significantly from that of the original films, but the derived adhesion force is stronger on the modified samples than on the untreated ones. Hence, the increased adhesion of the treated Teflon films is caused by the higher surface energy

  4. Graphene Oxide Conjugated Magnetic Beads for RNA Extraction.

    Science.gov (United States)

    Pham, Xuan-Hung; Baek, Ahruem; Kim, Tae Han; Lee, Sang Hun; Rho, Won-Yeop; Chung, Woo-Jae; Kim, Dong-Eun; Jun, Bong-Hyun

    2017-08-04

    A magnetic material that consists of silica-coated magnetic beads conjugated with graphene oxide (GO) was successfully prepared for facile ribonucleic acid (RNA) extraction. When the GO-modified magnetic beads were applied to separate the RNA from the lysed cell, the cellular RNAs were readily adsorbed to and readily desorbed from the surface of the GO-modified magnetic beads by urea. The amount of RNA extracted by the GO-modified magnetic beads was ≈170 % as much as those of the control extracted by a conventional phenol-based chaotropic solution. These results demonstrate that the facile method of RNA separation by using GO-modified magnetic beads as an adsorbent is an efficient and simple way to purify intact cellular RNAs and/or microRNA from cell lysates. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. tRNA's wobble decoding of the genome: 40 years of modification.

    Science.gov (United States)

    Agris, Paul F; Vendeix, Franck A P; Graham, William D

    2007-02-09

    The genetic code is degenerate, in that 20 amino acids are encoded by 61 triplet codes. In 1966, Francis Crick hypothesized that the cell's limited number of tRNAs decoded the genome by recognizing more than one codon. The ambiguity of that recognition resided in the third base-pair, giving rise to the Wobble Hypothesis. Post-transcriptional modifications at tRNA's wobble position 34, especially modifications of uridine 34, enable wobble to occur. The Modified Wobble Hypothesis proposed in 1991 that specific modifications of a tRNA wobble nucleoside shape the anticodon architecture in such a manner that interactions were restricted to the complementary base plus a single wobble pairing for amino acids with twofold degenerate codons. However, chemically different modifications at position 34 would expand the ability of a tRNA to read three or even four of the fourfold degenerate codons. One foundation of Crick's Wobble Hypothesis was that a near-constant geometry of canonical base-pairing be maintained in forming all three base-pairs between the tRNA anticodon and mRNA codon on the ribosome. In accepting an aminoacyl-tRNA, the ribosome requires maintenance of a specific geometry for the anticodon-codon base-pairing. However, it is the post-transcriptional modifications at tRNA wobble position 34 and purine 37, 3'-adjacent to the anticodon, that pre-structure the anticodon domain to ensure the correct codon binding. The modifications create both the architecture and the stability needed for decoding through restraints on anticodon stereochemistry and conformational space, and through selective hydrogen bonding. A physicochemical understanding of modified nucleoside contributions to the tRNA anticodon domain architecture and its decoding of the genome has advanced RNA world evolutionary theory, the principles of RNA chemistry, and the application of this knowledge to the introduction of new amino acids to proteins.

  6. TaxCollector: Modifying Current 16S rRNA Databases for the Rapid Classification at Six Taxonomic Levels

    Directory of Open Access Journals (Sweden)

    Eric W. Triplett

    2010-07-01

    Full Text Available The high level of conservation of 16S ribosomal RNA gene (16S rRNA in all Prokaryotes makes this gene an ideal tool for the rapid identification and classification of these microorganisms. Databases such as the Ribosomal Database Project II (RDP-II and the Greengenes Project offer access to sets of ribosomal RNA sequence databases useful in identification of microbes in a culture-independent analysis of microbial communities. However, these databases do not contain all of the taxonomic levels attached to the published names of the bacterial and archaeal sequences. TaxCollector is a set of scripts developed in Python language that attaches taxonomic information to all 16S rRNA sequences in the RDP-II and Greengenes databases. These modified databases are referred to as TaxCollector databases, which when used in conjunction with BLAST allow for rapid classification of sequences from any environmental or clinical source at six different taxonomic levels, from domain to species. The TaxCollector database prepared from the RDP-II database is an important component of a new 16S rRNA pipeline called PANGEA. The usefulness of TaxCollector databases is demonstrated with two very different datasets obtained using samples from a clinical setting and an agricultural soil. The six TaxCollector scripts are freely available on http://taxcollector.sourceforge.net and on http://www.microgator.org.

  7. Synthetic biology devices and circuits for RNA-based 'smart vaccines': a propositional review.

    Science.gov (United States)

    Andries, Oliwia; Kitada, Tasuku; Bodner, Katie; Sanders, Niek N; Weiss, Ron

    2015-02-01

    Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control the production of antigens and adjuvants in a desirable manner by administering small molecule drugs as chemical triggers. Advances in synthetic biology have resulted in the creation of highly predictable and modular genetic parts and devices that can be composed into synthetic gene circuits with complex behaviors. With the recent advent of modified RNA gene delivery methods and developments in the RNA replicon platform, we foresee a future in which mammalian synthetic biologists will create genetic circuits encoded exclusively on RNA. Here, we review the current repertoire of devices used in RNA synthetic biology and propose how programmable 'smart vaccines' will revolutionize the field of RNA vaccination.

  8. Enhancing siRNA-based cancer therapy using a new pH-responsive activatable cell-penetrating peptide-modified liposomal system

    Directory of Open Access Journals (Sweden)

    Xiang B

    2017-03-01

    Full Text Available Bai Xiang,1,* Xue-Li Jia,1,* Jin-Long Qi,2 Li-Ping Yang,1 Wei-Hong Sun,1 Xiao Yan,1 Shao-Kun Yang,1 De-Ying Cao,1 Qing Du,1 Xian-Rong Qi3 1Department of Pharmaceutics, School of Pharmaceutical Sciences, 2Department of Pharmacology, Hebei Medical University, Shijiazhuang, Hebei, 3School of Pharmaceutical Sciences, Peking University, Beijing, China *These authors contributed equally to this work Abstract: As a potent therapeutic agent, small interfering RNA (siRNA has been exploited to silence critical genes involved in tumor initiation and progression. However, development of a desirable delivery system is required to overcome the unfavorable properties of siRNA such as its high degradability, molecular size, and negative charge to help increase its accumulation in tumor tissues and promote efficient cellular uptake and endosomal/lysosomal escape of the nucleic acids. In this study, we developed a new activatable cell-penetrating peptide (ACPP that is responsive to an acidic tumor microenvironment, which was then used to modify the surfaces of siRNA-loaded liposomes. The ACPP is composed of a cell-penetrating peptide (CPP, an acid-labile linker (hydrazone, and a polyanionic domain, including glutamic acid and histidine. In the systemic circulation (pH 7.4, the surface polycationic moieties of the CPP (polyarginine are “shielded” by the intramolecular electrostatic interaction of the inhibitory domain. When exposed to a lower pH, a common property of solid tumors, the ACPP undergoes acid-catalyzed breakage at the hydrazone site, and the consequent protonation of histidine residues promotes detachment of the inhibitory peptide. Subsequently, the unshielded CPP would facilitate the cellular membrane penetration and efficient endosomal/lysosomal evasion of liposomal siRNA. A series of investigations demonstrated that once exposed to an acidic pH, the ACPP-modified liposomes showed elevated cellular uptake, downregulated expression of polo

  9. The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

    Science.gov (United States)

    Leontis, N B; Westhof, E

    1998-09-01

    A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings

  10. Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA

    DEFF Research Database (Denmark)

    Leihne, Vibeke; Kirpekar, Finn; Vågbø, Cathrine B

    2011-01-01

    Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we...... demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic...... activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8...

  11. Fast vertical growth of ZnO nanorods using a modified chemical bath deposition

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae-hyun [Department of Nano Systems Engineering, Center for Nano Manufacturing, Inje University, Obang-dong, Gimhae, Gyeongnam 621-749 (Korea, Republic of); Ryu, Hyukhyun, E-mail: hhryu@inje.ac.kr [Department of Nano Systems Engineering, Center for Nano Manufacturing, Inje University, Obang-dong, Gimhae, Gyeongnam 621-749 (Korea, Republic of); Lee, Won-Jae [Department of Materials and Components Engineering, Dong-Eui University, 995 Eomgwangno, Busanjin-gu, Busan 614-714 (Korea, Republic of)

    2014-06-01

    Highlights: • We grew vertical ZnO nanorods by a modified CBD process with a fast growth rate. • We studied the effects of the CBD process by varying growth temperature, time, and concentration. • The ZnO nanorods grown by the modified CBD showed good morphological and structural properties. - Abstract: In this study, we grew vertical ZnO nanorods on seeded Si (1 0 0) substrates using a modified chemical bath deposition (CBD). We investigated the effects of the growth temperature, growth time and concentration on the morphological and structural properties of the ZnO nanorods using field emission gun scanning electron microscopy (FEG-SEM) and X-ray diffraction. This modified CBD method shows improved results over conventional CBD. ZnO nanorods with good structural XRD properties were grown with a very fast growth rate in a wide range of growth conditions and did not require post-growth annealing.

  12. Direct, rapid RNA sequence analysis

    International Nuclear Information System (INIS)

    Peattie, D.A.

    1987-01-01

    The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

  13. A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNAHis pair.

    Science.gov (United States)

    Englert, Markus; Vargas-Rodriguez, Oscar; Reynolds, Noah M; Wang, Yane-Shih; Söll, Dieter; Umehara, Takuya

    2017-11-01

    Development of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNA His recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNA His pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus. E. coli was genetically engineered to use a C. crescentus HisRS•tRNA His pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo. A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNA His pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRS•tRNA His pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNA His CUA elevated its suppression efficiency by 2-fold. The C. crescentus HisRS•tRNA His pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNA His is orthogonal in MEOV1 cells. E. coli tRNA His CUA is an efficient amber suppressor in MEOV1. We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Chemical switches and logic gates based on surface modified semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Konrad, Szacilowski; Wojciech, Macyk [Jagiellonian Univ., Dept. of Chemistry, Krakow (Poland)

    2006-02-15

    Photoelectrochemical properties of multicomponent photo-electrodes based on titanium dioxide and cadmium sulfide powders modified with hexacyanoferrate complexes have been examined. Photocurrent responses were recorded as functions of applied potential and photon energy. Surprisingly, the photocurrent can be switched between positive and negative values as a result of potential or photon energy changes. This new effect called Photo Electrochemical Photocurrent Switching (PEPS) opens a possibility of new chemical switches and logic gates construction. Boolean logic analysis and a tentative mechanism of the device are discussed. (authors)

  15. Towards electron transport measurements in chemically modified graphene: effect of a solvent

    Energy Technology Data Exchange (ETDEWEB)

    Jacobsen, Arnhild; Ensslin, Klaus [Solid State Physics Laboratory, ETH Zurich (Switzerland); Koehler, Fabian M; Stark, Wendelin J, E-mail: arnhildj@phys.ethz.ch, E-mail: fabian.koehler@chem.ethz.ch [Institute for Chemical and Bioengineering, ETH Zurich (Switzerland)

    2010-12-15

    The chemical functionalization of graphene modifies the local electron density of carbon atoms and hence electron transport. Measuring these changes allows for a closer understanding of the chemical interaction and the influence of functionalization on the graphene lattice. However, not only chemistry, in this case diazonium chemistry, has an effect on electron transport. The latter is also influenced by defects and dopants resulting from different processing steps. Here, we show that the solvents used in the chemical reaction process change the transport properties. In more detail, the investigated combination of isopropanol and heating treatment reduces the doping concentration and significantly increases the mobility of graphene. Furthermore, isopropanol treatment alone increases the concentration of dopants and introduces an asymmetry between electron and hole transport, which might be difficult to distinguish from the effect of functionalization. The results shown in this work demand a closer look at the influence of solvents used for chemical modification in order to understand their influence.

  16. Towards electron transport measurements in chemically modified graphene: effect of a solvent

    International Nuclear Information System (INIS)

    Jacobsen, Arnhild; Ensslin, Klaus; Koehler, Fabian M; Stark, Wendelin J

    2010-01-01

    The chemical functionalization of graphene modifies the local electron density of carbon atoms and hence electron transport. Measuring these changes allows for a closer understanding of the chemical interaction and the influence of functionalization on the graphene lattice. However, not only chemistry, in this case diazonium chemistry, has an effect on electron transport. The latter is also influenced by defects and dopants resulting from different processing steps. Here, we show that the solvents used in the chemical reaction process change the transport properties. In more detail, the investigated combination of isopropanol and heating treatment reduces the doping concentration and significantly increases the mobility of graphene. Furthermore, isopropanol treatment alone increases the concentration of dopants and introduces an asymmetry between electron and hole transport, which might be difficult to distinguish from the effect of functionalization. The results shown in this work demand a closer look at the influence of solvents used for chemical modification in order to understand their influence.

  17. RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

    Science.gov (United States)

    Diener, Yvonne; Jurk, Marion; Kandil, Britta; Choi, Yeong-Hoon; Wild, Stefan; Bissels, Ute; Bosio, Andreas

    2015-01-01

    Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5′- and 3′-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs. PMID:26599627

  18. Effect of emulsifiers on complexation and retrogradation characteristics of native and chemically modified White sorghum (Sorghum bicolor) starch

    International Nuclear Information System (INIS)

    Ali, Tahira Mohsin; Hasnain, Abid

    2013-01-01

    Highlights: ► Sorghum starches were chemically modified. ► Starch–lipid complexes were studied in the presence of emulsifiers. ► Type II complexes were also detected in native and oxidized starches on adding GMS. ► Starch–lipid complexes sharply reduced retrogradation in modified starches. - Abstract: The effect of emulsifiers on complexation and retrogradation characteristics of native and chemically modified white sorghum starches was studied. Complex forming tendency of white sorghum starch with commercially available emulsifiers GMS and DATEM improved after acetylation. Presence of emulsifiers reduced λ max (wavelength of maximum absorbance) both for native and modified sorghum starches suggesting lower availability of amylose chains to complex with iodine. In native white sorghum starch (NWSS) and oxidized white sorghum starch (OWSS), both Type I and Type II starch–lipid complexes were observed on addition of 1.0% GMS prior to gelatinization. Acetylated-oxidized white sorghum starch (AOWSS) formed weakest complexes among all the modified starches. The results revealed that antistaling characteristics of modified sorghum starches were enhanced when used in combination with emulsifiers. The most prominent decline in reassociative capability among modified starches was observed for acetylated starches.

  19. A modified parallel artificial membrane permeability assay for evaluating the bioconcentration of highly hydrophobic chemicals in fish.

    Science.gov (United States)

    Kwon, Jung-Hwan; Escher, Beate I

    2008-03-01

    Low cost in vitro tools are needed at the screening stage of assessment of bioaccumulation potential of new and existing chemicals because the number of chemical substances that needs to be tested highly exceeds the capacity of in vivo bioconcentration tests. Thus, the parallel artificial membrane permeability assay (PAMPA) system was modified to predict passive uptake/ elimination rate in fish. To overcome the difficulties associated with low aqueous solubility and high membrane affinity of highly hydrophobic chemicals, we measured the rate of permeation from the donor poly(dimethylsiloxane)(PDMS) disk to the acceptor PDMS disk through aqueous and PDMS membrane boundary layers and term the modified PAMPA system "PDMS-PAMPA". Twenty chemicals were selected for validation of PDMS-PAMPA. The measured permeability is proportional to the passive elimination rate constant in fish and was used to predict the "minimum" in vivo elimination rate constant. The in vivo data were very close to predicted values except for a few polar chemicals and metabolically active chemicals, such as pyrene and benzo[a]pyrene. Thus, PDMS-PAMPA can be an appropriate in vitro system for nonmetabolizable chemicals. Combination with metabolic clearance rates using a battery of metabolic degradation assays would enhance the applicability for metabolizable chemicals.

  20. Chemically modified graphite felt as an efficient cathode in electro-Fenton for p-nitrophenol degradation

    International Nuclear Information System (INIS)

    Zhou, Lei; Zhou, Minghua; Hu, Zhongxin; Bi, Zhaoheng; Serrano, K. Groenen

    2014-01-01

    Highlights: • Chemically modified graphite felt was prepared using ethanol and hydrazine hydrate as reagents. • Carbon nanoparticles with functional groups were deposited on the surface after modification. • The electrochemical activity for ORR and H 2 O 2 generation on the modified electrode was improved. • The cathode modification effictively improved the EF performance for pollutant degradation. - Abstract: A simple method with low-cost chemical reagents ethanol and hydrazine hydrate was used to modify graphite felt as the cathode for electro-Fenton (EF) application, using p-nitrophenol (p-Np) as the model pollutant. Characterized by scanning electron microscope, contact angle, Raman spectrum and X-ray photoelectron spectroscopy, the morphology and surface physicochemical properties after modification were observed considerably changed. After modification, some nanoparticles and oxygen and nitrogen-containing functional groups appeared on the cathode surface, which greatly improved the surface hydrophilic property and the electrocatalytic activity for oxygen reduction reaction. The effects led to the hydrogen peroxide accumulation on the modified cathode markedly increased to 175.8 mg L −1 , while that on the unmodified one was only 67.5 mg L −1 . p-Np of initial 50 mg L −1 could be completely removed by EF using the modified cathode, and the mineralization ratio reached 51.4%, more than 2 times of the pristine one. After 10 cycles, the mineralization ratio of the modified cathode was still above 45%, suggesting that the modification method can provide an effective approach to improve EF performance, and thus benefits to promote its environmental applications

  1. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    Science.gov (United States)

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

    2013-01-01

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

  2. RNA and Proteins: Mutual Respect [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Kathleen B. Hall

    2017-03-01

    Full Text Available Proteins and RNA are often found in ribonucleoprotein particles (RNPs, where they function in cellular processes to synthesize proteins (the ribosome, chemically modify RNAs (small nucleolar RNPs, splice pre-mRNAs (the spliceosome, and, on a larger scale, sequester RNAs, degrade them, or process them (P bodies, Cajal bodies, and nucleoli. Each RNA–protein interaction is a story in itself, as both molecules can change conformation, compete for binding sites, and regulate cellular functions. Recent studies of Xist long non-coding RNP, the U4/5/6 tri-small nuclear RNP complex, and an activated state of a spliceosome reveal new features of RNA interactions with proteins, and, although their stories are incomplete, they are already fascinating.

  3. Fluorescence spectroscopic investigation of the interaction of citrinin with native and chemically modified cyclodextrins

    Energy Technology Data Exchange (ETDEWEB)

    Poór, Miklós, E-mail: poor.miklos@pte.hu [Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Szigeti út 12, Pécs H-7624 (Hungary); Matisz, Gergely; Kunsági-Máté, Sándor [Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624 (Hungary); János Szentágothai Research Center, Ifjúság útja 20, Pécs H-7624 (Hungary); Derdák, Diána [Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624 (Hungary); Szente, Lajos [CycloLab Cyclodextrin Research & Development Laboratory Ltd., Illatos út 7, Budapest H-1097 (Hungary); and others

    2016-04-15

    Citrinin (CIT) is a nephrotoxic mycotoxin produced by several Aspergillus, Penicillium and Monascus species. CIT is unavoidable contaminant of different foods and drinks due to its wide occurrence and high thermal stability. For this reason, development of new, more sensitive analytical methods and decontamination strategies has high importance. In our study, the complex formation of CIT with native and chemically modified cyclodextrins was investigated using fluorescence spectroscopy. Furthermore, thermodynamic and molecular modeling studies were also performed for the deeper understanding of these host-guest interactions. Our results show that among the tested compounds methylated β-cyclodextrins form the most stable complexes with CIT and these derivatives cause the highest fluorescence enhancement of CIT as well. These observations recommend that some of the chemically modified derivatives show more favourable properties than the native cyclodextrin, and suggesting more promising analytical applicability and higher affinity as potential toxin binders.

  4. 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...

  5. An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes.

    Science.gov (United States)

    Taoka, Masato; Yamauchi, Yoshio; Nobe, Yuko; Masaki, Shunpei; Nakayama, Hiroshi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Isobe, Toshiaki

    2009-11-01

    We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a approximately 21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

  6. Profiling the nucleobase and structure selectivity of anticancer drugs and other DNA alkylating agents by RNA sequencing.

    Science.gov (United States)

    Gillingham, Dennis; Sauter, Basilius

    2018-05-06

    Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically the chemical reactivity of DNA alkylators has been determined in vitro with short oligonucleotides. Here we use next generation RNA sequencing to report on the chemoselectivity of alkylating agents. We develop the method with the well-known clinically used DNA modifiying drugs streptozotocin and temozolomide, and then apply the technique to profile RNA modification with uncharacterized alkylation reactions such as with powerful electrophiles like trimethylsilyldiazomethane. The multiplexed and massively parallel format of NGS offers analyses of chemical reactivity in nucleic acids to be accomplished in less time with greater statistical power. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Effect of single base changes and the absence of modified bases in 16S RNA on the reconstitution and function of Escherichia coli 30S ribosomes

    International Nuclear Information System (INIS)

    Denman, R.; Krzyzosiak, W.; Nurse, K.; Ofengand, J.

    1987-01-01

    The gene coding for E. coli 16S rRNA was placed in pUC19 under the control of the strong class III T7 promoter, phi 10, by ligation of the 1490 bp BclI/BstEII fragment of the rrnB operon with appropriate synthetic oligodeoxynucleotides. Such constructs allowed efficient in vitro synthesis of full-length transcripts (up to 900 mol RNA/mol template) free of modified bases. The synthetic RNA could be assembled into 30S subunits upon addition of E. coli 30S ribosomal proteins. The particles co-sedimented with authentic 30S particles and were electron microscopically indistinguishable from them. Upon addition of 50S subunits, codon-dependent P-site binding of tRNA and codon-dependent polypeptide synthesis were >80% of 30S reconstituted from natural 16S RNA and >50% of isolated 30S. UV-induced crosslinking of P-site bound AcVal-tRNA to residue C 1400 was preserved. Changing C 1400 to A had little effect on reconstitution, P-site binding, or polypeptide synthesis. However, the substitution of C 1499 by G markedly inhibited assembly. The effect on P-site binding and polypeptide synthesis is under study. These results show (1) none of the modified bases of 16S RNA are essential for protein synthesis, (2) substitution of A for C 1400 has little functional effect, and (3) position 1400 may be important for ribosome assembly

  8. Transfer of Chemically Modified Graphene with Retention of Functionality for Surface Engineering.

    Science.gov (United States)

    Whitener, Keith E; Lee, Woo-Kyung; Bassim, Nabil D; Stroud, Rhonda M; Robinson, Jeremy T; Sheehan, Paul E

    2016-02-10

    Single-layer graphene chemically reduced by the Birch process delaminates from a Si/SiOx substrate when exposed to an ethanol/water mixture, enabling transfer of chemically functionalized graphene to arbitrary substrates such as metals, dielectrics, and polymers. Unlike in previous reports, the graphene retains hydrogen, methyl, and aryl functional groups during the transfer process. This enables one to functionalize the receiving substrate with the properties of the chemically modified graphene (CMG). For instance, magnetic force microscopy shows that the previously reported magnetic properties of partially hydrogenated graphene remain after transfer. We also transfer hydrogenated graphene from its copper growth substrate to a Si/SiOx wafer and thermally dehydrogenate it to demonstrate a polymer- and etchant-free graphene transfer for potential use in transmission electron microscopy. Finally, we show that the Birch reduction facilitates delamination of CMG by weakening van der Waals forces between graphene and its substrate.

  9. Covering chemical diversity of genetically-modified tomatoes using metabolomics for objective substantial equivalence assessment.

    Directory of Open Access Journals (Sweden)

    Miyako Kusano

    Full Text Available As metabolomics can provide a biochemical snapshot of an organism's phenotype it is a promising approach for charting the unintended effects of genetic modification. A critical obstacle for this application is the inherently limited metabolomic coverage of any single analytical platform. We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that % had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms.

  10. Covering Chemical Diversity of Genetically-Modified Tomatoes Using Metabolomics for Objective Substantial Equivalence Assessment

    Science.gov (United States)

    Hirai, Tadayoshi; Oikawa, Akira; Matsuda, Fumio; Fukushima, Atsushi; Arita, Masanori; Watanabe, Shin; Yano, Megumu; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi; Saito, Kazuki

    2011-01-01

    As metabolomics can provide a biochemical snapshot of an organism's phenotype it is a promising approach for charting the unintended effects of genetic modification. A critical obstacle for this application is the inherently limited metabolomic coverage of any single analytical platform. We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that % had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms. PMID:21359231

  11. 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

    Science.gov (United States)

    Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

    2018-03-01

    Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

  12. Identification of 5-hydroxycytidine at position 2501 concludes characterization of modified nucleotides in E. coli 23S rRNA

    DEFF Research Database (Denmark)

    Havelund, Jesper Foged; Giessing, Anders Michael Bernth; Hansen, Trine Møller

    2011-01-01

    rRNA-has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown...

  13. 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.

  14. Catalytic conversion of biomass pyrolysis-derived compounds with chemical liquid deposition (CLD) modified ZSM-5.

    Science.gov (United States)

    Zhang, Huiyan; Luo, Mengmeng; Xiao, Rui; Shao, Shanshan; Jin, Baosheng; Xiao, Guomin; Zhao, Ming; Liang, Junyu

    2014-03-01

    Chemical liquid deposition (CLD) with KH550, TEOS and methyl silicone oil as the modifiers was used to modify ZSM-5 and deposit its external acid sites. The characteristics of modified catalysts were tested by catalytic conversion of biomass pyrolysis-derived compounds. The effects of different modifying conditions (deposited amount, temperature, and time) on the product yields and selectivities were investigated. The results show KH550 modified ZSM-5 (deposited amount of 4%, temperature of 20°C and time of 6h) produced the maximum yields of aromatics (24.5%) and olefins (16.5%), which are much higher than that obtained with original ZSM-5 catalyst (18.8% aromatics and 9.8% olefins). The coke yield decreased from 44.1% with original ZSM-5 to 26.7% with KH550 modified ZSM-5. The selectivities of low-molecule-weight hydrocarbons (ethylene and benzene) decreased, while that of higher molecule-weight hydrocarbons (propylene, butylene, toluene, and naphthalene) increased comparing with original ZSM-5. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Methylated nucleosides in tRNA and tRNA methyltransferases

    Directory of Open Access Journals (Sweden)

    Hiroyuki eHori

    2014-05-01

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

  16. Deposition of carbon nanotubes onto aramid fibers using as-received and chemically modified fibers

    International Nuclear Information System (INIS)

    Rodríguez-Uicab, O.; Avilés, F.; Gonzalez-Chi, P.I; Canché-Escamilla, G.; Duarte-Aranda, S.; Yazdani-Pedram, M.; Toro, P.; Gamboa, F.; Mazo, M.A.; Nistal, A.; Rubio, J.

    2016-01-01

    Highlights: • The surface of aramid fibers was functionalized by two acid treatments. • The treatment based on HNO_3/H_2SO_4 reduced the mechanical properties of the fibers. • CNTs were deposited on the aramid fibers, reaching electrical conductivity. • Homogeneous CNT distribution was achieved by using pristine fibers or chlorosulfonic acid. - Abstract: Multiwall carbon nanotubes (MWCNTs) oxidized by an acid treatment were deposited on the surface of as-received commercial aramid fibers containing a surface coating (“sizing”), and fibers modified by either a chlorosulfonic treatment or a mixture of nitric and sulfuric acids. The surface of the aramid fiber activated by the chemical treatments presents increasing density of CO, COOH and OH functional groups. However, these chemical treatments reduced the tensile mechanical properties of the fibers, especially when the nitric and sulfuric acid mixture was used. Characterization of the MWCNTs deposited on the fiber surface was conducted by scanning electron microscopy, Raman spectroscopy mapping and X-ray photoelectron spectroscopy. These characterizations showed higher areal concentration and more homogeneous distribution of MWCNTs over the aramid fibers for as-received fibers and for those modified with chlorosulfonic acid, suggesting the existence of interaction between the oxidized MWCNTs and the fiber coating. The electrical resistance of the MWCNT-modified aramid yarns comprising ∼1000 individual fibers was in the order of MΩ/cm, which renders multifunctional properties.

  17. Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance

    DEFF Research Database (Denmark)

    Douthwaite, S; Powers, T; Lee, J Y

    1989-01-01

    The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected...... deletion mutants show a sensitive phenotype. Deletions that extend into the base-pairing between GCC1208 and GGU1240 result in non-functional 23 S RNAs, which consequently do not confer resistance. A number of phylogenetically conserved nucleotides have been shown to be non-essential for 23 S RNA function....... However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes...

  18. Inclusion of methoxy groups inverts the thermodynamic stabilities of DNA-RNA hybrid duplexes: A molecular dynamics simulation study.

    Science.gov (United States)

    Suresh, Gorle; Priyakumar, U Deva

    2015-09-01

    Modified nucleic acids have found profound applications in nucleic acid based technologies such as antisense and antiviral therapies. Previous studies on chemically modified nucleic acids have suggested that modifications incorporated in furanose sugar especially at 2'-position attribute special properties to nucleic acids when compared to other modifications. 2'-O-methyl modification to deoxyribose sugars of DNA-RNA hybrids is one such modification that increases nucleic acid stability and has become an attractive class of compounds for potential antisense applications. It has been reported that modification of DNA strands with 2'-O-methyl group reverses the thermodynamic stability of DNA-RNA hybrid duplexes. Molecular dynamics simulations have been performed on two hybrid duplexes (DR and RD) which differ from each other and 2'-O-methyl modified counterparts to investigate the effect of 2'-O-methyl modification on their duplex stability. The results obtained suggest that the modification drives the conformations of both the hybrid duplexes towards A-RNA like conformation. The modified hybrid duplexes exhibit significantly contrasting dynamics and hydration patterns compared to respective parent duplexes. In line with the experimental results, the relative binding free energies suggest that the introduced modifications stabilize the less stable DR hybrid, but destabilize the more stable RD duplex. Binding free energy calculations suggest that the increased hydrophobicity is primarily responsible for the reversal of thermodynamic stability of hybrid duplexes. Free energy component analysis further provides insights into the stability of modified duplexes. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Isolation of high-quality total RNA from leaves of Myrciaria dubia "CAMU CAMU".

    Science.gov (United States)

    Gómez, Juan Carlos Castro; Reátegui, Alina Del Carmen Egoavil; Flores, Julián Torres; Saavedra, Roberson Ramírez; Ruiz, Marianela Cobos; Correa, Sixto Alfredo Imán

    2013-01-01

    Myrciaria dubia is a main source of vitamin C for people in the Amazon region. Molecular studies of M. dubia require high-quality total RNA from different tissues. So far, no protocols have been reported for total RNA isolation from leaves of this species. The objective of this research was to develop protocols for extracting high-quality total RNA from leaves of M. dubia. Total RNA was purified following two modified protocols developed for leaves of other species (by Zeng and Yang, and by Reid et al.) and one modified protocol developed for fruits of the studied species (by Silva). Quantity and quality of purified total RNA were assessed by spectrophotometric and electrophoretic analysis. Additionally, quality of total RNA was evaluated with reverse-transcription polymerase chain reaction (RT-PCR). With these three modified protocols we were able to isolate high-quality RNA (A260nm/A280nm >1.9 and A260nm/A230nm >2.0). Highest yield was produced with the Zeng and Yang modified protocol (384±46µg ARN/g fresh weight). Furthermore, electrophoretic analysis showed the integrity of isolated RNA and the absence of DNA. Another proof of the high quality of our purified RNA was the successful cDNA synthesis and amplification of a segment of the M. dubia actin 1 gene. We report three modified protocols for isolation total RNA from leaves of M. dubia. The modified protocols are easy, rapid, low in cost, and effective for high-quality and quantity total RNA isolation suitable for cDNA synthesis and polymerase chain reaction.

  20. Adhesion mapping of chemically modified and poly(ethylene oxide)-grafted glass surfaces.

    Science.gov (United States)

    Jogikalmath, G; Stuart, J K; Pungor, A; Hlady, V

    1999-08-01

    Two-dimensional mapping of the adhesion pull-off forces was used to study the origin of surface heterogeneity in the grafted poly(ethylene oxide) (PEO) layer. The variance of the pull-off forces measured over the μm-sized regions after each chemical step of modifying glass surfaces was taken to be a measure of the surface chemical heterogeneity. The attachment of γ-glycidoxypropyltrimethoxy silane (GPS) to glass decreased the pull-off forces relative to the clean glass and made the surface more uniform. The subsequent hydrolysis of the terminal epoxide groups resulted in a larger surface heterogeneity which was modeled by two populations of the terminal hydroxyl groups, each with its own distribution of adhesion forces and force variance. The activation of the hydroxyls with carbonyldiimmidazole (CDI) healed the surface and lowered its adhesion, however, the force variance remained rather large. Finally, the grafting of the α,ω-diamino poly(ethyleneoxide) chains to the CDI-activated glass largely eliminated adhesion except at a few discrete regions. The adhesion on the PEO grafted layer followed the Poisson distribution of the pull-off forces. With the exception of the glass surface, a correlation between the water contact angles and the mean pull-off forces measured with the Si(3)N(4) tip surfaces was found for all modified glass surfaces.

  1. YibK is the 2'-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA(Leu) isoacceptors

    DEFF Research Database (Denmark)

    Benítez-Páez, Alfonso; Villarroya, Magda; Douthwaite, Stephen Roger

    2010-01-01

    to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2'-O-methylation at position 34 in both tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Loss of Yib...... of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N⁶-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.......Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most...

  2. Transfer RNA and human disease

    Directory of Open Access Journals (Sweden)

    Jamie A Abbott

    2014-06-01

    Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  3. Transfer RNA and human disease.

    Science.gov (United States)

    Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

    2014-01-01

    Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  4. Deposition of carbon nanotubes onto aramid fibers using as-received and chemically modified fibers

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Uicab, O. [Centro de Investigación Científica de Yucatán A.C., Unidad de Materiales, Calle 43 No.130, Col. Chuburna de Hidalgo, C.P. 97200 Mérida, Yucatán (Mexico); Avilés, F., E-mail: faviles@cicy.mx [Centro de Investigación Científica de Yucatán A.C., Unidad de Materiales, Calle 43 No.130, Col. Chuburna de Hidalgo, C.P. 97200 Mérida, Yucatán (Mexico); Gonzalez-Chi, P.I; Canché-Escamilla, G.; Duarte-Aranda, S. [Centro de Investigación Científica de Yucatán A.C., Unidad de Materiales, Calle 43 No.130, Col. Chuburna de Hidalgo, C.P. 97200 Mérida, Yucatán (Mexico); Yazdani-Pedram, M. [Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, S. Livingstone 1007, Independencia, Santiago (Chile); Toro, P. [Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Av. Beauchef 850, Santiago (Chile); Gamboa, F. [Centro de Investigacion y de Estudios Avanzados del IPN, Unidad Mérida, Depto. de Física Aplicada, Km. 6 Antigua Carretera a Progreso, 97310 Mérida, Yucatán (Mexico); Mazo, M.A.; Nistal, A.; Rubio, J. [Instituto de Cerámica y Vidrio (ICV-CSIC), Kelsen 5, 28049 Madrid (Spain)

    2016-11-01

    Highlights: • The surface of aramid fibers was functionalized by two acid treatments. • The treatment based on HNO{sub 3}/H{sub 2}SO{sub 4} reduced the mechanical properties of the fibers. • CNTs were deposited on the aramid fibers, reaching electrical conductivity. • Homogeneous CNT distribution was achieved by using pristine fibers or chlorosulfonic acid. - Abstract: Multiwall carbon nanotubes (MWCNTs) oxidized by an acid treatment were deposited on the surface of as-received commercial aramid fibers containing a surface coating (“sizing”), and fibers modified by either a chlorosulfonic treatment or a mixture of nitric and sulfuric acids. The surface of the aramid fiber activated by the chemical treatments presents increasing density of CO, COOH and OH functional groups. However, these chemical treatments reduced the tensile mechanical properties of the fibers, especially when the nitric and sulfuric acid mixture was used. Characterization of the MWCNTs deposited on the fiber surface was conducted by scanning electron microscopy, Raman spectroscopy mapping and X-ray photoelectron spectroscopy. These characterizations showed higher areal concentration and more homogeneous distribution of MWCNTs over the aramid fibers for as-received fibers and for those modified with chlorosulfonic acid, suggesting the existence of interaction between the oxidized MWCNTs and the fiber coating. The electrical resistance of the MWCNT-modified aramid yarns comprising ∼1000 individual fibers was in the order of MΩ/cm, which renders multifunctional properties.

  5. Surface characterization and free thyroid hormones response of chemically modified poly(ethylene terephthalate) blood collection tubes

    Science.gov (United States)

    Jalali Dil, Ebrahim; Kim, Samuel C.; Saffar, Amir; Ajji, Abdellah; Zare, Richard N.; Sattayapiwat, Annie; Esguerra, Vanessa; Bowen, Raffick A. R.

    2018-06-01

    The surface chemistry and surface energy of chemically modified polyethylene terephthalate (PET) blood collection tubes (BCTs) were studied and the results showed a significant increase in hydrophilicity and polarity of modified PET surface. The surface modification created nanometer-sized, needle-like asperities through molecular segregation at the surface. The surface dynamics of the modified PET was examined by tracking its surface properties over a 280-day period. The results showed surface rearrangement toward a surface with lower surface energy and fewer nanometer-sized asperities. Thromboelastography (TEG) was used to evaluate and compare the thrombogenicity of the inner walls of various types of BCTs. The TEG tracings and data from various types of BCTs demonstrated differences in the reactionand coagulation times but not in clot strength. The performance of the modified tubes in free triiodothyronine (FT3) and free thyroxine (FT4) hormone tests was examined, and it was found that the interference of modified PET tubes was negligible compared to that of commercially available PET BCTs.

  6. [Satellite RNA (RNA3) of tomato black ring virus is found with one of the 2 major RNAs (RNA2) in a new capsid nucleoprotein].

    Science.gov (United States)

    Doz, B; Dunez, J; Bove, J M

    1977-12-19

    Tomato Black Ring Virus (TBRV) like other NEPOviruses posseses two nucleoproteins M and B and two major RNAs, RNA1 and RNA2 respectively distributed in B and M. A new nucleoprotein has just been discovered and comprises one molecule of RNA2 associated with one molecule of RNA3. RNA3 is a small RNA of molecular weight 500,000 d considered to be a satellite RNA. Its level appears to depend on the infection stage, local or systemic. RNA3 is able to modify the relative proportions of nucleoproteins M and B and their respective RNAs. The satellite RNA, might be part of the genome and represent a monocistronic mRNA for protein capsid synthesis. However it seems perhaps more tempting to correlate TBRV-RNA3 with satellite RNA5 of certain strains of Cucumber mosaic virus.

  7. Removal of heavy metals from aqueous phases using chemically modified waste Lyocell fiber

    Energy Technology Data Exchange (ETDEWEB)

    Bediako, John Kwame; Wei, Wei; Kim, Sok; Yun, Yeoung-Sang, E-mail: ysyun@jbnu.ac.kr

    2015-12-15

    Highlights: • Waste Lyocell fiber was chemically modified into cellulose xanthate. • The sorbent showed high affinity for Pb(II), Cd(II) and Cu(II) ions. • The sorbent also showed strong Cu(II) selectivity in Pb(II)–Cd(II)–Cu(II) ternary metal solutions. - Abstract: In this study, an outstanding performance of chemically modified waste Lyocell for heavy metals treatment is reported. The sorbent, which was prepared by a simple and concise method, was able to bind heavy metals such as Pb(II), Cu(II) and Cd(II), with very high efficiencies. The binding mechanisms were studied through adsorption and standard characterization tests such as scanning electron microscopy, energy-dispersive spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analyses. Adsorption kinetics was very fast and attained equilibrium within 5 min in all metals studied. The maximum single metal uptakes were 531.29 ± 0.28 mg/g, 505.64 ± 0.21 mg/g, and 123.08 ± 0.26 mg/g for Pb(II), Cd(II) and Cu(II), respectively. In ternary metal systems, Cu(II) selectivity was observed and the underlying factors were discussed. The sorbent by its nature, could be very effective in treating large volumes of wastewater with the contact of very little amount.

  8. Pharmacomodulation of microRNA Expression in Neurocognitive Diseases: Obstacles and Future Opportunities.

    Science.gov (United States)

    Simion, Viorel; Nadim, Wissem Deraredj; Benedetti, Helene; Pichon, Chantal; Morisset-Lopez, Severine; Baril, Patrick

    2017-01-01

    Given the importance of microRNAs (miRNAs) in modulating brain functions and their implications in neurocognitive disorders there are currently significant efforts devoted in the field of miRNA-based therapeutics to correct and/or to treat these brain diseases. The observation that miRNA 29a/b-1 cluster, miRNA 10b and miRNA 7, for instance, are frequently deregulated in the brains of patients with neurocognitive diseases and in animal models of Alzheimer, Huntington's and Parkinson's diseases, suggest that correction of miRNA expression using agonist or antagonist miRNA oligonucleotides might be a promising approach to correct or even to cure such diseases. The encouraging results from recent clinical trials allow envisioning that pharmacological approaches based on miRNAs might, in a near future, reach the requirements for successful therapeutic outcomes and will improve the healthcare of patients with brain injuries or disorders. This review will focus on the current strategies used to modulate pharmacological function of miRNA using chemically modified oligonucleotides. We will then review the recent literature on strategies to improve nucleic acid delivery across the blood-brain barrier which remains a severe obstacle to the widespread application of miRNA therapeutics to treat brain diseases. Finally, we provide a state-of-art of current preclinical research performed in animal models for the treatment of neurocognitive disorders using miRNA as therapeutic agents and discuss future developments of miRNA therapeutics.

  9. Double-Layer Structured CO2 Adsorbent Functionalized with Modified Polyethyleneimine for High Physical and Chemical Stability.

    Science.gov (United States)

    Jeon, Sunbin; Jung, Hyunchul; Kim, Sung Hyun; Lee, Ki Bong

    2018-06-18

    CO 2 capture using polyethyleneimine (PEI)-impregnated silica adsorbents has been receiving a lot of attention. However, the absence of physical stability (evaporation and leaching of amine) and chemical stability (urea formation) of the PEI-impregnated silica adsorbent has been generally established. Therefore, in this study, a double-layer impregnated structure, developed using modified PEI, is newly proposed to enhance the physical and chemical stabilities of the adsorbent. Epoxy-modified PEI and diepoxide-cross-linked PEI were impregnated via a dry impregnation method in the first and second layers, respectively. The physical stability of the double-layer structured adsorbent was noticeably enhanced when compared to the conventional adsorbents with a single layer. In addition to the enhanced physical stability, the result of simulated temperature swing adsorption cycles revealed that the double-layer structured adsorbent presented a high potential working capacity (3.5 mmol/g) and less urea formation under CO 2 -rich regeneration conditions. The enhanced physical and chemical stabilities as well as the high CO 2 working capacity of the double-layer structured adsorbent were mainly attributed to the second layer consisting of diepoxide-cross-linked PEI.

  10. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

    Directory of Open Access Journals (Sweden)

    Isabel X. Wang

    2014-03-01

    Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

  11. Utilization of chemically modified citrus reticulata peels for biosorptive removal of acid yellow-73 dye from water

    International Nuclear Information System (INIS)

    Rehman, R.; Salman, M.; Mahmud, T.; Kanwal, F.; Zaman, W.

    2013-01-01

    Textile effluents contain several varieties of natural and synthetic dyes, which are non-biodegradable. Acid Yellow-73 is one of them. In this research work, adsorptive removal of this dye was investigated using chemically modified Citrus reticulata peels, in batch mode. It was noted that adsorption of dye on Citrus reticulata peels increased by increasing contact time and decreased in basic pH conditions. Langmuir and Freundlich isothermal models were followed by equilibrium data, but the first isotherm fitted the data better, showing that chemisorption occurred more as compared to physiosorption, showing maximum adsorption capacity 96.46 mg.g-1.L-1. The thermodynamic study showed that adsorption of Acid Yellow-73 on chemically modified Citrus reticulata peels was favorable in nature, following pseudo-second order kinetics. (author)

  12. A Study on Adsorption of Cadmium by Using Chemically Modified Salvinia

    International Nuclear Information System (INIS)

    Anies Suhaida Mohd Naspu; Anies Suhaida Mohd Naspu; Mohd Lias Kamal; Zaini Hamzah; Sharizal Hasan

    2014-01-01

    Salvinia was collected at the Tasik Melati, Perlis, Malaysia and it was modified using citric acid to increase the performance in removing heavy metals from standard heavy metal solution. Fourier Transform Infrared (FTIR) and Field Emission Scanning Electron Microscope (FESEM) were used to characterize Salvinia before and after chemical modification. Cadmium (Cd 2+ ) removal studies were carried out by using modified Salvinia and the effect of various parameters such as contact time, initial heavy metal concentration and biosorbent dosage were studied. The adsorption study was investigated by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to determine the removing of heavy metal concentration from the standard Cd 2+ solution. From the study, FTIR analysis was shown the increasing of intensity on hydroxyl group after modification. A clear and smooth surface morphology of Salvinia after treatment were observed by using FESEM. The adsorption of cadmium was increased as contact time and biosorbent dosage increased. In contrast, the percent of adsorption was slightly decreased when initial concentration of Cd 2+ increased. (author)

  13. Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.

    Science.gov (United States)

    Barragán-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D; Megat, Salim; Burton, Michael D; Price, Theodore J; Campbell, Zachary T

    2018-01-02

    Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

  14. Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.

    Science.gov (United States)

    Judge, Adam D; Robbins, Marjorie; Tavakoli, Iran; Levi, Jasna; Hu, Lina; Fronda, Anna; Ambegia, Ellen; McClintock, Kevin; MacLachlan, Ian

    2009-03-01

    siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

  15. Effective gene silencing activity of prodrug-type 2'-O-methyldithiomethyl siRNA compared with non-prodrug-type 2'-O-methyl siRNA.

    Science.gov (United States)

    Hayashi, Junsuke; Nishigaki, Misa; Ochi, Yosuke; Wada, Shun-Ichi; Wada, Fumito; Nakagawa, Osamu; Obika, Satoshi; Harada-Shiba, Mariko; Urata, Hidehito

    2018-07-01

    Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2'-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2'-O-MDTM siRNAs modified at the 5'-end side including 5'-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2'-O-methyl (2'-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2'-O-MDTM modifications. Our results indicate that 2'-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. A new method to study the change of miRNA-mRNA interactions due to environmental exposures.

    Science.gov (United States)

    Petralia, Francesca; Aushev, Vasily N; Gopalakrishnan, Kalpana; Kappil, Maya; W Khin, Nyan; Chen, Jia; Teitelbaum, Susan L; Wang, Pei

    2017-07-15

    Integrative approaches characterizing the interactions among different types of biological molecules have been demonstrated to be useful for revealing informative biological mechanisms. One such example is the interaction between microRNA (miRNA) and messenger RNA (mRNA), whose deregulation may be sensitive to environmental insult leading to altered phenotypes. The goal of this work is to develop an effective data integration method to characterize deregulation between miRNA and mRNA due to environmental toxicant exposures. We will use data from an animal experiment designed to investigate the effect of low-dose environmental chemical exposure on normal mammary gland development in rats to motivate and evaluate the proposed method. We propose a new network approach-integrative Joint Random Forest (iJRF), which characterizes the regulatory system between miRNAs and mRNAs using a network model. iJRF is designed to work under the high-dimension low-sample-size regime, and can borrow information across different treatment conditions to achieve more accurate network inference. It also effectively takes into account prior information of miRNA-mRNA regulatory relationships from existing databases. When iJRF is applied to the data from the environmental chemical exposure study, we detected a few important miRNAs that regulated a large number of mRNAs in the control group but not in the exposed groups, suggesting the disruption of miRNA activity due to chemical exposure. Effects of chemical exposure on two affected miRNAs were further validated using breast cancer human cell lines. R package iJRF is available at CRAN. pei.wang@mssm.edu or susan.teitelbaum@mssm.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  17. Effect of chemical heterogeneity on photoluminescence of graphite oxide treated with S-/N-containing modifiers

    Science.gov (United States)

    Ebrahim, Amani M.; Rodríguez-Castellón, Enrique; Montenegro, José María; Bandosz, Teresa J.

    2015-03-01

    Graphite oxide (GO) obtained using Hummers method was modified by hydrothermal treatment either with sulfanilic acid or polystyrene (3-ammonium) sulfonate at 100 °C or 85 °C, respectively. Both modifiers contain sulfur in the oxidized forms and nitrogen in the reduced forms. The materials were characterized using FTIR, XPS, thermal analysis, potentiometric titration and SEM. Their photoluminescent properties and their alteration with an addition of Ag+ were also measured. As a result of these modifications nitrogen was introduced to the graphene layers as amines, imides, amides, and sulfur as sulfones and sulfonic acids. Moreover, the presence of polyaniline was detected. This significantly affected the polarity, acid-base character, and conductivity of the materials. Apparently carboxylic groups of GO were involved in the surface reactions. The modified GOs lost their layered structure and the modifications resulted in the high degree of structural and chemical heterogeneity. Photoluminescence in visible light was recorded and linked to the presence of heteroatoms. For the polystyrene (3-ammonium) sulfonate modified sample addition of Ag+ quenched the photoluminescence at low wavelength showing sensitivity as a possible optical detector. No apparent effect was found for the sulfanilic acid modified sample.

  18. Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra.

    Directory of Open Access Journals (Sweden)

    Alison L McCormack

    Full Text Available The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left vs. untreated (right hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i the number and phenotype of nigral dopaminergic neurons, and (ii the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

  19. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

    Science.gov (United States)

    Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-05-01

    RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

  20. Studies on physico-chemical and mechanical properties of the irradiated latex modified mortar

    International Nuclear Information System (INIS)

    Yassene, A.A.M.A.

    2009-01-01

    This thesis contains three chapter; chapter(I): Introduction and literature review on:- Introduction to polymer. - Mechanism of polymer-cement co-matrix formation.-Sulphate attack. - Solidification /stabilization of heavy metal in cement mortar. chapter(II): Materials and experimental techniques that include: 1- Preparation of latex polymer films from different polymer latices of styrene butadine rubber latex (SBR), poly (styrene-acrylic ester) latex (SAE) and vinylacetate /versatic -ester copolymer latex (C2A). The effect of γ-irradiation dose on the physico - chemical and mechanical properties of different latex polymer films was studied.2- Preparation of latex polymer-modified cement mortar with different ratios of cement: latex polymer and different curing method.3- Solidification /stabilization (S/S) of electroplating heavy metal precipitate in latex polymer- modified mortar with different cement /electroplating heavy metal sludge ratio. chapter(III) results and discussion

  1. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG) transcripts

    Science.gov (United States)

    Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J.; Childs-Disney, Jessica; Sobczak, Krzysztof; Disney, Matthew D.

    2012-01-01

    Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)exp) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5’CAG/3’GAC motif found in r(CAG)exp hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)exp. The compound was identified by first studying the binding of RNA 1×1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5’CAG/3’GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate as small molecule capable of improving pre-mRNA splicing defects associated with the r(CAG)exp-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)exp and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)exp toxicity. The approach used in these studies, defining the small RNA motifs that bind known nucleic acid binders and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in human genomic sequence. PMID:22252896

  2. A Mechanistic Study of Chemically Modified Inorganic Membranes for Gas and Liquid Separations

    Energy Technology Data Exchange (ETDEWEB)

    Way, J. Douglas [Colorado School of Mines, Golden, CO (United States). Dept. of Chemical Engineering

    2011-01-21

    This final report will summarize the progress made during the period August 1, 1993 - October 31, 2010 with support from DOE grant number DE-FG03-93ER14363. The objectives of the research have been to investigate the transport mechanisms in micro- and mesoporous, metal oxide membranes and to examine the relationship between the microstructure of the membrane, the membrane surface chemistry, and the separation performance of the membrane. Examples of the membrane materials under investigation are the microporous silica hollow fiber membrane manufactured by PPG Industries, chemically modified mesoporous oxide membranes, and polymer membranes containing microporous oxides (mixed matrix membranes). Analytical techniques such as NMR, FTIR and Raman spectroscopy, thermal analysis, and gas adsorption were used to investigate membrane microstructure and to probe the chemical interactions occurring at the gas-membrane interface.

  3. MD SIMULATION STUDIES TO INVESTIGATE ISO-ENERGETIC CONFORMATIONAL BEHAVIOUR OF MODIFIED NUCLEOSIDES M2G AND M22G PRESENT IN tRNA

    Directory of Open Access Journals (Sweden)

    Rohit S Bavi

    2013-02-01

    Full Text Available Modified nucleic acid bases are most commonly found in tRNA. These may contain modifications from simple methylation to addition of bulky groups. Methylation of the four canonical nucleotide bases at a wide variety of positions is particularly prominent among the known modification. Methylation of N2 group of guanine is a relatively common modification in tRNA and rRNA. N2-methylguanosine (m2G is the second most often encountered nucleoside in E. coli tRNAs. N2, N2-dimethylguanosine (m22G is found in the majority of eukaryotic tRNAs and involved in forming base pair interactions with adjacent bases. Hence, in order to understand the structural significance of these methylated nucleic acid bases we have carried out molecular dynamics simulation to see the salvation effect. The results obtained shows iso-energetic conformational behaviors for m2G and m22G. The simulation trajectory of m2G shows regular periodical fluctuations suggesting that m2G is equally stable as either s-cis or s-trans rotamers. The two rotamers of m2G may interact canonically or non-canonically with opposite base as s-trans m2G26:C/A/U44 and s-cis m2G26:A/U44. The free rotations around the C-N bond could be the possible reason for these iso-energetic conformations. Dimethylation of G has almost no influence on base pairing with either A or U. Thus, these results reveal that modified nucleosides m2G and m22G may play an important role to prevent tRNA from adopting the unusual mitochondrial like conformation.

  4. Prediction of proton chemical shifts in RNA - Their use in structure refinement and validation

    International Nuclear Information System (INIS)

    Cromsigt, Jenny A.M.T.C.; Hilbers, Cees W.; Wijmenga, Sybren S.

    2001-01-01

    An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct.,76, 81-92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341-346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable analysis could be made of the structural dependencies of the 1 H chemical shifts revealing their physical origin. For example, a down-field shift of either H2' or H3' or both indicates a high-syn/syn χ-angle. In an A-helix it is essentially the 5'-neighbor that affects the chemical shifts of H5, H6 and H8 protons. The H5, H6 and H8 resonances can therefore be assigned in an A-helix on the basis of their observed chemical shifts. In general, the chemical shifts were found to be quite sensitive to structural changes. We therefore propose that a comparison between calculated and observed 1 H chemical shifts is a good tool for validation and refinement of structures derived from NOEs and J-couplings

  5. Physico-Chemical Properties and Biodegradability of Genetically Modified Populus trichocarpa and Pinus taeda

    Science.gov (United States)

    Edmunds, Charles Warren

    Increasing concerns over greenhouse gas emissions and the finite supply of fossil fuels lead to the goal of utilizing lignocellulosic feedstocks for biofuels, platform chemicals, and biocomposites. Lignin is responsible for the recalcitrance of lignocellulosic biomass and is a major barrier to its deconstruction. Great progress has been made in mapping and modifying the lignin biosynthetic pathway. However, the link between the genetic modification, resulting chemical and physical properties of the wood, and how these properties influence the thermomechanical and recalcitrance to biological and chemical degradation needs further investigation. In this dissertation, the study of modified Populus trichocarpa and Pinus taeda were utilized to accomplish this goal. Thermo-mechanical properties of genetically modified P. trichocarpa with altered lignin content and/or lignin structure were measured with a series of tools including; dynamic mechanical analysis, nuclear magnetic resonance, and wet chemistry techniques. Results demonstrated lignin content and lignin structure likely influence the glass transition temperature (Tg), and that decreased lignin content and the corresponding higher proportion of cell wall carbohydrates may contribute to increased molecular mobility in the wood polymer structure. The effect of lignin biosynthetic pathway modification on biological degradation of these transgenic wood specimens was of interest. However, experimental methods for fungal treatment on small young greenhouse-grown wood specimens are not well established. Therefore, a project was undertaken to develop a method for fungal inoculation and incubation for these unique specimens. Several parameters were tested, and a fungal treatment method was identified with sufficient weight loss after decay and significant reduction in variation of weight loss between replicates compared to previous experiments by direct inoculation of wood with liquid malt extract fungal culture

  6. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    International Nuclear Information System (INIS)

    Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin

    2006-01-01

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations

  7. Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

    Science.gov (United States)

    Park, Kiyun; Kwak, Inn-Sil

    2012-12-01

    The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

  8. Conjugation chemistry through acetals toward a dextran-based delivery system for controlled release of siRNA

    KAUST Repository

    Cui, Lina

    2012-09-26

    New conjugation chemistry for polysaccharides, exemplified by dextran, was developed to enable the attachment of therapeutic or other functional moieties to the polysaccharide through cleavable acetal linkages. The acid-lability of the acetal groups allows the release of therapeutics under acidic conditions, such as that of the endocytic compartments of cells, regenerating the original free polysaccharide in the end. The physical and chemical behavior of these acetal groups can be adjusted by modifying their stereoelectronic and steric properties, thereby providing materials with tunable degradation and release rates. We have applied this conjugation chemistry in the development of water-soluble siRNA carriers, namely acetal-linked amino-dextrans, with various amine structures attached through either slow- or fast-degrading acetal linker. The carriers with the best combination of amine moieties and structural composition of acetals showed high in vitro transfection efficiency and low cytotoxicity in the delivery of siRNA. © 2012 American Chemical Society.

  9. Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Frauke Hausburg

    2015-02-01

    Full Text Available Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC and pseudouridine-5'-triphosphate (Ψ. We then investigated their effect on i protein expression efficiencies and ii cell viability for human mesenchymal stem cells (hMSCs and fibroblasts from different origins. Results: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. Conclusion: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

  10. Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; Ambegia, Ellen; Choi, Catherine; Yaworski, Ed; Palmer, Lorne; McClintock, Kevin; MacLachlan, Ian

    2008-10-01

    Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

  11. Facile Synthesis of Mono-Dispersed Polystyrene (PS/Ag Composite Microspheres via Modified Chemical Reduction

    Directory of Open Access Journals (Sweden)

    Wen Zhu

    2013-12-01

    Full Text Available A modified method based on in situ chemical reduction was developed to prepare mono-dispersed polystyrene/silver (PS/Ag composite microspheres. In this approach; mono-dispersed PS microspheres were synthesized through dispersion polymerization using poly-vinylpyrrolidone (PVP as a dispersant at first. Then, poly-dopamine (PDA was fabricated to functionally modify the surfaces of PS microspheres. With the addition of [Ag(NH32]+ to the PS dispersion, [Ag(NH32]+ complex ions were absorbed and reduced to silver nanoparticles on the surfaces of PS-PDA microspheres to form PS/Ag composite microspheres. PVP acted both as a solvent of the metallic precursor and as a reducing agent. PDA also acted both as a chemical protocol to immobilize the silver nanoparticles at the PS surface and as a reducing agent. Therefore, no additional reducing agents were needed. The resulting composite microspheres were characterized by TEM, field emission scanning electron microscopy (FESEM, energy-dispersive X-ray spectroscopy (EDS, XRD, UV-Vis and surface-enhanced Raman spectroscopy (SERS. The results showed that Ag nanoparticles (NPs were homogeneously immobilized onto the PS microspheres’ surface in the presence of PDA and PVP. PS/Ag composite microspheres were well formed with a uniform and compact shell layer and were adjustable in terms of their optical property.

  12. Direct determination of cadmium in Orujo spirit samples by electrothermal atomic absorption spectrometry: Comparative study of different chemical modifiers

    International Nuclear Information System (INIS)

    Vilar Farinas, M.; Barciela Garcia, J.; Garcia Martin, S.; Pena Crecente, R.; Herrero Latorre, C.

    2007-01-01

    In this work, several analytical methods are proposed for cadmium determination in Orujo spirit samples using electrothermal atomic absorption spectrometry (ETAAS). Permanent chemical modifiers thermally coated on the platforms inserted in pyrolytic graphite tubes (such as W, Ir, Ru, W-Ir and W-Ru) were comparatively studied in relation to common chemical modifier mixtures [Pd-Mg(NO 3 ) 2 and (NH 4 )H 2 PO 4 -Mg(NO 3 ) 2 ] for cadmium stabilization. Different ETAAS Cd determination methods based on the indicated modifiers have been developed. In each case, pyrolysis and atomization temperatures, atomization shapes, characteristic masses and detection limits as well as other analytical characteristics have been determined. All the assayed modifiers (permanent and conventional) were capable of achieving the appropriate stabilization of the analyte, with the exception of Ru and W-Ru. Moreover, for all developed methods, recoveries (99-102%) and precision (R.S.D. lower than 10%) were acceptable. Taking into account the analytical performance (best detection limit LOD = 0.01 μg L -1 ), the ETAAS method based on the use of W as a permanent modifier was selected for further direct Cd determinations in Orujo samples from Galicia (NW Spain). The chosen method was applied in the determination of the Cd content in 38 representative Galician samples. The cadmium concentrations ranged -1

  13. Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Xiangyu He

    Full Text Available The phenotypic manifestations of mitochondrial DNA (mtDNA mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R or P(R 454 mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R, the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S, mto2(P(S and MTO2(P(R. The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

  14. Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions.

    NARCIS (Netherlands)

    Nolte-'t Hoen, E.N.M.; Buermans, H.P.; Waasdorp, M.; Stoorvogel, W.; Wauben, M.H.M.; `t Hoen, P.A.C.

    2012-01-01

    Cells release RNA-carrying vesicles and membrane-free RNA/protein complexes into the extracellular milieu. Horizontal vesicle-mediated transfer of such shuttle RNA between cells allows dissemination of genetically encoded messages, which may modify the function of target cells. Other studies used

  15. Methods and compositions for controlling gene expression by RNA processing

    Science.gov (United States)

    Doudna, Jennifer A.; Qi, Lei S.; Haurwitz, Rachel E.; Arkin, Adam P.

    2017-08-29

    The present disclosure provides nucleic acids encoding an RNA recognition sequence positioned proximal to an insertion site for the insertion of a sequence of interest; and host cells genetically modified with the nucleic acids. The present disclosure also provides methods of modifying the activity of a target RNA, and kits and compositions for carrying out the methods.

  16. Chemical modifiers in electrothermal atomic absorption determination of Platinum and Palladium containing preparations in blood serum

    Directory of Open Access Journals (Sweden)

    Аntonina Alemasova

    2012-11-01

    Full Text Available The biological liquids matrixes influence on the characteristic masses and repeatability of Pt and Pd electrothermal atomic absorption spectroscopy (ETAAS determination was studied. The chemical modifiers dimethylglyoxime and ascorbic acid for matrix interferences elimination and ETAAS results repeatability improvement were proposed while bioliquids ETAAS analysis, and their action mechanism was discussed.

  17. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

    Directory of Open Access Journals (Sweden)

    Alexey Berezhnoy

    2012-01-01

    Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

  18. Radiation modification of swollen and chemically modified cellulose

    International Nuclear Information System (INIS)

    Borsa, J.; Toth, T.

    2002-01-01

    Complete text of publication follows. Biodegradable hydrogel was produced by radiation-induced crosslinking of water soluble carboxymethyl cellulose. Mobility of the molecular chain was found to play an important role in the crosslinking reaction. In this work the role of cellulose chains' mobility in radiation-induced reactions of fibrous cellulose was studied. Mobility of chains was improved by swelling (in sodium hydroxide and tetramethylammonium hydroxide) and chemical modification (substitution of about 3 % of hydroxyl groups with carboxymethyl groups), respectively. All samples were neutralized after the treatments. Accessibility of cellulose characterized by water adsorption and retention was significantly improved by the treatments in the following order: sodium hydroxide < tetramethylammonium hydroxide < carboxymethylation. Less fibrillar structure of modified fibers was observed by electron microscope. Samples were irradiated in wet form in open air (10 kGy). Untreated sample coated with soluble CMC was also irradiated. Degree of polymerization, FTIR spectra, and water sorption of samples before and after irradiation are presented. Amount of water adsorbed on samples decreased after irradiation. It can be considered the consequence of crosslinks, which might improve the crease recovery ability of cotton fabric. High accessibility improved degradation rather than crosslinking of cellulose chains

  19. A molecular theory of chemically modified electrodes with self-assembled redox polyelectrolye thin films: Reversible cyclic voltammetry

    Energy Technology Data Exchange (ETDEWEB)

    Tagliazucchi, Mario; Calvo, Ernesto J. [INQUIMAE, DQIAyQF Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires (Argentina); Szleifer, Igal [Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208 (United States)

    2008-10-01

    A molecular theory of chemically modified electrodes is applied to study redox polyelectroyte modified electrodes. The molecular approach explicitly includes the size, shape, charge distribution, and conformations of all of the molecular species in the system as well as the chemical equilibria (redox and acid-base) and intermolecular interactions. An osmium pyridine-bipyridine complex covalently bound to poly(allyl-amine) backbone (PAH-Os) adsorbed onto mercapto-propane sulfonate (MPS) thiolated gold electrode is described. The potential and electrolyte composition dependent redox and nonredox capacitance can be calculated with the molecular theory in very good agreement with voltammetric experiments under reversible conditions without the use of freely adjustable parameter. Unlike existing phenomenological models the theory links the electrochemical behavior with the structure of the polymer layer. The theory predicts a highly inhomogeneous distribution of acid-base and redox states that strongly couples with the spatial arrangement of the molecular species in the nanometric redox film. (author)

  20. Spectroelectrochemical detection of microRNA-155 based on functional RNA immobilization onto ITO/GNP nanopattern.

    Science.gov (United States)

    Mohammadniaei, Mohsen; Yoon, Jinho; Lee, Taek; Choi, Jeong-Woo

    2018-05-20

    We fabricated a microRNA biosensor using the combination of surface enhanced Raman spectroscopy (SERS) and electrochemical (EC) techniques. For the first time, the weaknesses of each techniques for microRNA detection was compensated by the other ones to give rise to the specific and wide-range detection of miR-155. A single stranded 3' methylene blue (MB) and 5' thiol-modified RNA (MB-ssRNA-SH) was designed to detect the target miR-155 and immobilized onto the gold nanoparticle-modified ITO (ITO/GNP). Upon the invasion of target strand, the double-stranded RNA transformed rapidly to an upright structure resulting in a notable decrease in SERS and redox signals of the MB. For the first time, by combination of SERS and EC techniques in a single platform we extended the dynamic range of both techniques from 10 pM to 450 nM (SERS: 10 pM-5 nM and EC: 5 nM-450 nM). As well, the SERS technique improved the detection limit of the EC method from 100 pM to 100 fM, while the EC method covered single-mismatch detection which was the SERS deficiency. The fabricated single-step biosensor possessing a good capability of miRNA detection in human serum, could be employed throughout the broad ranges of biomedical and bioelectronics applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Distribution of phosphorus and hydroxypropyl groups within granules of modified sweet potato starches as determined after chemical peeling

    NARCIS (Netherlands)

    Zhao, J.; Schols, H.A.; Chen Zenghong,; Jin, Z.; Buwalda, P.; Gruppen, H.

    2015-01-01

    The distributions of phosphorus and hydroxypropyl groups within granules of cross-linked and hydroxypropylated sweet potato starches were investigated. Chemical surface peeling of starch granules was performed after sieving of native and modified starches into large-size (diameter = 20 µm) and

  2. Electrothermal atomic absorption spectrometric determination of copper in nickel-base alloys with various chemical modifiers*1

    Science.gov (United States)

    Tsai, Suh-Jen Jane; Shiue, Chia-Chann; Chang, Shiow-Ing

    1997-07-01

    The analytical characteristics of copper in nickel-base alloys have been investigated with electrothermal atomic absorption spectrometry. Deuterium background correction was employed. The effects of various chemical modifiers on the analysis of copper were investigated. Organic modifiers which included 2-(5-bromo-2-pyridylazo)-5-(diethylamino-phenol) (Br-PADAP), ammonium citrate, 1-(2-pyridylazo)-naphthol, 4-(2-pyridylazo)resorcinol, ethylenediaminetetraacetic acid and Triton X-100 were studied. Inorganic modifiers palladium nitrate, magnesium nitrate, aluminum chloride, ammonium dihydrogen phosphate, hydrogen peroxide and potassium nitrate were also applied in this work. In addition, zirconium hydroxide and ammonium hydroxide precipitation methods have also been studied. Interference effects were effectively reduced with Br-PADAP modifier. Aqueous standards were used to construct the calibration curves. The detection limit was 1.9 pg. Standard reference materials of nickel-base alloys were used to evaluate the accuracy of the proposed method. The copper contents determined with the proposed method agreed closely with the certified values of the reference materials. The recoveries were within the range 90-100% with relative standard deviation of less than 10%. Good precision was obtained.

  3. Influence of pyrolysis temperature on lead immobilization by chemically modified coconut fiber-derived biochars in aqueous environments.

    Science.gov (United States)

    Wu, Weidong; Li, Jianhong; Niazi, Nabeel Khan; Müller, Karin; Chu, Yingchao; Zhang, Lingling; Yuan, Guodong; Lu, Kouping; Song, Zhaoliang; Wang, Hailong

    2016-11-01

    Biochar has received widespread attention as an eco-friendly and efficient material for immobilization of toxic heavy metals in aqueous environments. In the present study, three types of coconut fiber-derived biochars were obtained by pyrolyzing at three temperatures, i.e., 300, 500, and 700 °C. In addition, nine types of biochars were prepared by chemical modification with ammonia, hydrogen peroxide, and nitric acid, respectively, which were used to investigate changes in physico-chemical properties by inter alia, Fourier transformation infrared spectrophotometry (FTIR), scanning electron microscope (SEM), and BET specific surface area analysis. Batch sorption experiments were carried out to determine the sorption capacity of the biochars for lead (Pb) in aqueous solutions. Results showed that the cation exchange capacity of biochar pyrolyzed at 300 °C and modified with nitric acid increased threefold compared to the control. Loosely corrugated carbon surface and uneven carbon surface of the biochar pyrolyzed at 300 °C were produced during ammonia and nitric acid modifications. Removal rate of Pb by the coconut biochar pyrolyzed at 300 °C and modified with ammonia was increased from 71.8 to 99.6 % compared to the untreated biochar in aqueous solutions containing 100 mg L -1 Pb. However, chemical modification did not enhance adsorption of Pb of the biochars pyrolyzed at higher temperatures (e.g., 500 or 700 °C), indicating that resistance of biochars to chemical treatment increased with pyrolysis temperature.

  4. RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

    Science.gov (United States)

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

    2013-01-01

    T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

  5. Direct determination of cadmium in Orujo spirit samples by electrothermal atomic absorption spectrometry: Comparative study of different chemical modifiers

    Energy Technology Data Exchange (ETDEWEB)

    Vilar Farinas, M. [Departamento de Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo (Spain); Barciela Garcia, J. [Departamento de Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo (Spain); Garcia Martin, S. [Departamento de Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo (Spain); Pena Crecente, R. [Departamento de Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo (Spain); Herrero Latorre, C. [Departamento de Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo (Spain)]. E-mail: cherrero@lugo.usc.es

    2007-05-22

    In this work, several analytical methods are proposed for cadmium determination in Orujo spirit samples using electrothermal atomic absorption spectrometry (ETAAS). Permanent chemical modifiers thermally coated on the platforms inserted in pyrolytic graphite tubes (such as W, Ir, Ru, W-Ir and W-Ru) were comparatively studied in relation to common chemical modifier mixtures [Pd-Mg(NO{sub 3}){sub 2} and (NH{sub 4})H{sub 2}PO{sub 4}-Mg(NO{sub 3}){sub 2}] for cadmium stabilization. Different ETAAS Cd determination methods based on the indicated modifiers have been developed. In each case, pyrolysis and atomization temperatures, atomization shapes, characteristic masses and detection limits as well as other analytical characteristics have been determined. All the assayed modifiers (permanent and conventional) were capable of achieving the appropriate stabilization of the analyte, with the exception of Ru and W-Ru. Moreover, for all developed methods, recoveries (99-102%) and precision (R.S.D. lower than 10%) were acceptable. Taking into account the analytical performance (best detection limit LOD = 0.01 {mu}g L{sup -1}), the ETAAS method based on the use of W as a permanent modifier was selected for further direct Cd determinations in Orujo samples from Galicia (NW Spain). The chosen method was applied in the determination of the Cd content in 38 representative Galician samples. The cadmium concentrations ranged

  6. Translation of satellite tobacco necrosis virus RNA modified by (not equal to)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is inhibited in a wheat germ cell-free system

    International Nuclear Information System (INIS)

    Haas, R.; Pulkrabek, P.; Takanami, Y.; Grunberger, D.

    1983-01-01

    It has been shown that (not equal to)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) modification of rabbit globin mRNA results in inhibition of translational initiation. In order to explore the possibility that modification of the 5' cap structure was responsible for this inhibition, the naturally non-capped mRNA from satellite tobacco necrosis virus (STNV) was reacted with BPDE and translated in a wheat germ cell-free system. The extent of modification was 1.3 and 2.9 BPDE residues/molecule. High performance liquid chromatography of the modified nucleosides from enzymatically hydrolyzed STNV RNA revealed that greater than 90% of the nucleoside adducts were substituted at the exocyclic amino group of guanosine. The translational ability of the lower and higher modified STNV, measured by incorporation of [ 14 C]amino acids into acid-precipitable polypeptides is inhibited by 55% and 63%, respectively. Polyacrylamide gel electrophoretic analyses of the translation products indicate that predominantly full-length coat proteins are synthesized but with the carcinogen-modified STNV the amount is reduced. On the other hand, 80S initiation complex formation is not inhibited as measured by binding of the BPDE-modified STNV to ribosomes and followed by glycerol gradient centrifugation. Under these conditions, aurintricarboxylic acid completely inhibits 80S initiation complex formation in the presence of either modified or native STNV. These results suggest that inhibition of in vitro translation of BPDE-modified STNV, in contrast to that of globin mRNA, is not at the level of initiation complex formation but possibly by premature termination of growing polypeptides

  7. Circularly polarized luminescence of helically assembled pyrene π-stacks on RNA and DNA duplexes.

    Science.gov (United States)

    Nakamura, Mitsunobu; Ota, Fuyuki; Takada, Tadao; Akagi, Kazuo; Yamana, Kazushige

    2018-05-01

    In this report, we describe the circularly polarized luminescence (CPL) of the RNA duplexes having one to four 2'-O-pyrene modified uridines (Upy) and the DNA duplexes having two, four, and six pyrene modified non-nucleosidic linkers (Py). Both the pyrene π-stack arrays formed on the RNA and DNA double helical structures exhibited pyrene excimer fluorescence. In the pyrene-modified RNA systems, the RNA duplex having four Upys gives CPL emission with g lum value of <0.01 at 480 nm. The structure of pyrene stacks on the RNA duplex may be rigidly regulated with increase in the Upy domains, which resulted in the CPL emission. In the DNA systems, the pyrene-modified duplexes containing two and four Pys exhibited CPL emission with g lum values of <0.001 at 505 nm. The pyrene π-stack arrays presented here show CPL emission. However, the g lum values are relatively small when compared with our previous system consisting of the pyrene-zipper arrays on RNA. © 2018 Wiley Periodicals, Inc.

  8. Pharmacological profiling of zebrafish behavior using chemical and genetic classification of sleep-wake modifiers.

    Science.gov (United States)

    Nishimura, Yuhei; Okabe, Shiko; Sasagawa, Shota; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2015-01-01

    Sleep-wake states are impaired in various neurological disorders. Impairment of sleep-wake states can be an early condition that exacerbates these disorders. Therefore, treating sleep-wake dysfunction may prevent or slow the development of these diseases. Although many gene products are likely to be involved in the sleep-wake disturbance, hypnotics and psychostimulants clinically used are limited in terms of their mode of action and are not without side effects. Therefore, there is a growing demand for developing new hypnotics and psychostimulants with high efficacy and few side effects. Toward this end, animal models are indispensable for use in genetic and chemical screens to identify sleep-wake modifiers. As a proof-of-concept study, we performed behavioral profiling of zebrafish treated with chemical and genetic sleep-wake modifiers. We were able to demonstrate that behavioral profiling of zebrafish treated with hypnotics or psychostimulants from 9 to 10 days post-fertilization was sufficient to identify drugs with specific modes of action. We were also able to identify behavioral endpoints distinguishing GABA-A modulators and hypocretin (hcrt) receptor antagonists and between sympathomimetic and non-sympathomimetic psychostimulants. This behavioral profiling can serve to identify genes related to sleep-wake disturbance associated with various neuropsychiatric diseases and novel therapeutic compounds for insomnia and excessive daytime sleep with fewer adverse side effects.

  9. Efficiency of modified chemical remediation techniques for soil contaminated by organochlorine pesticides

    Science.gov (United States)

    Correa-Torres, S. N.; Kopytko, M.; Avila, S.

    2016-07-01

    This study reports the optimization of innovation chemical techniques in order to improve the remediation of soils contaminated with organochloride pesticides. The techniques used for remediation were dehalogenation and chemical oxidation in soil contaminated by pesticides. These techniques were applied sequentially and combined to evaluate the design optimize the concentration and contact time variables. The soil of this study was collect in cotton crop zone in Agustin Codazzi municipality, Colombia, and its physical properties was measure. The modified dehalogenation technique of EPA was applied on the contaminated soil by adding Sodium Bicarbonate solution at different concentrations and rates during 4, 7 and 14 days, subsequently oxidation technique was implemented by applying a solution of KMnO4 at different concentration and reaction times. Organochlorine were detected by Gas Chromatography analysis coupled Mass Spectrometry and its removals were between 85.4- 90.0% of compounds such as 4, 4’-DDT, 4,4’-DDD, 4,4-DDE, trans-Clordane y Endrin. These results demonstrate that the technique of dehalogenation with oxidation chemistry can be used for remediation soils contaminated by organochloride pesticides.

  10. Chemically and compositionally modified solid solution disordered multiphase nickel hydroxide positive electrode for alkaline rechargeable electrochemical cells

    Science.gov (United States)

    Ovshinsky, Stanford R.; Corrigan, Dennis; Venkatesan, Srini; Young, Rosa; Fierro, Christian; Fetcenko, Michael A.

    1994-01-01

    A high capacity, long cycle life positive electrode for use in an alkaline rechargeable electrochemical cell comprising: a solid solution nickel hydroxide material having a multiphase structure that comprises at least one polycrystalline .gamma.-phase including a polycrystalline .gamma.-phase unit cell comprising spacedly disposed plates with at least one chemical modifier incorporated around the plates, the plates having a range of stable intersheet distances corresponding to a 2.sup.+ oxidation state and a 3.5.sup.+, or greater, oxidation state; and at least one compositional modifier incorporated into the solid solution nickel hydroxide material to promote the multiphase structure.

  11. Comparison of Escherichia coli tRNAPhe in the free state, in the ternary complex and in the ribosomal A and P sites by chemical probing

    DEFF Research Database (Denmark)

    Douthwaite, S; Garrett, R A; Wagner, R

    1983-01-01

    tRNAPheE.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679-4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex...

  12. Backbone and sidechain methyl Ile (δ1), Leu and Val chemical shift assignments of RDE-4 (1-243), an RNA interference initiation protein in C. elegans.

    Science.gov (United States)

    Chiliveri, Sai Chaitanya; Kumar, Sonu; Marelli, Udaya Kiran; Deshmukh, Mandar V

    2012-10-01

    The RNAi pathway of several organisms requires presence of double stranded RNA binding proteins for functioning of Dicer in gene regulation. In C. elegans, a double stranded RNA binding protein, RDE-4 (385 aa, 44 kDa) recognizes long exogenous dsRNA and initiates the RNAi pathway. We have achieved complete backbone and stereospecific methyl sidechain Ile (δ1), Leu and Val chemical shifts of first 243 amino acids of RDE-4, namely RDE-4ΔC.

  13. Transfecting Human Monocytes with RNA.

    Science.gov (United States)

    Dannull, Jens; Nair, Smita K

    2016-01-01

    Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

  14. Influence of citric acid as chemical modifier for lead determination in dietary calcium supplement samples by graphite furnace atomic absorption spectrometry

    International Nuclear Information System (INIS)

    Cezar Paz de Mattos, Julio; Medeiros Nunes, Adriane; Figueiredo Martins, Ayrton; Luiz Dressler, Valderi; Marlon de Moraes Flores, Erico

    2005-01-01

    Citric acid was used as a chemical modifier for Pb determination by graphite furnace atomic absorption spectrometry in dietary supplement samples (calcium carbonate, dolomite and oyster shell samples) and its efficiency was compared to the use of palladium. Pyrolysis and atomization curves were established without use of chemical modifier, with the addition of 20, 100 and 200 μg of citric acid, and with 3 μg of palladium. The citric acid modifier made possible the interference-free Pb determination in the presence of high concentrations of Ca and Mg nitrates. Acid sample digestion involving closed vessels (microwave-assisted and conventional heating) and acid attack using polypropylene vessels at room temperature were compared. All digestion procedures presented similar results for calcium carbonate and dolomite samples. However, for oyster shell samples accurate results were obtained only with the use of closed vessel systems. Analyte addition and matrix-matched standards were used for calibration. The characteristic mass for Pb using citric acid and palladium were 16 and 25 pg, respectively. The relative standard deviation (RSD) was always less than 5% when citric acid was used. The relative and absolute limits of detection were 0.02 μg g -1 and 8 pg with citric acid and 0.1 μg g -1 and 44 pg with the Pd modifier, respectively (n = 10, 3σ). The recovery of Pb in spiked calcium supplement samples (10 μg l -1 ) was between 98% and 105%. With the use of 100 μg of citric acid as chemical modifier, problems such as high background absorption and high RSD values were minimized in comparison to the addition of 3 μg of palladium

  15. Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization.

    Science.gov (United States)

    Forsgren, Eva; Locke, Barbara; Semberg, Emilia; Laugen, Ane T; Miranda, Joachim R de

    2017-08-01

    Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling-transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at -80°C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder™ homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15min. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-10-01

    Full Text Available We generated human induced pluripotent stem cells (KSCBi002-B and KSCBi002-B-1 from the dermal fibroblasts of a donor using a modified RNA-based gene delivery method. According to GTG-banding analysis, the generated KSCBi002-B line has a cytogenetic abnormality (46,XY, t(1;4(q21;q25 that is distinct from that of the donor, whereas KSCBi002-B-1 has a normal karyotype (46,XY. These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model. These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  17. The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

    Science.gov (United States)

    Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

    2011-02-01

    Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    Science.gov (United States)

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Hybridization Properties of RNA Containing 8-Methoxyguanosine and 8-Benzyloxyguanosine.

    Directory of Open Access Journals (Sweden)

    Daniel Sylwester Baranowski

    Full Text Available Modified nucleobase analogues can serve as powerful tools for changing physicochemical and biological properties of DNA or RNA. Guanosine derivatives containing bulky substituents at 8 position are known to adopt syn conformation of N-glycoside bond. On the contrary, in RNA the anti conformation is predominant in Watson-Crick base pairing. In this paper two 8-substituted guanosine derivatives, 8-methoxyguanosine and 8-benzyloxyguanosine, were synthesized and incorporated into oligoribonucleotides to investigate their influence on the thermodynamic stability of RNA duplexes. The methoxy and benzyloxy substituents are electron-donating groups, decreasing the rate of depurination in the monomers, as confirmed by N-glycoside bond stability assessments. Thermodynamic stability studies indicated that substitution of guanosine by 8-methoxy- or 8-benzyloxyguanosine significantly decreased the thermodynamic stability of RNA duplexes. Moreover, the presence of 8-substituted guanosine derivatives decreased mismatch discrimination. Circular dichroism spectra of modified RNA duplexes exhibited patterns typical for A-RNA geometry.

  20. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain

    Directory of Open Access Journals (Sweden)

    Mehran Nikan

    2016-01-01

    Full Text Available The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA, the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6–60 μg. Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73% and cortex (up to 51% after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg, we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders.

  1. Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo

    NARCIS (Netherlands)

    Mook, Olaf R.; Baas, Frank; de Wissel, Marit B.; Fluiter, Kees

    2007-01-01

    RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered

  2. Auto-Regulatory RNA Editing Fine-Tunes mRNA Re-Coding and Complex Behaviour in Drosophila

    Science.gov (United States)

    Savva, Yiannis A.; Jepson, James E.C; Sahin, Asli; Sugden, Arthur U.; Dorsky, Jacquelyn S.; Alpert, Lauren; Lawrence, Charles; Reenan, Robert A.

    2014-01-01

    Auto-regulatory feedback loops are a common molecular strategy used to optimize protein function. In Drosophila many mRNAs involved in neuro-transmission are re-coded at the RNA level by the RNA editing enzyme dADAR, leading to the incorporation of amino acids that are not directly encoded by the genome. dADAR also re-codes its own transcript, but the consequences of this auto-regulation in vivo are unclear. Here we show that hard-wiring or abolishing endogenous dADAR auto-regulation dramatically remodels the landscape of re-coding events in a site-specific manner. These molecular phenotypes correlate with altered localization of dADAR within the nuclear compartment. Furthermore, auto-editing exhibits sexually dimorphic patterns of spatial regulation and can be modified by abiotic environmental factors. Finally, we demonstrate that modifying dAdar auto-editing affects adaptive complex behaviors. Our results reveal the in vivo relevance of auto-regulatory control over post-transcriptional mRNA re-coding events in fine-tuning brain function and organismal behavior. PMID:22531175

  3. 2'-O-methyl-modified RNAs act as TLR7 antagonists.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; Liang, Lisa; McClintock, Kevin; Yaworski, Ed; MacLachlan, Ian

    2007-09-01

    RNA molecules such as single-stranded RNA (ssRNA) and small interfering RNA (siRNA) duplexes induce Toll-like receptor (TLR)-mediated immune stimulation after intracellular delivery. We have previously shown that selective incorporation of 2'-O-methyl (2'OMe) residues into siRNA abrogates cytokine production without reduction of gene silencing activity. Here we show that 2'OMe-modified RNA acts as a potent inhibitor of RNA-mediated cytokine induction in both human and murine systems. This activity does not require the direct incorporation of 2'OMe nucleotides into the immunostimulatory RNA or that the 2'OMe nucleotide-containing RNA be annealed as a complementary strand to form a duplex. Our results indicate that 2'OMe RNA acts as a potent antagonist of immunostimulatory RNA. We further show that 2'OMe RNA is able significantly to reduce both interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) induction by the small-molecule TLR7 agonist loxoribine in human peripheral blood mononuclear cells (human PBMCs), in murine Flt3L dendritic cells (Flt3L DCs), and in vivo in mice. These results indicate that 2'OMe-modified RNA may have utility as an inhibitor of TLR7 with potential applications in the treatment of inflammatory and autoimmune diseases that involve TLR7-mediated immune stimulation.

  4. Chemical stability and osteogenic activity of plasma-sprayed boron-modified calcium silicate-based coatings.

    Science.gov (United States)

    Lu, Xiang; Li, Kai; Xie, Youtao; Huang, Liping; Zheng, Xuebin

    2016-11-01

    In recent years, CaSiO 3 bio-ceramic coatings have attracted great attention because of their good bioactivity. However, their high degradation rates in physiological environment restrict their practical applications. In this work, boron-modified CaSiO 3 ceramic (Ca 11 Si 4 B 2 O 22 , B-CS) coating was developed on Ti substrates by plasma-spraying technique attempting to obtain enhanced chemical stability and osteogenic activity. The B-CS coating possessed significantly increased chemical stability due to the introduction of boron and consequently the modified crystal structure, while maintaining good bioactivity. Scanning electron microscope and immunofluorescence studies showed that better cellular adhesion and extinctive filopodia-like processes were observed on the B-CS coating. Compared with the pure CaSiO 3 (CS) coating, the B-CS coating promoted MC3T3-E1 cells attachment and proliferation. In addition, enhanced collagen I (COL-I) secretion, alkaline phosphatase activity, and extracellular matrix mineralization levels were detected from the B-CS coating. According to RT-PCR results, notable up-regulation expressions of mineralized tissue-related genes, such as runt-related transcription factor 2 (Runx2), bone sialoprotein and osteocalcin, and bone morphogenetic protein 7 (BMP-7) were observed on the B-CS coating compared with the CS coating. The above results suggested that Ca 11 Si 4 B 2 O 22 coatings possess excellent osteogenic activity and might be a promising candidate for orthopedic applications.

  5. The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

    Science.gov (United States)

    Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

    2010-01-01

    Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

  6. [Research progress in chemical communication among insect-resistant genetically modified plants, insect pests and natural enemies].

    Science.gov (United States)

    Liu, Qing-Song; Li, Yun-He; Chen, Xiu-Ping; Peng, Yu-Fa

    2014-08-01

    Semiochemicals released by plants or insects play an important role in the communication among plants, phytophagous insects and their natural enemies. They thus form a chemical information network which regulates intra- and inter-specific behaviors and sustains the composition and structure of plant and insect communities. The application of insect-resistant genetically modified (IRGM) crops may affect the chemical communication within and among the tritrophic levels, and thus cause disturbances to the biotic community structure and the stability of the farmland ecosystem. This has raised concerns about the environmental safety of IRGM crops and triggered research worldwide. In the current article we provided a brief summary of the chemical communication among plants, herbivores and natural enemies; analyzed the potential of IRGM crops to affect the chemical communication between plants and arthropods and the related mechanisms; and discussed the current research progress and the future prospects in this field. We hope that this will promote the research in this field by Chinese scientists and increase our understanding of the potential effects of growing of IRGM crops on the arthropod community structure.

  7. Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer.

    Science.gov (United States)

    Voutila, Jon; Reebye, Vikash; Roberts, Thomas C; Protopapa, Pantelitsa; Andrikakou, Pinelopi; Blakey, David C; Habib, Robert; Huber, Hans; Saetrom, Pal; Rossi, John J; Habib, Nagy A

    2017-12-06

    Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells. A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity. This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Singular anti-RNA virus-directed proteins.

    Directory of Open Access Journals (Sweden)

    Rayanade R

    2000-07-01

    Full Text Available AIMS: To additionally purify and characterise the anti-RNA virus-directed protein termed p14. MATERIALS AND METHODS: Antiviral assays of p14 against RNA and DNA viruses were carried out and its antigenic similarities with chicken interferon (CIFN were studied. HPLC-Reverse Phase of p14 was performed to further purify p14. RESULTS: p14 showed antiviral activity against RNA viruses only and not against DNA viruses. It was antigenically distinct from CIFN. Purification of p14 yielded three proteins with antiviral activity, which had different physico-chemical properties than those described for interferons. CONCLUSIONS: The data presented on the antiviral, immunological and physico-chemical properties, establish the unique nature of p14 vis-á-vis those of interferons.

  9. Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

    Directory of Open Access Journals (Sweden)

    Takeshi Kubota

    Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

  10. A dsRNA-binding protein MdDRB1 associated with miRNA biogenesis modifies adventitious rooting and tree architecture in apple.

    Science.gov (United States)

    You, Chun-Xiang; Zhao, Qiang; Wang, Xiao-Fei; Xie, Xing-Bin; Feng, Xiao-Ming; Zhao, Ling-Ling; Shu, Huai-Rui; Hao, Yu-Jin

    2014-02-01

    Although numerous miRNAs have been already isolated from fruit trees, knowledge about miRNA biogenesis is largely unknown in fruit trees. Double-strand RNA-binding (DRB) protein plays an important role in miRNA processing and maturation; however, its role in the regulation of economically important traits is not clear yet in fruit trees. EST blast and RACE amplification were performed to isolate apple MdDRB1 gene. Following expression analysis, RNA binding and protein interaction assays, MdDRB1 was transformed into apple callus and in vitro tissue cultures to characterize the functions of MdDRB1 in miRNA biogenesis, adventitious rooting, leaf development and tree growth habit. MdDRB1 contained two highly conserved DRB domains. Its transcripts existed in all tissues tested and are induced by hormones. It bound to double-strand RNAs and interacted with AtDCL1 (Dicer-Like 1) and MdDCL1. Chip assay indicated its role in miRNA biogenesis. Transgenic analysis showed that MdDRB1 controls adventitious rooting, leaf curvature and tree architecture by modulating the accumulation of miRNAs and the transcript levels of miRNA target genes. Our results demonstrated that MdDRB1 functions in the miRNA biogenesis in a conserved way and that it is a master regulator in the formation of economically important traits in fruit trees. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. In vivo silencing of alpha-synuclein using naked siRNA

    Directory of Open Access Journals (Sweden)

    Charisse Klaus

    2008-11-01

    Full Text Available Abstract Background Overexpression of α-synuclein (SNCA in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD, possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. Results We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked, murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. Conclusion We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression.

  12. In vivo silencing of alpha-synuclein using naked siRNA

    Science.gov (United States)

    Lewis, Jada; Melrose, Heather; Bumcrot, David; Hope, Andrew; Zehr, Cynthia; Lincoln, Sarah; Braithwaite, Adam; He, Zhen; Ogholikhan, Sina; Hinkle, Kelly; Kent, Caroline; Toudjarska, Ivanka; Charisse, Klaus; Braich, Ravi; Pandey, Rajendra K; Heckman, Michael; Maraganore, Demetrius M; Crook, Julia; Farrer, Matthew J

    2008-01-01

    Background Overexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. Results We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. Conclusion We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression. PMID:18976489

  13. Coronary Heart Disease Alters Intercellular Communication by Modifying Microparticle-Mediated MicroRNA Transport

    Science.gov (United States)

    Finn, Nnenna A.; Eapen, Danny; Manocha, Pankaj; Kassem, Hatem Al; Lassegue, Bernard; Ghasemzadeh, Nima; Quyyumi, Arshed; Searles, Charles D.

    2013-01-01

    Coronary heart disease (CHD) is characterized by abnormal intercellular communication and circulating microRNAs (miRNAs) are likely involved in this process. Here, we show that CHD was associated with changes in the transport of circulating miRNA, particularly decreased miRNA enrichment in microparticles (MPs). Additionally, MPs from CHD patients were less efficient at transferring miRNA to cultured HUVECs, which correlated with their diminished capacity to bind developmental endothelial locus-1 (Del-1). In summary, CHD was associated with distinct changes in circulating miRNA transport and these changes may contribute to the abnormal intercellular communication that underlies CHD initiation and progression. PMID:24042051

  14. Chemical Sensors Based on IR Spectroscopy and Surface-Modified Waveguides

    Science.gov (United States)

    Lopez, Gabriel P.; Niemczyk, Thomas

    1999-01-01

    Sol-gel processing techniques have been used to apply thin porous films to the surfaces of planar infrared (IR) waveguides to produce widely useful chemical sensors. The thin- film coating serves to diminish the concentration of water and increase the concentration of the analyte in the region probed by the evanescent IR wave. These porous films are composed of silica, and therefore, conventional silica surface modification techniques can be used to give the surface a specific functional character. The sol-gel film was surface-modified to make the film highly hydrophobic. These sensors were shown to be capable of detecting non-polar organic analytes, such as benzonitrile, in aqueous solution with detection limits in the ppb range. Further, these porous sol-gel structures allow the analytes to diffuse into and out of the films rapidly, thus reaching equilibrium in less than ten seconds. These sensors are unique because of the fact that their operation is based on the measurement of an IR absorption spectrum. Thus, these sensors are able to identify the analytes as well as measure concentration with high sensitivity. These developments have been documented in previous reports and publications. Recently, we have also targeted detection of the polar organic molecules acetone and isopropanol in aqueous solution. Polar organics are widely used in industrial and chemical processes, hence it is of interest to monitor their presence in effluents or decontamination process flows. Although large improvements in detection limits were expected with non-polar organic molecules in aqueous solutions using very hydrophobic porous sol-gel films on silicon attenuated total reflectance (Si ATR) waveguides, it was not as clear what the detection enhancements might be for polar organic molecules. This report describes the use of modified sol-gel-coated Si ATR sensors for trace detection and quantitation of small polar organic molecules in aqueous solutions. The detection of both acetone

  15. Bioreducible poly(amido amine)s for siRNA delivery

    NARCIS (Netherlands)

    van der Aa, L.J.

    2011-01-01

    Successes in RNA interference based therapies are still limited due to the lack of efficient delivery of the mediator, small interfering RNA (siRNA), to the targeted site. The key to success can be the delivery of the siRNA molecules by polymer-based carrier systems, since they can be chemically

  16. The chemical structure of DNA sequence signals for RNA transcription

    Science.gov (United States)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  17. Thermo-chemical characterization of a Al nanoparticle and NiO nanowire composite modified by Cu powder

    International Nuclear Information System (INIS)

    Bohlouli-Zanjani, Golnaz; Wen, John Z.; Hu, Anming; Persic, John; Ringuette, Sophie; Zhou, Y. Norman

    2013-01-01

    Highlights: • First study on the copper modified powder-type Al nanoparticle and NiO nanowire composites. • Experimental findings were unique in identifying the AlNi formation and comparing with the Al/CuO thermite. • Potential applications in material joining and bonding. - Abstract: Thermo-chemical properties of the Al nanoparticle and NiO nanowire composites modified by the micro-sized copper additive were investigated experimentally. Their onset temperatures of ignition and energy release data per mass were characterized using differential thermal analysis measurements. These microstructures and chemical compositions of reaction products were analyzed using scanning electron microscopy, energy dispersive X-ray spectroscopy and X-ray diffraction. The fuel-rich Al/NiO/Cu composites produced two types of metallic spheres. Copper spheres were formed from melting and solidification of the copper additive, while AlNi composite spheres were identified by the energy dispersive X-ray spectroscopy and X-ray diffraction analyses. It was found that the amount of the copper additive did not significantly influence the onset temperature of thermite peaks, but caused a dramatic change in energy release. The aforementioned ignition and energetic properties were compared with these from the Al nanoparticle and CuO nanowire composites

  18. Methods to enable the design of bioactive small molecules targeting RNA.

    Science.gov (United States)

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

  19. Mechanical and chemical properties of polyvinyl alcohol modified cement mortar with silica fume used as matrix including radioactive waste

    International Nuclear Information System (INIS)

    Dakroury, A. M.

    2007-01-01

    This paper discussed the mechanical and chemical properties of polyvinyl alcohol - modified cement mortar with silica fume to assess the safety for disposal of radioactive waste. The modified cement mortars containing polyvinyl alcohol (PVA) in the presence of 10 % silica fume (SF) .The chemical reaction between polymer and cement - hydrated product were investigated by the Infrared Spectral Technology, Differential Thermal Analysis and X-ray diffraction. The leaching of 137Cs from a waste composite into a surrounding fluid has been studied .The results shown that PVA increases the strength and decreases the porosity. The increase in strength duo to the interaction of PVA with cement , may be forming some new compound that fill the pores or improve the bond between the cement . The pozzolanic reaction of the SF increases the calcium silicate hydrates in the hardening matrix composites. There is distinct change in the refinement of the pore structure in cement composites giving fewer capillary pores and more of the finer gel pores

  20. Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

    Science.gov (United States)

    Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

    2006-02-01

    The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

  1. Structure-function analysis of human TYW2 enzyme required for the biosynthesis of a highly modified Wybutosine (yW base in phenylalanine-tRNA.

    Directory of Open Access Journals (Sweden)

    Virginia Rodriguez

    Full Text Available Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNA(Phe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2 is active in yeast and can synthesize the yW of yeast tRNA(Phe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet, and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNA(Phe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis.

  2. Bis-pyrene-modified unlocked nucleic acids: synthesis, hybridization studies, and fluorescent properties

    DEFF Research Database (Denmark)

    Perlíková, Pavla; Ejlersen, Maria; Langkjaer, Niels

    2014-01-01

    Efficient synthesis of a building block for the incorporation of a bis-pyrene-modified unlocked nucleic acid (UNA) into oligonucleotides (DNA*) was developed. The presence of bis-pyrene-modified UNA within a duplex leads to duplex destabilization that is more profound in DNA*/RNA and less distinc......)uracil:pyrene exciplex emission in the single-stranded form. Such fluorescent properties enable the application of bis-pyrene-modified UNA in the development of fluorescence probes for DNA/RNA detection and for detection of deletions at specific positions....

  3. Conditional RNA interference achieved by Oct-1 POU/rtTA fusion protein activator and a modified TRE-mouse U6 promoter

    International Nuclear Information System (INIS)

    Fei Zhaoliang; Chen Zheng; Wang Zhugang; Fei Jian

    2007-01-01

    RNA interference (RNAi) is a powerful technique and is widely used to down-regulate expression of specific genes in cultured cells and in vivo. In this paper, we report our development of a new tetracycline-inducible RNAi expression using a modified TRE-mouse U6 promoter in which the distal sequence element (DSE) was replaced by the tetracycline-responsive element (TRE). The modified TRE-mouse U6 promoter can be activated by a Tet-on version tetracycline-regulated artificial activator rTetOct which was constructed by fusing the rtTA DNA binding domain with the Oct-1 POU activation domain. This rTetOct/TRE-U6 system was successfully applied to conditionally and reversibly down-regulate the expression of endogenous p53 gene in MCF7 cells, and the expression of β-defensin gene (mBin1b) either transiently expressed in COS7 cells or stably expressed in CHO cells

  4. RNA damage in biological conflicts and the diversity of responding RNA repair systems

    Science.gov (United States)

    Burroughs, A. Maxwell; Aravind, L.

    2016-01-01

    RNA is targeted in biological conflicts by enzymatic toxins or effectors. A vast diversity of systems which repair or ‘heal’ this damage has only recently become apparent. Here, we summarize the known effectors, their modes of action, and RNA targets before surveying the diverse systems which counter this damage from a comparative genomics viewpoint. RNA-repair systems show a modular organization with extensive shuffling and displacement of the constituent domains; however, a general ‘syntax’ is strongly maintained whereby systems typically contain: a RNA ligase (either ATP-grasp or RtcB superfamilies), nucleotidyltransferases, enzymes modifying RNA-termini for ligation (phosphatases and kinases) or protection (methylases), and scaffold or cofactor proteins. We highlight poorly-understood or previously-uncharacterized repair systems and components, e.g. potential scaffolding cofactors (Rot/TROVE and SPFH/Band-7 modules) with their respective cognate non-coding RNAs (YRNAs and a novel tRNA-like molecule) and a novel nucleotidyltransferase associating with diverse ligases. These systems have been extensively disseminated by lateral transfer between distant prokaryotic and microbial eukaryotic lineages consistent with intense inter-organismal conflict. Components have also often been ‘institutionalized’ for non-conflict roles, e.g. in RNA-splicing and in RNAi systems (e.g. in kinetoplastids) which combine a distinct family of RNA-acting prim-pol domains with DICER-like proteins. PMID:27536007

  5. Reproducible pattern of microRNA in normal human skin

    DEFF Research Database (Denmark)

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease....

  6. Reproducible pattern of microRNA in normal human skin

    DEFF Research Database (Denmark)

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease.......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...

  7. Chemical and semisynthesis of modified histones.

    Science.gov (United States)

    Maity, Suman Kumar; Jbara, Muhammad; Brik, Ashraf

    2016-05-01

    Post-translational modifications (PTMs) of histones play critical roles in the epigenetic regulation of eukaryotic genome by directly altering the biophysical properties of chromatin or by recruiting effector proteins. The large number of PTMs and the inherent complexity in their population and signaling processes make it highly challenging to understand epigenetics-related processes. To address these challenges, accesses to homogeneously modified histones are obligatory. Over the last decade, synthetic protein chemists have been devising novel synthetic tools and applying state-of-the-art chemoselective ligation strategies to prepare precious materials useful in answering fundamental questions in this area. In this short review, we cover some of the recent breakthroughs in these directions in particular the synthesis and semi-synthesis of modified histones and their use to unravel the mysteries of epigenetics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  8. 40 CFR 721.3710 - Polyether modified fatty acids (generic).

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Polyether modified fatty acids... Specific Chemical Substances § 721.3710 Polyether modified fatty acids (generic). (a) Chemical substance... Polyether modified fatty acids (PMN P-99-0435) is subject to reporting under this section for the...

  9. 40 CFR 721.9513 - Modified magnesium silicate polymer (generic).

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Modified magnesium silicate polymer... Specific Chemical Substances § 721.9513 Modified magnesium silicate polymer (generic). (a) Chemical... as modified magnesium silicate polymer (PMN P-98-604) is subject to reporting under this section for...

  10. Orotidine-Containing RNA: Implications for the Hierarchical Selection (Systems Chemistry Emergence) of RNA.

    Science.gov (United States)

    Kim, Eun-Kyong; Martin, Vincent; Krishnamurthy, Ramanarayanan

    2017-09-12

    The prebiotic synthesis of canonical nucleobases from HCN is a cornerstone for the RNA world hypothesis. However, their role in the primordial pathways to RNA is still debated. The very same process starting from HCN also gives rise to orotic acid, which (via orotidine) plays a crucial role in extant biology in the de novo synthesis of uridine and cytidine, the informational base-pairs in RNA. However, orotidine itself is absent in RNA. Given the prebiotic and biological relevance of orotic acid vis-à-vis uracil, we investigated orotidine-containing RNA oligonucleotides and show that they have severely compromised base-pairing properties. While not unexpected, these results suggest that the emergence of extant RNA cannot just be a consequence of the plausible prebiotic formation of its chemical constituents/building blocks. In combination with other investigations on alternative prebiotic nucleobases, sugars, and linkers, these findings imply that the selection of the components of extant RNA occurred at a higher hierarchical level of an oligomer/polymer based on its functional properties-pointing to a systems chemistry emergence of RNA from a library of precursors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The RNA 5 of Prunus necrotic ringspot virus is a biologically inactive copy of the 3'-UTR of the genomic RNA 3.

    Science.gov (United States)

    Di Terlizzi, B; Skrzeczkowski, L J; Mink, G I; Scott, S W; Zimmerman, M T

    2001-01-01

    In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3' termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3' untranslated terminal region (3'-UTR) of the genomic RNA 3.

  12. I prokaryota si chrání svoji RNA – nové typy 5'-RNA čepiček

    Czech Academy of Sciences Publication Activity Database

    Cahová, Hana

    2017-01-01

    Roč. 111, č. 7 (2017), s. 414-423 ISSN 0009-2770 R&D Projects: GA ČR(CZ) GJ16-16358Y Institutional support: RVO:61388963 Keywords : 5'-RNA cap * NAD-RNA * RNA chemical modification Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 0.387, year: 2016 http://www.chemicke-listy.cz/docs/full/2017_07_414-423.pdf

  13. Sensitive and label-free detection of miRNA-145 by triplex formation.

    Science.gov (United States)

    Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

    2016-01-01

    The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

  14. Mutagenicity of chemicals in genetically modified animals

    NARCIS (Netherlands)

    Willems MI; van Benthem J; LEO

    2001-01-01

    The strategy for assessing human health risks of chemicals consists of a large number of tests in different research disciplines. Tests include acute and chronic toxicity, genotoxicity, reproduction toxicity and carcinogenicity. Genotoxic properties of chemicals are assessed in short-term in vitro

  15. Chemical Peeling with a Modified Phenol Formula for the Treatment of Facial Freckles on Asian Skin.

    Science.gov (United States)

    Sun, Hua-Feng; Lu, Hai-Shan; Sun, Le-Qi; Ping, Wei-Dong; Mao, Dong-Sheng; Li, Dan

    2018-04-01

    Chemical peeling is an efficient method for the treatment of pigment disorders. For freckles, medium-depth to deep peeling using a phenol solution is one of the most effective chemical peels, and modifications of facial skin can be observed up to 20 years after peeling. However, applying phenol to the skin may cause serious side effects. Phenol peeling has been rarely used in Asia due to its tendency to cause permanent pigmentary changes and hypertrophic scars. In total, 896 Chinese inpatients with facial freckles were enrolled in this study. The phenol formula was modified with crystalline phenol, dyclonine, camphor, anhydrous alcohol and glycerin and adjusted to a concentration of 73.6-90.0%. The entire peeling treatment was divided into two procedures performed separately on 2 days. All patients exhibited 26% or greater improvement, and 99.66% of patients exhibited 51% or greater improvement (good and excellent). Scarring and systemic complications were not observed in any patient. The modified phenol formula is very effective and safe for the treatment of facial freckles in Asian patients. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

  16. A study on the chemical stability and electrode performance of modified NiO cathodes for molten carbonate fuel cells

    International Nuclear Information System (INIS)

    Kim, Seung-Goo; Yoon, Sung Pil; Han, Jonghee; Nam, Suk Woo; Lim, Tae Hoon; Oh, In-Hwan; Hong, Seong-Ahn

    2004-01-01

    The chemical stabilities of modified NiO cathodes doped with 1.5 mol% CoO and 1.5 mol% LiCoO 2 fabricated by a conventional tape casting method were evaluated through the real MCFC single cell operation. The heat-treated samples before oxidation had proper porosities and microstructures for a MCFC cathode. At 150 mA cm -2 in current density, the MCFC single cell using a CoO-doped NiO cathode showed stable cell voltages in the range of 0.833-0.843 V for 1000 h. In contrast, the cell using a LiCoO 2 -doped NiO cathode with a maximum of 0.836 V at 500 h degraded to 0.826 V at 1000 h due to a wet seal breakdown at the cathode side. The amounts of nickel precipitated in the electrolytes of the cells using modified NiO cathodes doped with CoO and LiCoO 2 after the operation for 1000 h were 1.2 and 1.4 wt.%, respectively, which were about 60% lower than that of the standard cells using pure NiO cathodes. The enhanced chemical stability of modified NiO cathodes seems to be attributed to the fact that the presence of cobalt increases the lithium content in the cathodes by converting Ni 2+ to Ni 3+ , resulting in stabilizing the layered crystal structure

  17. Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

    Science.gov (United States)

    Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

    2011-01-01

    Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

  18. Circular RNA (circRNA) was an important bridge in the switch from the RNA world to the DNA world.

    Science.gov (United States)

    Soslau, Gerald

    2018-06-14

    The concept that life on Earth began as an RNA world has been built upon extensive experimentation demonstrating that many of the building blocks required for living cells could be synthesized in the laboratory under conditions approximating our primordial world. Many of the building blocks for life have also been found in meteorites indicating that meteors may have been a source for these molecules, or more likely, that they represent the chemical library present in most/all bodies in the universe after the big bang. Perhaps the most important support for the concept comes from the fact that some RNA species possess catalytic activity, ribozymes, and that RNA could be reverse transcribe to DNA. The thrust of numerous papers on this topic has been to explore how the available molecules on Earth, at its birth, gave rise to life as we know it today. This paper focuses more on a reverse view of the topic. The "how" molecular building blocks were synthesized is not addressed nor how the "first" RNA molecules were synthesized. We can clearly speculate on the variable environmental conditions and chemistry available on Earth billions of years ago. However, we can never truly replicate the changing conditions or know the chemical composition of Earth at the beginning of time. We can, however, confirm that over millions, perhaps billions of years the basic building blocks for life accumulated sufficiently to initiate evolution to an RNA world followed by our RNA/DNA world. Here we are attempting to take the information from our current knowledge of biology and by inference and extrapolation work backward to hypothesize biological events in the march forward from RNA to DNA. It is proposed that the primordial replicating RNA cell, the ribocyte, evolved from liposomes encompassing required reactants and products for "life" and that ribonucleopeptide complexes formed membrane pores to support bidirectional ion and molecular transport to maintain biological functions and

  19. In Silico and In Vitro Anticancer Activity of Isolated Novel Marker Compound from Chemically Modified Bioactive Fraction from Curcuma longa (NCCL).

    Science.gov (United States)

    Naqvi, Arshi; Malasoni, Richa; Gupta, Swati; Srivastava, Akansha; Pandey, Rishi R; Dwivedi, Anil Kumar

    2017-10-01

    Turmeric ( Curcuma longa ) is reported to possess wide array of biological activities. Herbal Medicament (HM) is a standardized hexane-soluble fraction of C. longa and is well known for its neuroprotective effect. In this study, we attempted to synthesize a novel chemically modified bioactive fraction from HM (NCCL) along with isolation and characterization of a novel marker compound (I). NCCL was prepared from HM. The chemical structure of the marker compound isolated from NCCL was determined from 1D/2D nuclear magnetic resonance, mass spectroscopy, and Fourier transform infrared. The compound so isolated was subjected to in silico and in vitro screenings to test its inhibitory effect on estrogen receptors. Molecular docking studies revealed that the binding poses of the compound I was energetically favorable. Among NCCL and compound I taken for in vitro studies, NCCL had exhibited good anti-cancer activity over compound I against MCF-7, MDA-MB-231, DU-145, and PC-3 cells. This is the first study about the synthesis of a chemically modified bioactive fraction which used a standardized extract since the preparation of the HM. It may be concluded that NCCL fraction having residual components induce more cell death than compound I alone. Thus, NCCL may be used as a potent therapeutic drug. In the present paper, a standardized hexane soluble fraction of Curcuma longa (HM) was chemically modified to give a novel bioactive fraction (NCCL). A novel marker compound was isolated from NCCL and was characerized using various spectral techniques. The compound so isolated was investigated for in-silico screenings. NCCL and isolated compound was subjected to in-vitro anti-cancer screenings against MCF 7, MDA MB 231 (breast adenocarcinoma) and DU 145 and PC 3 cell lines (androgen independent human prostate cancer cells). The virtual screenings reveals that isolated compound has shown favourable drug like properties. NCCL fraction having residual components induces more cell

  20. Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

    Science.gov (United States)

    Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

    2015-05-06

    RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

  1. Comparative study of ZnSe thin films deposited from modified chemical bath solutions with ammonia-containing and ammonia-free precursors

    International Nuclear Information System (INIS)

    Chen Liangyan; Zhang Daoli; Zhai Guangmei; Zhang Jianbing

    2010-01-01

    Ammonia is one of the complexing agents which are the most commonly used in the precursors of ZnSe thin films by chemical bath deposition, but its high volatility may be harmful to human beings and environments. In our experiments, ZnSe films were obtained from modified chemical solutions with ammonia-containing and ammonia-free precursors. X-ray diffraction, field-emission scanning electron microscope (FSEM), and absorption spectrum were applied to investigate the microstructure, morphology and optical properties of the samples obtained from both growth conditions, which were investigated in this work. The ammonia-free chemical bath deposited ZnSe films showed comparable properties with the ammonia-containing ones, indicating that ZnSe films from ammonia-free chemical solution may be preferred buffer layer in thin film solar cells with less environmental contamination.

  2. Characteristics of Chemical and Functional Properties of Modified Cassava Flour (Manihot esculenta) by Autoclaving-Cooling Cycles Method

    Science.gov (United States)

    Cecep Erwan Andriansyah, Raden; Rahman, Taufik; Herminiati, Ainia; Rahman, Nurhaidar; Luthfiyanti, Rohmah

    2017-12-01

    The modified cassava flour can be made using the method of the autoclaving cooling cycle (AAC). The stability of the warming can be seen from the decreasing value of breakdown viscosity, while the stability of the stirring process can be seen by the decreasing value of setback viscosity. The stages of research include: (1) the making of cassava flour, (2) the making of modified cassava flour by the method of treatment of ACC with a variety of flour concentration and autoclaving time, (3) chemical analysis of the moisture, ash, fat, protein, carbohydrate; The functional properties of the pasting characteristics to the initial temperature of the pasting, peak viscosity, hot paste viscosity, breakdown viscosity, cold paste viscosity and setback viscosity. The result shows that cassava flour modified by treatment of flour concentration 16% and autoclaving time 41 minutes having pasting code and pasting viscosity which is resistant to high temperature. Flour with this character is flour that is expected to maintain the texture of processed products with a paste form that remains stable. Utilization of modified cassava flour by the ACC method can be applied to the pasting product such as noodle and spaghetti, hoping to support for food diversification program to reduce dependence on wheat flour in Indonesia.

  3. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.

    Science.gov (United States)

    Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

    2015-11-10

    In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. siRNA and innate immunity.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; MacLachlan, Ian

    2009-06-01

    Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of inflammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modification approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

  5. The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells

    Science.gov (United States)

    Pickard, Mark R.; Williams, Gwyn T.

    2016-01-01

    Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and –insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies. PMID:26862727

  6. Synthesis and characterization of amino acid-functionalized calcium phosphate nanoparticles for siRNA delivery.

    Science.gov (United States)

    Bakan, Feray; Kara, Goknur; Cokol Cakmak, Melike; Cokol, Murat; Denkbas, Emir Baki

    2017-10-01

    Small interfering RNAs (siRNA) are short nucleic acid fragments of about 20-27 nucleotides, which can inhibit the expression of specific genes. siRNA based RNAi technology has emerged as a promising method for the treatment of a variety of diseases. However, a major limitation in the therapeutic use of siRNA is its rapid degradation in plasma and cellular cytoplasm, resulting in short half-life. In addition, as siRNA molecules cannot penetrate into the cell efficiently, it is required to use a carrier system for its delivery. In this work, chemically and morphologically different calcium phosphate (CaP) nanoparticles, including spherical-like hydroxyapatite (HA-s), needle-like hydroxyapatite (HA-n) and calcium deficient hydroxyapatite (CDHA) nanoparticles were synthesized by the sol-gel technique and the effects of particle characteristics on the binding capacity of siRNA were investigated. In order to enhance the gene loading efficiency, the nanoparticles were functionalized with arginine and the morphological and their structural characteristics were analyzed. The addition of arginine did not significantly change the particle sizes; however, it provided a significantly increased binding of siRNA for all types of CaP nanoparticles, as revealed by spectrophotometric measurements analysis. Arginine functionalized HA-n nanoparticles showed the best binding behavior with siRNA among the other nanoparticles due to its high, positive zeta potential (+18.8mV) and high surface area of Ca ++ rich "c" plane. MTT cytotoxicity assays demonstrated that all the nanoparticles tested herein were biocompatible. Our results suggest that high siRNA entrapment in each of the three modified non-toxic CaP nanoparticles make them promising candidates as a non-viral vector for delivering therapeutic siRNA molecules to treat cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

    OpenAIRE

    Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

    2015-01-01

    In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA c...

  8. Chemical Ligation Reactions of Oligonucleotides for Biological and Medicinal Applications.

    Science.gov (United States)

    Abe, Hiroshi; Kimura, Yasuaki

    2018-01-01

    Chemical ligation of oligonucleotides (ONs) is the key reaction for various ON-based technologies. We have tried to solve the problems of RNA interference (RNAi) technology by applying ON chemical ligation to RNAi. We designed a new RNAi system, called intracellular buildup RNAi (IBR-RNAi), where the RNA fragments are built up into active small-interference RNA (siRNA) in cells through a chemical ligation reaction. Using the phosphorothioate and iodoacetyl groups as reactive functional groups for the ligation, we achieved RNAi effects without inducing immune responses. Additionally, we developed a new chemical ligation for IBR-RNAi, which affords a more native-like structure in the ligated product. The new ligation method should be useful not only for IBR-RNAi but also for the chemical synthesis of biofunctional ONs.

  9. Can balneotherapy modify microRNA expression levels in osteoarthritis? A comparative study in patients with knee osteoarthritis

    Science.gov (United States)

    Giannitti, C.; De Palma, A.; Pascarelli, N. A.; Cheleschi, S.; Giordano, N.; Galeazzi, M.; Fioravanti, Antonella

    2017-12-01

    The aim of this study was to evaluate the whole-blood levels of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e in patients with bilateral knee osteoarthritis (OA) after a cycle of mud-bath therapy (MBT). Thirty-two patients with knee OA defined by the ACR criteria were included. Twenty-one patients (MBT group) were daily treated with a combination of local mud-packs at 42 °C and baths in mineral water, at 37 °C for 15 min, for 12 applications over a period of 2 weeks, in addition to standard therapy; 11 patients (control group) continued their conventional treatment alone. Global pain score evaluated by visual analog scale (VAS), WOMAC subscores, and microRNA expression were evaluated at baseline and after 2 weeks. Peripheral whole blood was collected into PAXgene™ Blood RNA tubes, stored at - 80 °C, and total RNA was extracted. The expression of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e was determined by qRT-PCR. After MBT, we observed a statistically significant improvement of clinical parameters and a significant decrease of miR-155, miR-181a, miR-146a ( p < 0.001), and miR-223 ( p < 0.01) expression levels. No clinical and biochemical modifications were detected in the control group. No significant variations of miR-let-7e were shown in both groups after 2 weeks. In conclusion, MBT can modify the expression of miR-155, miR-181a, miR-146a, and miR-223, which are upregulated in OA. It could be due to the heat stress and the hydrostatic pressure, since some miRNAs were found to be temperature- and mechano-responsive. Further studies are needed to better explain the mechanism of action of MBT and the role of miRNAs in OA.

  10. Movement of regulatory RNA between animal cells.

    Science.gov (United States)

    Jose, Antony M

    2015-07-01

    Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions. © 2015 Wiley Periodicals, Inc.

  11. Chemically-modified graphene sheets as an active layer for eco-friendly metal electroplating on plastic substrates

    International Nuclear Information System (INIS)

    Oh, Joon-Suk; Hwang, Taeseon; Nam, Gi-Yong; Hong, Jung-Pyo; Bae, Ah-Hyun; Son, Sang-Ik; Lee, Geun-Ho; Sung, Hak kyung; Choi, Hyouk Ryeol; Koo, Ja Choon; Nam, Jae-Do

    2012-01-01

    Eco-friendly nickel (Ni) electroplating was carried out on a plastic substrate using chemically modified graphene sheets as an active and conductive layer to initiate electroplating without using conventional pre-treatment or electroless metal-seeding processes. A graphene oxide (GO) solution was self-assembled on a polyethylene terephthalate (PET) film followed by evaporation to give GO layers (thickness around 6.5 μm) on PET (GO/PET) film. Then, the GO/PET film was chemically and thermally reduced to convert the GO layers to reduced graphene oxide (RGO) layers on the PET substrate. The RGO-coated PET (RGO/PET) film showed the sheet resistance of 100 Ω per square. On RGO/PET film, Ni electroplating was conducted under the constant-current condition and the entire surface of the PET film was completely metalized with Ni without any voids.

  12. 40 CFR 721.10089 - Modified salicylic acid, zirconium complex (generic).

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Modified salicylic acid, zirconium... Specific Chemical Substances § 721.10089 Modified salicylic acid, zirconium complex (generic). (a) Chemical... as modified salicylic acid, zirconium complex (PMN P-00-552) is subject to reporting under this...

  13. [Effects of Different Modifier Concentrations on Lead-Zinc Tolerance, Subcellular Distribution and Chemical Forms for Four Kinds of Woody Plants].

    Science.gov (United States)

    Chen, Yong-hua; Zhang, Fu-yun; Wu, Xiao-fu; Liang, Xi; Yuan, Si-wen

    2015-10-01

    Four kinds of lead-zinc tolerant woody plants: Nerium oleander, Koelreuteria paniculata, Paulownia and Boehmeria were used as materials to estimate their enrichment and transferable capacity of lead (Pb) and zinc (Zn) and analyze the subcellular distribution and chemical speciation of Zn and Ph in different parts of plants, under different modifier concentrations (CK group: 100% lead-zinc slag plus a small amount of phosphate fertilizer, improved one: 85% of lead-zinc slag ± 10% peat ± 5% bacterial manure plus a small amount of phosphate fertilizer, improved two: 75% lead-zinc slag ± 20% peat ± 5% bacterial manure ± a small amount of phosphate). Results showed that: (1) The content of Pb, Zn in matrix after planting four kinds of plants was lower than before, no significant difference between improved one and improved two of Nerium oleander and Boehmeria was found, but improved two was better than improved one of Paulownia, while improved one was better than improved two of Koelreuteria paniculata; Four plants had relatively low aboveground enrichment coefficient of Pb and Zn, but had a high transfer coefficient, showed that the appropriate modifier concentration was able to improve the Pb and Zn enrichment and transfer ability of plants. (2) In subcellular distribution, most of Pb and Zn were distributed in plant cell wall components and soluble components while the distribution in cell organelles such as mitochondria, chloroplasts and nucleus component were less. Compared with CK group, two improved group made soluble components of the cell walls of Pb fixation and retention of zinc role in the enhancement. (3) As for the chemical forms of Pb and Zn in plants, the main chemical forms of Pb were hydrochloric acid, sodium chloride and ethanol extractable forms, while other chemical form contents were few, the main chemical forms of Zn were different based on plant type. Compared with CK group, the proportion of the active Pb chemical form in different plant

  14. DETERMINING LIGHTFASTNESS PROPERTIES OF VEGETABLE TANNINS AND CHEMICAL PROPERTIES OF THE LEATHERS TANNED WITH MODIFIED MIMOSA AND QUEBRACHO

    Directory of Open Access Journals (Sweden)

    OMUR Sukru

    2016-05-01

    Full Text Available The vegetable tannins are the oldest tanning agents used in leather industry. They give their natural character and colour to the leathers which they are applied to, but they have the disadvantage of colour change when they are exposed to light for prolonged times. In this study light fastness properties of leathers tanned with mimosa, quebracho, valonea and chestnut tannins were measured. Lightfastness properties of mimosa and quebracho tannins were found lower. Then these tannins were modified with sulphitation, novalac synthesis and sulphomethylation processes. Lightfastness and determination of volatile matter, determination of matter soluble in dichloromethane, determination of sulphated total ash and sulphated water-insoluble ash, determination of water soluble matter, water soluble inorganic matter and water soluble organic matter, determination of nitrogen content and hide substance, calculation of degree of tannage determination of formaldehyde content analyses were performed to the leathers tanned with modified mimosa and quebracho tannins. From comparison of results, it was understood that sulpmethylation process can be used for production of leathers with higher lightfastness and without major change on chemical properties. When chemical properties of leathers tanned with modified quebracho and mimosa are considered: volatile matter, sulphated total ash and sulphated water- insoluble ash, water soluble matter, water soluble inorganic matter and water soluble organic matter, hide substance and formaldehyde contents were found compatible with standard mimosa and quebracho. However degree of tannage and matter soluble in dichloromethane values were found lower, which means some enhancements in modification or fatliquoring process should be considered.

  15. Comparison of Chemical Modifiers for Simultaneous Determination of Different Selenium-Compounds in Serum and Urine by Zeeman-Effect Electrothermal Atomic-Absorption Spectrometry

    DEFF Research Database (Denmark)

    Johannessen, J.K.; Gammelgaard, Bente; Jons, O.

    1993-01-01

    The thermal stability of selenite, selenate, selenomethionine and trimethylselenonium was studied using different chemical modifiers in various amounts. The normally recommended amounts of nickel nitrate, magnesium nitrate, copper nitrate, copper nitrate mixed with magnesium nitrate, palladium ni...

  16. Uranium (Vi) sorption onto zirconium diphosphate chemically modified

    International Nuclear Information System (INIS)

    Garcia G, N.; Ordonez R, E.

    2010-10-01

    This work deals with the uranium (Vi) speciation after sorption onto zirconium diphosphate (ZrP 2 O 7 ) surface, hydrated and in a surface modified with organic acids. Oxalic and citric acids were chosen to modify the ZrP 2 O 7 surface because they have poly carboxylic groups and they mimic the organic matter in nature. Thus the interest of this work is to evaluate the uranium (Vi) sorption edge at different s ph values in natural and modified surfaces. The luminescence technique (fluorescence and phosphorescence, respectively) was used for the quantification and speciation of uranyl sorbed at the zirconium diphosphate interface. The fluorescence experiment, showed that adsorption of uranyl on surface of zirconium diphosphate tends to 100%. The speciation shows that there are different complexes in surface which were formed between zirconium diphosphate and uranyl, since it is produced a displacement of wavelength in fluorescence spectra of each system. (Author)

  17. The True Story and Advantages of RNA Phage Capsids as Nanotools.

    Science.gov (United States)

    Pumpens, Paul; Renhofa, Regina; Dishlers, Andris; Kozlovska, Tatjana; Ose, Velta; Pushko, Peter; Tars, Kaspars; Grens, Elmars; Bachmann, Martin F

    2016-01-01

    RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools. © 2016 S. Karger AG, Basel.

  18. Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

    Science.gov (United States)

    Conde, João; Oliva, Nuria; Atilano, Mariana; Song, Hyun Seok; Artzi, Natalie

    2016-03-01

    The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs--a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)--provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.

  19. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

  20. Chemically modified carboxypeptidase Y with increased amidase activity

    International Nuclear Information System (INIS)

    Breddam, K.

    1984-01-01

    Treatment of carboxypeptidase Y with 14 C-iodoacetamide caused a drastic reduction in the peptidase activity towards FA-Phe-Leu-OH while the esterase activity towards FA-Phe-OMe, the amidase activity towards FA-Phe-NH 2 and the peptidyl amino acid amide hydrolase activity towards FA-Phe-Gly-NH 2 were much less affected. The loss of peptidase activity could be correlated with the incorporation of a single equivalent of reagent and it was demonstrated that the site of reaction was a methionyl residue, thus forming a sulfonium derivative. Analogous methionyl modifications were performed: carboxypeptidase Y modified with phenacylbromide hydrolysed substrates with bulky leaving groups in the P position, i.e. -OEt, -OBzl, -Gly-NH 2 ,-Gly-OH, and -Leu-OH, at reduced rates while substrates with small groups in that position, i.e. -OMe and -NH 2 , were hydrolysed with increased rates. These results indicate that the methionyl residue modified by phenacylbromide is located in the S binding site of the enzyme. Similar results were obtained with carboxypeptidase Y modified with m-nitrophen- acylbromide and p-nitrophenacylbromide. The increase in amidase activity and decrease in peptidyl amino acid amide hydrolase activity of carboxypeptidase Y following modification with phenacylbromide, m-nitrophenacylbromide, and p-nitrophenacylbromide was exploited in deamidation of peptide amides. These modified enzymes deamidated peptide amides with the exception of those containing a C-terminal glycyl or seryl residue in yields of 80-100% which is significantly higher than with unmodified carboxypeptidase Y. (author)

  1. Electrocatalytic miRNA Detection Using Cobalt Porphyrin-Modified Reduced Graphene Oxide

    Directory of Open Access Journals (Sweden)

    Camille De Souza

    2014-06-01

    Full Text Available Metalated porphyrins have been described to bind nucleic acids. Additionally, cobalt porphyrins present catalytic properties towards oxygen reduction. In this work, a carboxylic acid-functionalized cobalt porphyrin was physisorbed on reduced graphene oxide, then immobilized on glassy carbon electrodes. The carboxylic groups were used to covalently graft amino-terminated oligonucleotide probes which are complementary to a short microRNA target. It was shown that the catalytic oxygen electroreduction on cobalt porphyrin increases upon hybridization of miRNA strand (“signal-on” response. Current changes are amplified compared to non-catalytic amperometric system. Apart from oxygen, no added reagent is necessary. A limit of detection in the sub-nanomolar range was reached. This approach has never been described in the literature.

  2. Computational analysis of siRNA recognition by the Ago2 PAZ domain and identification of the determinants of RNA-induced gene silencing.

    Directory of Open Access Journals (Sweden)

    Mahmoud Kandeel

    Full Text Available RNA interference (RNAi is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.

  3. Growth of aligned ZnO nanowires via modified atmospheric pressure chemical vapor deposition

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yuping; Li, Chengchen [Faculty of Science, Jiangsu University, Zhenjiang, Jiangsu, 212013 (China); Chen, Mingming, E-mail: andychain@live.cn [Faculty of Science, Jiangsu University, Zhenjiang, Jiangsu, 212013 (China); Yu, Xiao; Chang, Yunwei [Faculty of Science, Jiangsu University, Zhenjiang, Jiangsu, 212013 (China); Chen, Anqi [State Key Laboratory of Optoelectronic Materials and Technologies, School of Electronics & Information Technology, Sun Yat-Sen University, Guangzhou Higher Education Mega Center (University Town), Guangzhou, 510006 (China); Zhu, Hai, E-mail: zhuhai5@mail.sysu.edu.cn [State Key Laboratory of Optoelectronic Materials and Technologies, School of Electronics & Information Technology, Sun Yat-Sen University, Guangzhou Higher Education Mega Center (University Town), Guangzhou, 510006 (China); Tang, Zikang, E-mail: zktang@umac.mo [State Key Laboratory of Optoelectronic Materials and Technologies, School of Electronics & Information Technology, Sun Yat-Sen University, Guangzhou Higher Education Mega Center (University Town), Guangzhou, 510006 (China); The Institute of Applied Physics and Materials Engineering, University of Macau, Avenida da Universidade, Taipa, Macau (China)

    2016-12-09

    In this work, we report the growth of high-quality aligned ZnO nanowires via a facile atmospheric pressure chemical vapor deposition (CVD) method. The CVD reactor chamber used was more complicated than a conventional one due to the quartz boats loaded with sources (ZnO/C) and substrates being inserted into a semi-open quartz tube, and then placed inside the CVD reactor. The semi-open quartz tube played a very important role in growing the ZnO nanowires, and demonstrated that the transportation properties of Zn and O vapor differ from those in the conventional CVD reactor chamber. Aligned ZnO nanowires were successfully obtained, though they were only found at substrates located upstream. The very high crystalline quality of the obtained ZnO nanowires was demonstrated by high-resolution transmission electron microscopy and room temperature photoluminescence investigations. Such ZnO nanowires with high crystalline quality may provide opportunities for the fabrication of ZnO-based nano-devices in future. - Highlights: • High-quality aligned ZnO nanowires were obtained via modified chemical vapor deposition under atmospheric pressure. • The semi-open quartz tube plays very important roles in growing ZnO nanowires. • The transportation properties of Zn and O vapor differ from those in the conventional CVD reactor chamber.

  4. Growth of aligned ZnO nanowires via modified atmospheric pressure chemical vapor deposition

    International Nuclear Information System (INIS)

    Zhao, Yuping; Li, Chengchen; Chen, Mingming; Yu, Xiao; Chang, Yunwei; Chen, Anqi; Zhu, Hai; Tang, Zikang

    2016-01-01

    In this work, we report the growth of high-quality aligned ZnO nanowires via a facile atmospheric pressure chemical vapor deposition (CVD) method. The CVD reactor chamber used was more complicated than a conventional one due to the quartz boats loaded with sources (ZnO/C) and substrates being inserted into a semi-open quartz tube, and then placed inside the CVD reactor. The semi-open quartz tube played a very important role in growing the ZnO nanowires, and demonstrated that the transportation properties of Zn and O vapor differ from those in the conventional CVD reactor chamber. Aligned ZnO nanowires were successfully obtained, though they were only found at substrates located upstream. The very high crystalline quality of the obtained ZnO nanowires was demonstrated by high-resolution transmission electron microscopy and room temperature photoluminescence investigations. Such ZnO nanowires with high crystalline quality may provide opportunities for the fabrication of ZnO-based nano-devices in future. - Highlights: • High-quality aligned ZnO nanowires were obtained via modified chemical vapor deposition under atmospheric pressure. • The semi-open quartz tube plays very important roles in growing ZnO nanowires. • The transportation properties of Zn and O vapor differ from those in the conventional CVD reactor chamber.

  5. BioVLAB-MMIA-NGS: microRNA-mRNA integrated analysis using high-throughput sequencing data.

    Science.gov (United States)

    Chae, Heejoon; Rhee, Sungmin; Nephew, Kenneth P; Kim, Sun

    2015-01-15

    It is now well established that microRNAs (miRNAs) play a critical role in regulating gene expression in a sequence-specific manner, and genome-wide efforts are underway to predict known and novel miRNA targets. However, the integrated miRNA-mRNA analysis remains a major computational challenge, requiring powerful informatics systems and bioinformatics expertise. The objective of this study was to modify our widely recognized Web server for the integrated mRNA-miRNA analysis (MMIA) and its subsequent deployment on the Amazon cloud (BioVLAB-MMIA) to be compatible with high-throughput platforms, including next-generation sequencing (NGS) data (e.g. RNA-seq). We developed a new version called the BioVLAB-MMIA-NGS, deployed on both Amazon cloud and on a high-performance publicly available server called MAHA. By using NGS data and integrating various bioinformatics tools and databases, BioVLAB-MMIA-NGS offers several advantages. First, sequencing data is more accurate than array-based methods for determining miRNA expression levels. Second, potential novel miRNAs can be detected by using various computational methods for characterizing miRNAs. Third, because miRNA-mediated gene regulation is due to hybridization of an miRNA to its target mRNA, sequencing data can be used to identify many-to-many relationship between miRNAs and target genes with high accuracy. http://epigenomics.snu.ac.kr/biovlab_mmia_ngs/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. A noninterventional study documenting use and success of implants with a new chemically modified titanium surface in daily dental practice.

    Science.gov (United States)

    Luongo, Giuseppe; Oteri, Giacomo

    2010-01-01

    A new chemically modified titanium surface, SLActive, has recently been developed. The results obtained in controlled clinical trials indicate that this implant can be safely used and that it offers predictable results. The goal of this noninterventional study was to verify that the success rates of implants used in daily dental practice are comparable to those reported in controlled clinical trials. This study was a prospective, noninterventional study using implants with a chemically modified surface according to the daily dental practice procedures applied by private practitioners. The choice of the implantation procedure and the loading protocol were the responsibility of the investigator and were chosen according to the patient's needs. Thirty clinical centers actively participated in this study, and 226 patients were treated, of which, 8 patients were lost to follow-up. Because of the noninterventional design of the study, the patients were not selected according to strictly defined inclusion/exclusion criteria. Thus, the study included individuals with risk factors such as smoking (24%), untreated gingivitis or periodontitis (9%), and bruxism (6%). The implants were equally distributed between mandible (46%) and maxilla (54%). A bone augmentation procedure was done in 31% of the cases. Early loading (functional loading between 48 hours and 3 months after implant insertion) was applied most frequently (48%), followed by the conventional loading protocol (3 to 6 months after implant placement, 34%). Immediate restoration and immediate loading were rare (7% and 2%, respectively). Of 276 implants inserted and documented, 5 implants failures were reported, all of which were associated with a sinus floor augmentation procedure. The survival rate was 98.2% at the 1-year follow-up visit. The results showed that implants with a chemically modified surface can be successfully restored with success rates similar to those reported in formal clinical trials under more

  7. The European Regulatory Environment of RNA-Based Vaccines.

    Science.gov (United States)

    Hinz, Thomas; Kallen, Kajo; Britten, Cedrik M; Flamion, Bruno; Granzer, Ulrich; Hoos, Axel; Huber, Christoph; Khleif, Samir; Kreiter, Sebastian; Rammensee, Hans-Georg; Sahin, Ugur; Singh-Jasuja, Harpreet; Türeci, Özlem; Kalinke, Ulrich

    2017-01-01

    A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist that specifically address mRNA-based vaccines. The existing regulatory framework, however, clearly defines that mRNA-based vaccines in most cases have to be centrally approved. Interestingly, depending on whether RNA-based vaccines are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively. Besides an overview on the current clinical use of mRNA vaccines in various therapeutic areas a detailed discussion of the current regulatory situation is provided and regulatory perspectives are discussed.

  8. 40 CFR 721.10151 - Modified styrene, divinylbenzene polymer (generic).

    Science.gov (United States)

    2010-07-01

    ... polymer (generic). 721.10151 Section 721.10151 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10151 Modified styrene, divinylbenzene polymer (generic). (a) Chemical... as modified styrene, divinylbenzene polymer (PMN P-07-642) is subject to reporting under this section...

  9. Nuclear RNA quantification in protoplast cell-cycle phases.

    Science.gov (United States)

    Bergounioux, C; Perennes, C; Brown, S C; Gadal, P

    1988-01-01

    Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.

  10. Interleukin-6 modifies mRNA expression in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Hassing, Helle Adser; Wojtaszewski, Jørgen; Jakobsen, Anne Hviid

    2011-01-01

    Aim: The aim of the present study was to test the hypothesis that interleukin-6 plays a role in exercise-induced PGC-1a and TNFa mRNA responses in skeletal muscle and to examine the potential IL-6 mediated AMPK regulation in these responses. Methods: Whole body IL-6 knockout and wildtype (WT) mal...

  11. Removal of pharmaceutical pollutants from synthetic wastewater using chemically modified biomass of green alga Scenedesmus obliquus.

    Science.gov (United States)

    Ali, Mohamed E M; Abd El-Aty, Azza M; Badawy, Mohamed I; Ali, Rizka K

    2018-04-30

    Pharmaceutical compounds are considered emerging environmental pollutants that have a potential harmful impact on environment and human health. In this study, the biomass of alga (Scenedesmus obliquus) was modified using alkaline solution, and used for the biosorption of tramadol (TRAM) and other pharmaceuticals. The adsorption kinetics and isotherms were investigated. The obtained results reveal high adsorption capacity of tramadol over modified algal biomass (MAB) after 45min with removal percentage of 91%. Pseudo-second order model was well fitted with the experimental data with correlation coefficient (0.999). Biosorption of tramadol on modified algal biomass proceeds with Freundlich isotherm model with correlation coefficient (0.942) that emphasized uptake of TRAM by MAB is driven by chemisorption. FTIR spectra of MAB before and after the adsorption were analyzed; some IR bands were detected with slight shift and low intensity suggesting their involving in adsorption. The tramadol biosorption by MAB is a chemical process as confirmed by Dubinin-Radushkevich. The adsorption of pharmaceutical over MAB is mainly preceded by hydrophilic interactions between amino and carbonyl groups in pharmaceutical molecules and hydroxyl and carbonyl functional groups on surface of biosorbent. It was emphasized by disappearance O-H and C-O from biomass IR spectra after adsorption. In matrix of pharmaceutical, the recorded adsorption capacities for CEFA, PARA, IBU, TRAM and CIP are 68, 58, 42, 42 and 39mg/g over MAB at natural pH and MAB dose of 0.5g/L. Furthermore, oxygen uptake by bacteria was applied for estimate the toxicity of pharmaceutical. The recorded result concluded the efficient reusability of modified algal biomass for biosorption of pharmaceuticals, as well only the adsorption efficiency decreased by 4.5% after three runs. Subsequently, the modified algal biomass is a promising reusable adsorbent for decontamination of wastewater from pharmaceuticals. Copyright

  12. Canonical A-to-I and C-to-U RNA editing is enriched at 3'UTRs and microRNA target sites in multiple mouse tissues.

    Directory of Open Access Journals (Sweden)

    Tongjun Gu

    Full Text Available RNA editing is a process that modifies RNA nucleotides and changes the efficiency and fidelity of the central dogma. Enzymes that catalyze RNA editing are required for life, and defects in RNA editing are associated with many diseases. Recent advances in sequencing have enabled the genome-wide identification of RNA editing sites in mammalian transcriptomes. Here, we demonstrate that canonical RNA editing (A-to-I and C-to-U occurs in liver, white adipose, and bone tissues of the laboratory mouse, and we show that apparent non-canonical editing (all other possible base substitutions is an artifact of current high-throughput sequencing technology. Further, we report that high-confidence canonical RNA editing sites can cause non-synonymous amino acid changes and are significantly enriched in 3' UTRs, specifically at microRNA target sites, suggesting both regulatory and functional consequences for RNA editing.

  13. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

    Energy Technology Data Exchange (ETDEWEB)

    Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Hong, Li [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States); LeBlanc, Gerald A., E-mail: Gerald_LeBlanc@ncsu.edu [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States)

    2011-01-25

    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  14. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

    International Nuclear Information System (INIS)

    Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Li Hong; LeBlanc, Gerald A.

    2011-01-01

    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  15. Methods for Determination of 2′-O-Me in RNA

    DEFF Research Database (Denmark)

    Birkedal, Ulf; Krogh, Nicolai; Andersen, Kasper Langebjerg

    2016-01-01

    mechanism of ribose methylation is found in rRNA of Archaea and Eukarya where a methyltransferase (fibrillarin) use sRNA (Archaea) or box C/D snoRNA (Eukarya) as guide RNAs to specify the site of modification. The general function of these modifications is to promote ribosome biogenesis, in particular...... for mapping modifications and quantitating the fraction of RNA molecules modified in a population until recently remained poorly developed. Here, we review the methods that have been used to study 2′-O-Me in RNA starting with the original approach employing in vivo isotope labeling followed by paper......Ribose methylation is one of the most abundant RNA modifications and is found in all kingdoms of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. The most abundant...

  16. Extraction of metal ions using chemically modified silica gel: a PIXE analysis.

    Science.gov (United States)

    Jal, P K; Dutta, R K; Sudarshan, M; Saha, A; Bhattacharyya, S N; Chintalapudi, S N; K Mishra, B

    2001-08-30

    Organic ligand with carboxyhydrazide functional group was immobilised on the surface of silica gel and the metal binding capacity of the ligand-embedded silica was investigated. The functional group was covalently bonded to the silica matrix through a spacer of methylene groups by sequential reactions of silica gel with dibromobutane, malonic ester and hydrazine in different media. Surface area value of the modified silica was determined. The changes in surface area were correlated with the structural change of the silica surface due to chemical modifications. A mixture solution of metal ions [K(I),Cr(III),Co(II),Ni(II),Cu(II),Zn(II),Hg(II) and U(VI)] was treated with the ligand-embedded silica in 10(-3) M aqueous solution. The measurement of metal extraction capacity of the silica based ligand was done by multielemental analysis of the metal complexes thus formed by using Proton Induced X-ray Emission (PIXE) technique.

  17. Recent advances in developing small molecules targeting RNA.

    Science.gov (United States)

    Guan, Lirui; Disney, Matthew D

    2012-01-20

    RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void.

  18. EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

    Science.gov (United States)

    Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

    2016-11-01

    Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

  19. miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity

    DEFF Research Database (Denmark)

    Moore, Michael J; Scheel, Troels K H; Luna, Joseph M

    2015-01-01

    microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modifie...

  20. Nuclease-resistant c-di-AMP derivatives that differentially recognize RNA and protein receptors

    Science.gov (United States)

    Meehan, Robert E.; Torgerson, Chad D.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

    2016-01-01

    The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways. PMID:26789423

  1. Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: characterization of the oxidation and oxidative desulfurization of 2-thiouridine.

    Science.gov (United States)

    Sochacka, E

    2001-01-01

    In order to efficiently assess the chemical stability of modified nucleosides to the reagents and conditions of automated oligonucleotide synthesis, we designed, developed and tested a scheme in which the modified nucleoside, directly attached to a solid support, is exposed to the cyclic chemistry of the instrument. Stability of 2-thiouridine against different oxidizers was investigated. Tertbutyl hydroperoxide (1 M) in anhydrous acetonitrile was a more effective oxidizer for the incorporation of 2-thiouridine into oligonucleotide chains than the same oxidizer in methylene chloride. Carbon tetrachloride/water in the presence of a basic catalyst was superior in maintaining the thiocarbonyl function, but its utility for RNA synthesis has yet to be fully tested, whereas 2-phenylsulfonyloxaziridine was a very efficient reagent for oxidative desulfurization of 2-thiouridine.

  2. Biosorption of Cu (II onto chemically modified waste mycelium of Aspergillus awamori: Equilibrium, kinetics and modeling studies

    Directory of Open Access Journals (Sweden)

    ZDRAVKA VELKOVA

    2012-01-01

    Full Text Available The biosorption potential of chemically modified waste mycelium of industrial xylanase-producing strain Aspergillus awamori for Cu (II removal from aqueous solutions was evaluated. The influence of pH, contact time and initial Cu (II concentration on the removal efficiency was evaluated. Maximum biosorption capacity was reached by sodium hydroxide treated waste fungal mycelium at pH 5.0. The Langmuir adsorption equation matched very well the adsorption equilibrium data in the studied conditions. The process kinetic followed the pseudo-firs order model.

  3. Stress-dependent cardiac remodeling occurs in the absence of microRNA-21 in mice

    DEFF Research Database (Denmark)

    Patrick, David M; Montgomery, Rusty L; Qi, Xiaoxia

    2010-01-01

    MicroRNAs inhibit mRNA translation or promote mRNA degradation by binding complementary sequences in 3' untranslated regions of target mRNAs. MicroRNA-21 (miR-21) is upregulated in response to cardiac stress, and its inhibition by a cholesterol-modified antagomir has been reported to prevent card...

  4. The successes and future prospects of the linear antisense RNA amplification methodology.

    Science.gov (United States)

    Li, Jifen; Eberwine, James

    2018-05-01

    It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

  5. Investigation of chemical modifiers for sulfur determination in diesel fuel samples by high-resolution continuum source graphite furnace molecular absorption spectrometry using direct analysis

    Energy Technology Data Exchange (ETDEWEB)

    Huber, Charles S. [Instituto Federal Sul-rio-grandense, Câmpus Pelotas, Pelotas, RS (Brazil); Universidade Federal do Rio Grande do Sul, Instituto de Química, Porto Alegre, RS (Brazil); Vale, Maria Goreti R. [Universidade Federal do Rio Grande do Sul, Instituto de Química, Porto Alegre, RS (Brazil); Instituto Nacional de Ciência e Tecnologia do CNPq, INCT de Energia e Ambiente, Universidade Federal da Bahia, Salvador, BA (Brazil); Welz, Bernhard [Instituto Nacional de Ciência e Tecnologia do CNPq, INCT de Energia e Ambiente, Universidade Federal da Bahia, Salvador, BA (Brazil); Universidade Federal de Santa Catarina, Departamento de Química, Florianópolis, SC (Brazil); Andrade, Jailson B. [Instituto Nacional de Ciência e Tecnologia do CNPq, INCT de Energia e Ambiente, Universidade Federal da Bahia, Salvador, BA (Brazil); Dessuy, Morgana B., E-mail: mbdessuy@ufrgs.br [Universidade Federal do Rio Grande do Sul, Instituto de Química, Porto Alegre, RS (Brazil); Instituto Nacional de Ciência e Tecnologia do CNPq, INCT de Energia e Ambiente, Universidade Federal da Bahia, Salvador, BA (Brazil)

    2015-06-01

    High-resolution continuum source graphite furnace molecular absorption spectrometry has been applied for sulfur determination in diesel fuel. The sharp rotational lines of the carbon monosulfide molecule (formed during the vaporization step) were used to measure the absorbance. The analytical line at 258.056 nm was monitored using the sum of three pixels. Different chemical modifiers were investigated and the mixture of palladium and magnesium was used as chemical modifier in combination with iridium as permanent modifier. L-Cysteine was chosen as sulfur standard and the calibration was done against aqueous standard solutions. The proposed method was applied for the analyses of four diesel samples: two S10 samples and two S500 samples. The trueness of the method was checked with a certified reference material (CRM) of sulfur in diesel fuel (NIST 2724b). Accurate results, for samples and CRM, were achieved after a dilution with propan-1-ol. The following figures of merit were obtained: characteristic mass of 17 ± 3 ng, limit of detection and limit of quantification of 1.4 mg kg{sup −1} and 4.7 mg kg{sup −1}, respectively. - Highlights: • Ir, Ru and Zr were investigated as permanent modifiers. • Ca, Mg, Pd and Pd/Mg were investigated as modifiers in solution. • Indirect determination of sulfur monitoring the molecular absorbance of the CS • Direct analysis of diesel samples using a dilution in propan-1-ol.

  6. Assay reproducibility in clinical studies of plasma miRNA.

    Directory of Open Access Journals (Sweden)

    Jonathan Rice

    Full Text Available There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas and 16 patients without any neoplasm (controls. Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy. We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4% documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ≤12 h provided consistent ΔCt. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively. A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was

  7. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    Science.gov (United States)

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. © 2015 Lama and Ryan; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Polypropylene-modified kaolinite composites: Effect of chemical ...

    African Journals Online (AJOL)

    PP/kaolinite compounds were prepared by the melt intercalation method. The effects of modified clay on properties of the prepared composites were studied. The XRD results showed that the treatment with the ammonium salt caused the return to the initial state of the clay. The thermogravimetric analysis thermograms (TGA) ...

  9. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

    Directory of Open Access Journals (Sweden)

    Randall Marcelo Chin

    2018-03-01

    Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

  10. An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing

    DEFF Research Database (Denmark)

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria

    2016-01-01

    /2 12 min (naked) to t1/2 45 min (single cholesteryl) and t1/2 71 min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing......HSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies...

  11. Investigation of chemical modifiers for phosphorus in a graphite furnace using high-resolution continuum source atomic absorption spectrometry

    International Nuclear Information System (INIS)

    Lepri, Fabio G.; Dessuy, Morgana B.; Vale, Maria Goreti R.; Borges, Daniel L.G.; Welz, Bernhard; Heitmann, Uwe

    2006-01-01

    Phosphorus is not one of the elements that are typically determined by atomic absorption spectrometry, but this technique nevertheless offers several advantages that make it attractive, such as the relatively great freedom from interferences. As the main resonance lines for phosphorus are in the vacuum-ultraviolet, inaccessible by conventional atomic absorption spectrometry equipment, L'vov and Khartsyzov proposed to use the non-resonance doublet at 213.5 / 213.6 nm. Later it turned out that with conventional equipment it is necessary to use a chemical modifier in order to get reasonable sensitivity, and lanthanum was the first one suggested for that purpose. In the following years more than 30 modifiers have been proposed for the determination of this element, and there is no consensus about the best one. In this work high-resolution continuum source atomic absorption spectrometry has been used to investigate the determination of phosphorus without a modifier and with the addition of selected modifiers of very different nature, including the originally recommended lanthanum modifier, several palladium-based modifiers and sodium fluoride. As high-resolution continuum source atomic absorption spectrometry is revealing the spectral environment of the analytical line at high resolution, it became obvious that without the addition of a modifier essentially no atomic phosphorus is formed, even at 2700 deg. C . The absorption measured with line source atomic absorption spectrometry in this case is due to the PO molecule, the spectrum of which is overlapping with the atomic line. Palladium, with or without the addition of calcium or ascorbic acid, was found to be the only modifier to produce almost exclusively atomic phosphorus. Lanthanum and particularly sodium fluoride produced a mixture of P and PO, depending on the atomization temperature. This fact can explain at least some of the discrepancies found in the literature and some of the phenomena observed in the

  12. Implant materials modified by colloids

    Directory of Open Access Journals (Sweden)

    Zboromirska-Wnukiewicz Beata

    2016-03-01

    Full Text Available Recent advances in general medicine led to the development of biomaterials. Implant material should be characterized by a high biocompatibility to the tissue and appropriate functionality, i.e. to have high mechanical and electrical strength and be stable in an electrolyte environment – these are the most important properties of bioceramic materials. Considerations of biomaterials design embrace also electrical properties occurring on the implant-body fluid interface and consequently the electrokinetic potential, which can be altered by modifying the surface of the implant. In this work, the surface of the implants was modified to decrease the risk of infection by using metal colloids. Nanocolloids were obtained using different chemical and electrical methods. It was found that the colloids obtained by physical and electrical methods are more stable than colloids obtained by chemical route. In this work the surface of modified corundum implants was investigated. The implant modified by nanosilver, obtained by electrical method was selected. The in vivo research on animals was carried out. Clinical observations showed that the implants with modified surface could be applied to wounds caused by atherosclerotic skeleton, for curing the chronic and bacterial inflammations as well as for skeletal reconstruction surgery.

  13. Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex.

    Science.gov (United States)

    Cukras, Anthony R; Southworth, Daniel R; Brunelle, Julie L; Culver, Gloria M; Green, Rachel

    2003-08-01

    Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.

  14. Chemical shift assignments of the first and second RRMs of Nrd1, a fission yeast MAPK-target RNA binding protein.

    Science.gov (United States)

    Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

    2017-10-01

    Negative regulator differentiation 1 (Nrd1), a fission yeast RNA binding protein, modulates cytokinesis and sexual development and contributes to stress granule formation in response to environmental stresses. Nrd1 comprises four RRM domains and binds and stabilizes Cdc4 mRNA that encodes the myosin II light chain. Nrd1 binds the Cpc2 fission-yeast RACK1 homolog, and the interaction promotes Nrd1 localization to stress granules. Interestingly, Pmk1 mitogen-activated protein kinase phosphorylates Thr40 in the unstructured N-terminal region and Thr126 in the first RRM domain of Nrd1. Phosphorylation significantly reduces RNA-binding activity and likely modulates Nrd1 function. To reveal the relationship between the structure and function of Nrd1 and how phosphorylation affects structure, we used heteronuclear NMR techniques to investigate the three-dimensional structure of Nrd1. Here we report the 1 H, 13 C, and 15 N resonance assignments of RRM1-RRM2 (residues 108-284) comprising the first and second RRMs obtained using heteronuclear NMR techniques. Secondary structures derived from the chemical shifts are reported. These data should contribute to the understanding of the three-dimensional structure of the RRM1-RRM2 region of Nrd1 and the perturbation caused by phosphorylation.

  15. The Long-Term Study of Some mechanical and Chemical Properties of Polymer Modified Cement Waste Forms

    International Nuclear Information System (INIS)

    Shatta, H.A.; Eskander, S.B.

    2002-01-01

    The present work is a new experimental trend to study the long-term durability of polymer/cement/borate waste forms in storage and disposal sites during the long period of time. The data obtained indicate that all the samples of polymer modified cement waste forms immersed in both distilled and ground water (for more than 2700 days) show a slight increase in the compressive strength values. The chemical stability of the immobilized borate waste forms in distilled and ground water was studied. The cumulative leach fraction in percentage, of Cs-137 isotope from the final solidified waste forms in case of using ground water was lower than that in distilled water for all samples used and the presence of polymer lowered the percent release of radioactive Cs-137 isotope in all samples studied . The leach coefficients (Lc) and the leach indices (Li) of radioactive cesium-137 isotope for cement borate and polymer modified cement waste forms were also calculated. It was found that the leach indices values greater than 6

  16. Evidence of pestivirus RNA in human virus vaccines.

    Science.gov (United States)

    Harasawa, R; Tomiyama, T

    1994-01-01

    We examined live virus vaccines against measles, mumps, and rubella for the presence of pestivirus RNA or of pestiviruses by reverse transcription PCR. Pestivirus RNA was detected in two measles-mumps-rubella combined vaccines and in two monovalent vaccines against mumps and rubella. Nucleotide sequence analysis of the PCR products indicated that a modified live vaccine strain used for immunization of cattle against bovine viral diarrhea is not responsible for the contamination of the vaccines. Images PMID:8077414

  17. Developing electrodes chemically modified with cucurbit[6]uril to detect 3,4-methylenedioxymethamphetamine (MDMA) by voltammetry

    International Nuclear Information System (INIS)

    Tadini, Maraine Catarina; Balbino, Marco Antonio; Eleoterio, Izabel Cristina; Siqueirade Oliveira, Laura; Dias, Luis Gustavo; Jean-François Demets, Grégoire; Firmino de Oliveira, Marcelo

    2014-01-01

    Graphical abstract: - Highlights: • A new stand in forensic chemistry. • Voltammetric method for the determination of MDMA in seized samples. • A new voltammetric sensor for MDMA. - Abstract: This study aimed to develop an electrode chemically modified with cucurbit[6]uril to detect 3,4-methylenedioxymethamphetamine (MDMA), the main active principle of ecstasy samples, by voltammetry. We modified the electrode surface with a film containing cucurbit[6]uril, Nafion, and methanol, using the dip coating or the spin coating technique. During analysis, we employed an electrochemical cell with a conventional three-electrode system and KCl solution (0.1 mol L −1 ) as the supporting electrolyte. We conducted cyclic voltammetry at concentrations ranging from 4.2 × 10 −6 to 4.8 × 10 −5 mol L −1 . We also accomplished scanning electron microscopy, to investigate the structural behavior of the film that originated on the electrode surface. We obtained the following results when we used dip coating to prepare the modified electrode: standard deviation (SD) = 0.024 μA, limit of detection (LOD) = 3.5 μmol L −1 , limit of quantification (LOQ) = 11.7 μmol L −1 , and amperometric sensitivity (m) = 20.9 × 10 3 μA L mol −1 . As for spin coating, we obtained SD = 0.024 μA, LOD = 2.7 μmol L −1 , LOQ = 9.1 μmol L −1 and m = 25.9 × 10 3 μA mol L −1 . These are very promising data: the modified electrode is more sensitive than the conventional glassy carbon electrode under the studied experimental conditions

  18. Interaction of tRNA with Eukaryotic Ribosome

    Directory of Open Access Journals (Sweden)

    Dmitri Graifer

    2015-03-01

    Full Text Available This paper is a review of currently available data concerning interactions of tRNAs with the eukaryotic ribosome at various stages of translation. These data include the results obtained by means of cryo-electron microscopy and X-ray crystallography applied to various model ribosomal complexes, site-directed cross-linking with the use of tRNA derivatives bearing chemically or photochemically reactive groups in the CCA-terminal fragment and chemical probing of 28S rRNA in the region of the peptidyl transferase center. Similarities and differences in the interactions of tRNAs with prokaryotic and eukaryotic ribosomes are discussed with concomitant consideration of the extent of resemblance between molecular mechanisms of translation in eukaryotes and bacteria.

  19. Directed self-assembly of nanogold using a chemically modified nanopatterned surface

    Science.gov (United States)

    Nidetz, Robert; Kim, Jinsang

    2012-02-01

    Electron-beam lithography (EBL) was used to define an aminosilane nanopatterned surface in order to electrostatically self-assemble gold nanoparticles (Au NPs). The chemically modified nanopatterned surfaces were immersed into a Au NP solution to allow the Au NPs to self-assemble. Equilibrium self-assembly was achieved in only 20 min. The number of Au NPs that self-assembled on an aminosilane dot was controlled by manipulating the diameters of both the Au NPs and the dots. Adding salt to the Au NP solution enabled the Au NPs to self-assemble in greater numbers on the same sized dot. However, the preparation of the Au NP solution containing salt was sensitive to spikes in the salt concentration. These spikes led to aggregation of the Au NPs and non-specific deposition of Au NPs on the substrate. The Au NP patterned surfaces were immersed in a sodium hydroxide solution in order to lift-off the patterned Au NPs, but no lift-off was observed without adequate physical agitation. The van der Waals forces are too strong to allow for lift-off despite the absence of electrostatic forces.

  20. Directed self-assembly of nanogold using a chemically modified nanopatterned surface

    International Nuclear Information System (INIS)

    Nidetz, Robert; Kim, Jinsang

    2012-01-01

    Electron-beam lithography (EBL) was used to define an aminosilane nanopatterned surface in order to electrostatically self-assemble gold nanoparticles (Au NPs). The chemically modified nanopatterned surfaces were immersed into a Au NP solution to allow the Au NPs to self-assemble. Equilibrium self-assembly was achieved in only 20 min. The number of Au NPs that self-assembled on an aminosilane dot was controlled by manipulating the diameters of both the Au NPs and the dots. Adding salt to the Au NP solution enabled the Au NPs to self-assemble in greater numbers on the same sized dot. However, the preparation of the Au NP solution containing salt was sensitive to spikes in the salt concentration. These spikes led to aggregation of the Au NPs and non-specific deposition of Au NPs on the substrate. The Au NP patterned surfaces were immersed in a sodium hydroxide solution in order to lift-off the patterned Au NPs, but no lift-off was observed without adequate physical agitation. The van der Waals forces are too strong to allow for lift-off despite the absence of electrostatic forces. (paper)

  1. REDIdb 3.0: A Comprehensive Collection of RNA Editing Events in Plant Organellar Genomes.

    Science.gov (United States)

    Lo Giudice, Claudio; Pesole, Graziano; Picardi, Ernesto

    2018-01-01

    RNA editing is an important epigenetic mechanism by which genome-encoded transcripts are modified by substitutions, insertions and/or deletions. It was first discovered in kinetoplastid protozoa followed by its reporting in a wide range of organisms. In plants, RNA editing occurs mostly by cytidine (C) to uridine (U) conversion in translated regions of organelle mRNAs and tends to modify affected codons restoring evolutionary conserved aminoacid residues. RNA editing has also been described in non-protein coding regions such as group II introns and structural RNAs. Despite its impact on organellar transcriptome and proteome complexity, current primary databases still do not provide a specific field for RNA editing events. To overcome these limitations, we developed REDIdb a specialized database for RNA editing modifications in plant organelles. Hereafter we describe its third release containing more than 26,000 events in a completely novel web interface to accommodate RNA editing in its genomics, biological and evolutionary context through whole genome maps and multiple sequence alignments. REDIdb is freely available at http://srv00.recas.ba.infn.it/redidb/index.html.

  2. REDIdb 3.0: A Comprehensive Collection of RNA Editing Events in Plant Organellar Genomes

    Directory of Open Access Journals (Sweden)

    Claudio Lo Giudice

    2018-04-01

    Full Text Available RNA editing is an important epigenetic mechanism by which genome-encoded transcripts are modified by substitutions, insertions and/or deletions. It was first discovered in kinetoplastid protozoa followed by its reporting in a wide range of organisms. In plants, RNA editing occurs mostly by cytidine (C to uridine (U conversion in translated regions of organelle mRNAs and tends to modify affected codons restoring evolutionary conserved aminoacid residues. RNA editing has also been described in non-protein coding regions such as group II introns and structural RNAs. Despite its impact on organellar transcriptome and proteome complexity, current primary databases still do not provide a specific field for RNA editing events. To overcome these limitations, we developed REDIdb a specialized database for RNA editing modifications in plant organelles. Hereafter we describe its third release containing more than 26,000 events in a completely novel web interface to accommodate RNA editing in its genomics, biological and evolutionary context through whole genome maps and multiple sequence alignments. REDIdb is freely available at http://srv00.recas.ba.infn.it/redidb/index.html

  3. Ion beam-induced topographical and chemical modification on the poly(styrene-co-allyl alcohol) and its effect on the molecular interaction between the modified surface and liquid crystals

    International Nuclear Information System (INIS)

    Jeong, Hae-Chang; Park, Hong-Gyu; Lee, Ju Hwan; Jang, Sang Bok; Oh, Byeong-Yun; Seo, Dae-Shik

    2016-01-01

    We demonstrated uniform liquid crystal (LC) alignment on ion beam (IB)-irradiated poly(styrene-co-allyl alcohol) by modifying the chemical bonding on the surface. An IB-irradiated copolymer was used for the alignment layer. We used physico-chemical analysis to determine the IB-irradiated surface modification and LC alignment mechanism on the surface. During IB treatment on poly(styrene-co-allyl alcohol), IB irradiation induces breaking of chemical bonds on the surface to give rise to new bonds with oxygen atoms. This causes a strong Van der Waals interaction between LCs and the modified surface, thereby resulting in uniform LC alignment. The results of contact angle (CA) studies of the copolymer support the chemical bonding changes that were investigated by XPS. We achieved uniform homogeneous LC alignment and obtained stable electro-optical performance by controlling the IB energy. Therefore, the LC cells with IB-irradiated poly(styrene-co-allyl alcohol) exhibited a good potential for alternative alignment of layers in LC applications.

  4. Ion beam-induced topographical and chemical modification on the poly(styrene-co-allyl alcohol) and its effect on the molecular interaction between the modified surface and liquid crystals

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Hae-Chang [Information Display Device Laboratory, Department of Electrical and Electronic Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749 (Korea, Republic of); Park, Hong-Gyu [School of Electrical, Electronic & Control Engineering, Changwon National University, 20 Changwondaehak-ro, Uichang-gu, Changwon-si, Gyeongsangnam-do, 51140 (Korea, Republic of); Lee, Ju Hwan; Jang, Sang Bok [Information Display Device Laboratory, Department of Electrical and Electronic Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749 (Korea, Republic of); Oh, Byeong-Yun [ZeSHTech Co., Ltd., Business Incubator, Gwangju Institute of Science and Technology, 123, Cheomdangwagi-ro, Buk-gu, Gwangju, 500-712 (Korea, Republic of); Seo, Dae-Shik, E-mail: dsseo@yonsei.ac.kr [Information Display Device Laboratory, Department of Electrical and Electronic Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749 (Korea, Republic of)

    2016-10-01

    We demonstrated uniform liquid crystal (LC) alignment on ion beam (IB)-irradiated poly(styrene-co-allyl alcohol) by modifying the chemical bonding on the surface. An IB-irradiated copolymer was used for the alignment layer. We used physico-chemical analysis to determine the IB-irradiated surface modification and LC alignment mechanism on the surface. During IB treatment on poly(styrene-co-allyl alcohol), IB irradiation induces breaking of chemical bonds on the surface to give rise to new bonds with oxygen atoms. This causes a strong Van der Waals interaction between LCs and the modified surface, thereby resulting in uniform LC alignment. The results of contact angle (CA) studies of the copolymer support the chemical bonding changes that were investigated by XPS. We achieved uniform homogeneous LC alignment and obtained stable electro-optical performance by controlling the IB energy. Therefore, the LC cells with IB-irradiated poly(styrene-co-allyl alcohol) exhibited a good potential for alternative alignment of layers in LC applications.

  5. An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

    Science.gov (United States)

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria; Dagnæs-Hansen, Frederik; Wengel, Jesper; Malle, Birgitte Mølholm; Kragh-Hansen, Ulrich; Cameron, Jason; Bukrinski, Jens Thostrup; Howard, Kenneth A

    2016-06-28

    Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. A radiobiological approach to cancer treatment. Possible chemical and physical agents modifying radiosensitivity in comparison with high LET radiations

    International Nuclear Information System (INIS)

    Sugahara, T.

    1982-01-01

    Biological characteristics of high LET radiations are summarized to be low oxygen enhancement ratio, high RBE, low repair and low cell cycle dependency of radiosensitivity. Various chemical modifiers of radiosensitivity and radiological effect of hyperthermia are classified into these four properties. It is evident that we have now various means to mimic high LET radiations as far as biological response is concerned though some of them are still in experimental stage. Among them, the means to cope with hypoxia and repair which are assumed to be the most important causes of radioresistance of human tumors are discussed in some detail. It is expected that through the present seminar we would have consensus to concentrate our effort of development for new modifying means available and useful in developing countries. (author)

  7. Functional RNA structures throughout the Hepatitis C Virus genome.

    Science.gov (United States)

    Adams, Rebecca L; Pirakitikulr, Nathan; Pyle, Anna Marie

    2017-06-01

    The single-stranded Hepatitis C Virus (HCV) genome adopts a set of elaborate RNA structures that are involved in every stage of the viral lifecycle. Recent advances in chemical probing, sequencing, and structural biology have facilitated analysis of RNA folding on a genome-wide scale, revealing novel structures and networks of interactions. These studies have underscored the active role played by RNA in every function of HCV and they open the door to new types of RNA-targeted therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins.

    Science.gov (United States)

    Schiera, Gabriella; Di Liegro, Carlo Maria; Puleo, Veronica; Colletta, Oriana; Fricano, Anna; Cancemi, Patrizia; Di Cara, Gianluca; Di Liegro, Italia

    2016-11-01

    Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2.

  9. Functional properties of unmodified and modified Jack bean ...

    African Journals Online (AJOL)

    The native Jack bean (Canavalia eniformis) starch was chemically modified through oxidation and acetylation. Proximate composition analysis revealed higher moisture, protein, fat and ash contents 'native unmodified than modified starches and higher yield in modified starches. Swelling capacity and solubility of all the ...

  10. Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

    Science.gov (United States)

    Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

    2014-12-01

    Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

  11. Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

    DEFF Research Database (Denmark)

    Fisker, Niels; Westergaard, Majken; Hansen, Henrik Frydenlund

    2007-01-01

    We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced ...

  12. Intracellular Delivery of siRNA by Polycationic Superparamagnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Betzaida Castillo

    2012-01-01

    Full Text Available The siRNA transfection efficiency of nanoparticles (NPs, composed of a superparamagnetic iron oxide core modified with polycationic polymers (poly(hexamethylene biguanide or branched polyethyleneimine, were studied in CHO-K1 and HeLa cell lines. Both NPs demonstrated to be good siRNA transfection vehicles, but unmodified branched polyethyleneimine (25 kD was superior on both cell lines. However, application of an external magnetic field during transfection (magnetofection increased the efficiency of the superparamagnetic NPs. Furthermore, our results reveal that these NPs are less toxic towards CHO-K1 cell lines than the unmodified polycationic-branched polyethyleneimine (PEI. In general, the external magnetic field did not alter the cell’s viability nor it disrupted the cell membranes, except for the poly(hexamethylene biguanide-modified NP, where it was observed that in CHO-K1 cells application of the external magnetic field promoted membrane damage. This paper presents new polycationic superparamagnetic NPs as promising transfection vehicles for siRNA and demonstrates the advantages of magnetofection.

  13. The use of chemical modified chitosan with succinic anhydride in the methylene blue adsorption

    International Nuclear Information System (INIS)

    Lima, Ilauro S.; Ribeiro, Emerson S.; Airoldi, Claudio

    2006-01-01

    The adsorption capacity of a-chitosan and its modified form with succinic anhydride was compared with the traditional adsorbent active carbon by using the dye methylene blue, employed in the textile industry. The isotherms for both biopolymers were classified as SSA systems in the Giles model, more specifically in L class and subgroup 3. The dye concentration in the supernatant in the adsorption assay was determined through electronic spectroscopy. By calorimetric titration thermodynamic data of the interaction between methylene blue and the chemically modified chitosan at the solid/liquid interface were obtained. The enthalpy of the dye/chitosan interaction gave 2.47 ± 0.02 kJ mol-1 with an equilibrium constant of 7350 ± 10 and for the carbon/dye interaction this constant gave 5951 ± 8. The spontaneity of these adsorptions are reflected by the free Gibbs energies of -22.1 ± 0.4 and -21.5 ± 0.2 kJ mol-1, respectively, found for these systems. This new adsorbent derived from a natural polysaccharide is as efficient as activated carbon. However 97% of the bonded dye can be eluted by sodium chloride solution, while this same operation elutes only 42% from carbon. Chitosan is efficient in dye removal with the additional advantage of being cheap, non-toxic, biocompatible and biodegradable. (author)

  14. Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

    Directory of Open Access Journals (Sweden)

    Pablo Cordero

    2015-11-01

    Full Text Available Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2 and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10% and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

  15. Progress in chemical treatment of LEU targets by the modified Cintichem process

    International Nuclear Information System (INIS)

    Wu, D.; Landsberger, S.; Vandegrift, G.F.

    1996-01-01

    Presented here are recent experimental results on tests of a modified Cintichem process for producing 99 Mo from low enriched uranium (LEU). Studies were focused in three areas: (1) testing the effects on 99 Mo recovery and purity of dissolving LEU foil in nitric acid alone, rather than in the sulfuric/nitric acid mixture currently used, (2) measuring decontamination factors for radionuclide impurities in each purification step, and (3) testing the effects on processing of adding barrier materials to the LEU metal-foil target. The experimental results show that switching from dissolving the target in the sulfuric/nitric mixture to using nitric acid alone should cause no significant difference in 99 Mo product yield or purity. Further, the results show that overall decontamination factors for gamma emitters in the LEU-target processing are high enough to meet the purity requirements for the 99 Mo product. The results also show that the selected barrier materials, Cu, Fe, and Ni, do not interfere with 99 Mo recovery and can be removed during chemical processing of the LEU target

  16. A chemically modified [alpha]-amylase with a molten-globule state has entropically driven enhanced thermal stability

    Energy Technology Data Exchange (ETDEWEB)

    Siddiqui, Khawar Sohail; Poljak, Anne; De Francisci, Davide; Guerriero, Gea; Pilak, Oliver; Burg, Dominic; Raftery, Mark J.; Parkin, Don M.; Trewhella, Jill; Cavicchioli, Ricardo (Sydney); (New South)

    2010-11-15

    The thermostability properties of TAA were investigated by chemically modifying carboxyl groups on the surface of the enzyme with AMEs. The TAA{sub MOD} exhibited a 200% improvement in starch-hydrolyzing productivity at 60 C. By studying the kinetic, thermodynamic and biophysical properties, we found that TAA{sub MOD} had formed a thermostable, MG state, in which the unfolding of the tertiary structure preceded that of the secondary structure by at least 20 C. The X-ray crystal structure of TAA{sub MOD} revealed no new permanent interactions (electrostatic or other) resulting from the modification. By deriving thermodynamic activation parameters of TAA{sub MOD}, we rationalised that thermostabilisation have been caused by a decrease in the entropy of the transition state, rather than being enthalpically driven. Far-UV CD shows that the origin of decreased entropy may have arisen from a higher helical content of TAA{sub MOD}. This study provides new insight into the intriguing properties of an MG state resulting from the chemical modification of TAA.

  17. Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs.

    Science.gov (United States)

    Mandal, Debabrata; Köhrer, Caroline; Su, Dan; Babu, I Ramesh; Chan, Clement T Y; Liu, Yuchen; Söll, Dieter; Blum, Paul; Kuwahara, Masayasu; Dedon, Peter C; Rajbhandary, Uttam L

    2014-02-01

    Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2(Ile)) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2(Ile) binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.

  18. Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity.

    Science.gov (United States)

    Wyman, Stacia K; Knouf, Emily C; Parkin, Rachael K; Fritz, Brian R; Lin, Daniel W; Dennis, Lucas M; Krouse, Michael A; Webster, Philippa J; Tewari, Muneesh

    2011-09-01

    Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called "isomiRs" adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here, we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including MTPAP, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes-MTPAP, ZCCHC6, and TUT1-have not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next-generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo.

  19. Development and characterization of chitosan-PEG-TAT nanoparticles for the intracellular delivery of siRNA

    Directory of Open Access Journals (Sweden)

    Malhotra M

    2013-05-01

    Full Text Available Meenakshi Malhotra,1 Catherine Tomaro-Duchesneau,1 Shyamali Saha,2 Imen Kahouli,3 Satya Prakash11Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Faculty of Medicine, 2Faculty of Dentistry, 3Department of Experimental Medicine, McGill University, Montreal, QC, CanadaAbstract: Recently, cell-penetrating peptides have been proposed to translocate antibodies, proteins, and other molecules in targeted drug delivery. The proposed study presents the synthesis and characterization of a peptide-based chitosan nanoparticle for small interfering RNA (siRNA delivery, in-vitro. Specifically, the synthesis included polyethylene glycol (PEG, a hydrophilic polymer, and trans-activated transcription (TAT peptide, which were chemically conjugated on the chitosan polymer. The conjugation was achieved using N-Hydroxysuccinimide-PEG-maleimide (heterobifunctional PEG as a cross-linker, with the bifunctional PEG facilitating the amidation reaction through its N-Hydroxysuccinimide group and reacting with the amines on chitosan. At the other end of PEG, the maleimide group was chemically conjugated with the cysteine-modified TAT peptide. The degree of substitution on chitosan with PEG and on PEG with TAT was confirmed using colorimetric assays. The resultant polymer was used to form nanoparticles complexing siRNA, which were then characterized for particle size, morphology, cellular uptake, and cytotoxicity. The nanoparticles were tested in-vitro on mouse neuroblastoma cells (Neuro2a. Particle size and surface charge were characterized and an optimal pH condition and PEG molecular weight were determined to form sterically stable nanoparticles. Results indicate 7.5% of the amines in chitosan polymer were conjugated to the PEG and complete conjugation of TAT peptide was observed on the synthesized PEGylated chitosan polymer. Compared with unmodified chitosan nanoparticles, the nanoparticles formed at pH 6 were

  20. Transdermal Delivery of siRNA through Microneedle Array

    Science.gov (United States)

    Deng, Yan; Chen, Jiao; Zhao, Yi; Yan, Xiaohui; Zhang, Li; Choy, Kwongwai; Hu, Jun; Sant, Himanshu J.; Gale, Bruce K.; Tang, Tao

    2016-02-01

    Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively.

  1. Peptidomimetics with beta-peptoid resudies as carriers for intracellular delivery of small interfering RNA (siRNA)

    DEFF Research Database (Denmark)

    Foged, Camilla

    cytometry. We conclude that simple complex formation via electrostatic interactions between siRNA and the cationic peptidomimetics is not sufficient for the delivery of siRNA to the RNA interference (RNAi) pathway in the cytoplasm. We are currently testing chemical conjugates of si...... prepared by mixing and characterized with respect to size and surface charge. At ratios of peptide nitrogen to siRNA phosphate (N/P) of 1 and below, particles with narrow size distributions (poly dispersity indexes lower than 0.11) ranging from approximately 100 to 350 nm were formed, and they showed...... a negative zeta potential (-24 to -31 mV). At higher N/P ratios, larger aggregates with zeta potential close to neutral were formed. However, the complexes were not able to silence the expression of enhanced green fluorescent protein (EGFP) in HeLa-cells stably expressing EGFP, which was measured by flow...

  2. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Science.gov (United States)

    Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

    2016-01-01

    The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  3. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Directory of Open Access Journals (Sweden)

    Venkatesh Kota

    Full Text Available The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO, but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP elements from the 5' untranslated regions (UTR of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1 mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  4. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    Science.gov (United States)

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  5. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    Science.gov (United States)

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  6. Fatigue behavior of Ti–6Al–4V alloy in saline solution with the surface modified at a micro- and nanoscale by chemical treatment

    Energy Technology Data Exchange (ETDEWEB)

    Claros, Cesar Adolfo Escobar; Oliveira, Diego Pedreira; Campanelli, Leonardo Contri; Pereira da Silva, Paulo Sergio Carvalho; Bolfarini, Claudemiro

    2016-10-01

    This work evaluated the influence of the surface modification using acid etching combined with alkaline treatment on the fatigue strength of Ti–6Al–4V ELI alloy. The topography developed by chemical surface treatments (CST) was examined by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Increased roughness and effective surface area were investigated and compared with the Ti–6Al–4V samples without modification. Surface composition was analyzed by energy dispersive X-ray spectroscopy (EDS). Axial fatigue resistance of polished and modified surfaces was determined by stepwise load increase tests and staircase test method. Light microscopy and SEM were employed to examine the fracture surface of the tested specimens. According to the results, a similar fatigue behavior was found and a negligible difference in the fatigue crack nucleation was observed for the Ti–6Al–4V with CST in comparison to the samples without treatment. - Highlights: • Fatigue behavior of Ti–6Al–4V with the surface modified by chemical treatments • The topography developed did not induce differences in the fatigue resistance. • Untreated and chemically treated surfaces presented fractographic similarities.

  7. Fatigue behavior of Ti–6Al–4V alloy in saline solution with the surface modified at a micro- and nanoscale by chemical treatment

    International Nuclear Information System (INIS)

    Claros, Cesar Adolfo Escobar; Oliveira, Diego Pedreira; Campanelli, Leonardo Contri; Pereira da Silva, Paulo Sergio Carvalho; Bolfarini, Claudemiro

    2016-01-01

    This work evaluated the influence of the surface modification using acid etching combined with alkaline treatment on the fatigue strength of Ti–6Al–4V ELI alloy. The topography developed by chemical surface treatments (CST) was examined by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Increased roughness and effective surface area were investigated and compared with the Ti–6Al–4V samples without modification. Surface composition was analyzed by energy dispersive X-ray spectroscopy (EDS). Axial fatigue resistance of polished and modified surfaces was determined by stepwise load increase tests and staircase test method. Light microscopy and SEM were employed to examine the fracture surface of the tested specimens. According to the results, a similar fatigue behavior was found and a negligible difference in the fatigue crack nucleation was observed for the Ti–6Al–4V with CST in comparison to the samples without treatment. - Highlights: • Fatigue behavior of Ti–6Al–4V with the surface modified by chemical treatments • The topography developed did not induce differences in the fatigue resistance. • Untreated and chemically treated surfaces presented fractographic similarities.

  8. The chemical juggernaut.

    Science.gov (United States)

    Cadbury, D

    1997-01-01

    Man-made chemicals pervade and support every aspect of modern living. The chemical industry has become such a powerful force in the global economy, sales of synthetic chemicals and products derived from them constitute well in excess of a third of the world's gross national product. But, these man-made chemicals are also 'elixirs of death,' the symbol of human destruction. Laboratory tests have shown that a number of chemicals in common use possess a remarkable property: they can weakly mimic or modify the action of human hormones. It has been proven that some chemicals found in plastics, pesticides, and industrial products are weakly estrogenic, modifying the action of the female hormone. In addition, other chemicals affect the male hormones, androgens, or anti-androgens; others are thought to target different hormone systems, such as thyroid and adrenal glands. Many research studies are being conducted to establish the impact of chemicals on human health. Of special concern are the rising incidence of testicular cancer, decline in human sperm counts, and the sharp rise of breast cancer. In conclusion, although there is a worldwide debate on the effects of chemical exposure on humans, the significance of findings for human health, concerning testicular and breast cancer, are still unknown. An international treaty is called for to control the use of the persistent hormonally active chemicals.

  9. Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

    Science.gov (United States)

    He, Bifang; Tjhung, Katrina F; Bennett, Nicholas J; Chou, Ying; Rau, Andrea; Huang, Jian; Derda, Ratmir

    2018-01-19

    Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

  10. RNA-Targeted Therapies and Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Stéphane Mathis

    2018-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal motor disease in adults. Its pathophysiology remains mysterious, but tremendous advances have been made with the discovery of the most frequent mutations of its more common familial form linked to the C9ORF72 gene. Although most cases are still considered sporadic, these genetic mutations have revealed the role of RNA production, processing and transport in ALS, and may be important players in all ALS forms. There are no disease-modifying treatments for adult human neurodegenerative diseases, including ALS. As in spinal muscular atrophy, RNA-targeted therapies have been proposed as potential strategies for treating this neurodegenerative disorder. Successes achieved in various animal models of ALS have proven that RNA therapies are both safe and effective. With careful consideration of the applicability of such therapies in humans, it is possible to anticipate ongoing in vivo research and clinical trial development of RNA therapies for treating ALS.

  11. RNA-Targeted Therapies and Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Mathis, Stéphane; Le Masson, Gwendal

    2018-01-15

    Amyotrophic lateral sclerosis (ALS) is a fatal motor disease in adults. Its pathophysiology remains mysterious, but tremendous advances have been made with the discovery of the most frequent mutations of its more common familial form linked to the C9ORF72 gene. Although most cases are still considered sporadic, these genetic mutations have revealed the role of RNA production, processing and transport in ALS, and may be important players in all ALS forms. There are no disease-modifying treatments for adult human neurodegenerative diseases, including ALS. As in spinal muscular atrophy, RNA-targeted therapies have been proposed as potential strategies for treating this neurodegenerative disorder. Successes achieved in various animal models of ALS have proven that RNA therapies are both safe and effective. With careful consideration of the applicability of such therapies in humans, it is possible to anticipate ongoing in vivo research and clinical trial development of RNA therapies for treating ALS.

  12. RNA Detection Based on Graphene Field-Effect Transistor Biosensor

    Directory of Open Access Journals (Sweden)

    Meng Tian

    2018-01-01

    Full Text Available Graphene has attracted much attention in biosensing applications due to its unique properties. In this paper, the monolayer graphene was grown by chemical vapor deposition (CVD method. Using the graphene as the electric channel, we have fabricated a graphene field-effect transistor (G-FET biosensor that can be used for label-free detection of RNA. Compared with conventional method, the G-FET RNA biosensor can be run in low cost, be time-saving, and be miniaturized for RNA measurement. The sensors show high performance and achieve the RNA detection sensitivity as low as 0.1 fM, which is two orders of magnitude lower than the previously reports. Moreover, the G-FET biosensor can readily distinguish target RNA from noncomplementary RNA, showing high selectivity for RNA detection. The developed G-FET RNA biosensor with high sensitivity, fast analysis speed, and simple operation may provide a new feasible direction for RNA research and biosensing.

  13. Modified Polyadenylation-Based RT-qPCR Increases Selectivity of Amplification of 3′-MicroRNA Isoforms

    Directory of Open Access Journals (Sweden)

    Charlotte Nejad

    2018-01-01

    Full Text Available MicroRNA (miRNA detection by reverse transcription (RT quantitative real-time PCR (RT-qPCR is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3′-end length variants (“isomiRs”, little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3′-end miRNA isoforms can exhibit 3′-length specific regulatory functions, underlining the need to develop strategies to differentiate 3′-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20–21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3′-isomiRs.

  14. A Two-Piece Derivative of a Group I Intron RNA as a Platform for Designing Self-Assembling RNA Templates to Promote Peptide Ligation

    Directory of Open Access Journals (Sweden)

    Takahiro Tanaka

    2012-01-01

    Full Text Available Multicomponent RNA-peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems. We have developed self-folding and self-assembling RNAs that serve as templates to assist chemical ligation between two reactive peptides with RNA-binding capabilities. The design principle of previous templates, however, can be applied only to limited classes of RNA-binding peptides. In this study, we employed a two-piece derivative of a group I intron RNA from the Tetrahymena large subunit ribosomal RNA (LSU rRNA as a platform for new template RNAs. In this group I intron-based self-assembling platform, modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure. The new self-assembling platform allows us to expand the repertoire of substrate peptides in template RNA design.

  15. Extracellular and circulating redox- and metalloregulated eRNA and eRNP: copper ion-structured RNA cytokines (angiotropin ribokines) and bioaptamer targets imparting RNA chaperone and novel biofunctions to S100-EF-hand and disease-associated proteins.

    Science.gov (United States)

    Wissler, Josef H

    2004-06-01

    Bioassays for cellular differentiation and tissue morphogenesis were used to design methods for isolation of bioactive redox- and metalloregulated nucleic acids and copper ion complexes with proteins from extracellular, circulating, wound, and supernatant fluids of cultured cells. In extracellular biospheres, diversities of nucleic acids were found to be secreted by cells upon activation. They may reflect nucleic acid biolibraries with molecular imprints of cellular history. After removal of protein components, eRNA prototypes exuded by activated cells were sequenced. They are small, endogenous, highly modified and edited, redox- and metalloregulated 5'-end phosphorylated extracellular eRNA (approximately 2-200 bases) with cellular, enzymic, and bioaptamer functions. Fenton-type OH* radical redox reactions may form modified nucleotides in RNA as wobbles eRNA per se, or as copper ion-complex with protein (e.g., S100A12-EF-hand protein, angiotropin-related protein, calgranulin-C, hippocampal neurite differentiation factor) are shown to be bioactive in vivo and in vitro as cytokines (ribokines) and as nonmitogenic angiomorphogens for endothelial cell differentiation in the formation of organoid supracellular capillary structures. As bioaptamers, copper ion-structured eRNA imparts novel biofunctions to proteins that they do not have on their own. The origin of extracellular RNA and intermediate precursors (up to 500 bases) was traced to intracellular parent nucleic acids. Intermediate precursors with and without partial homology were found. This suggests that bioaptamers are not directly retranslatable gene products. Metalloregulated eRNA bioaptamer function was investigated by domains (e.g. 5'...CUG...3' hairpin loop) for folding, bioactivity, and binding of protein with copper, calcium, and alkali metal ion affinity. Vice versa, metalloregulated nucleic acid-binding domains (K3H, R3H) in proteins were identified. Interaction of protein and eRNA docking potentials

  16. Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

    Science.gov (United States)

    Kirpekar, F; Douthwaite, S; Roepstorff, P

    2000-02-01

    We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

  17. THE USE OF CHEMICALS AS PLANT REGULATORS. AGRICULTURAL CHEMICALS TECHNOLOGY, NUMBER 8.

    Science.gov (United States)

    Ohio State Univ., Columbus. Center for Vocational and Technical Education.

    ONE OF A SERIES DESIGNED TO ASSIST TEACHERS IN PREPARING POST-SECONDARY STUDENTS FOR AGRICULTURAL CHEMICAL OCCUPATIONS, THIS MODULE IS SPECIFICALLY CONCERNED WITH CHEMICALS AS PLANT REGULATORS. IT WAS DEVELOPED BY A NATIONAL TASK FORCE ON THE BASIS OF DATA FROM STATE STUDIES. SECTIONS INCLUDE -- (1) CHEMICALS AS MODIFIERS OF PLANT GROWTH, (2)…

  18. Supported liquid membrane stability in chiral resolution by chemically and physically modified membranes

    Energy Technology Data Exchange (ETDEWEB)

    Molinari, R.; Argurio, P. [Arcavata di Rende Univ. of Calabria, Arcavata di Rende, CS (Italy). Dept. of Chemical and Materials Engineering

    2001-04-01

    In the present work some stability studies on Supported Liquid Membranes (SLMs) to be used for chiral separations were realized. In particular, primary aim was to determine how a modification of the support surface influences the SLM stability. First, the procedure for support modification was optimised, making a screening of various compounds (sulphuric acid, nitric acid, chromic acid, sodium dodecyl sulphate (SDS), glycerol, oleic alcohol, propylene glycol (PPG), bovine serum albumin (BSA)) and testing their performance by means of contact angle measurements. Next, a second screening was realized by permeation tests in a stirred cell. Finally, to compare the stability of modified with unmodified support in a process of interest for chemical and/or biochemical industries, some permeation tests for resolution of DNB-DL-Leucine were realized in a re-circulation system. Results showed a better surface hydrophilization of chemically modified support and better stability of the sulphonated support. However, in operating conditions a little high stability of the unmodified support was obtained. [Italian] Nel presente lavoro sono stati realizzati degli studi di stabilita' di Membrane Liquide Supportate (SLMs) da impiegare in separazioni chirali. In particolare, obiettivo principale e' stato quello di determinare l'influenza che una modifica della superficie del supporto ha sulla stabilita' della SLM. Cosi', in un primo momento, e' stata ottimizzata le procedura di modifica del supporto, facendo una selezione tra vari composti (acido solforico, acido nitrico, acido cromico, sodio dodecil solfato (SDS), glicerolo, alcool oleico, glicole propilenico (PPG), siero di albumina bovina (BSA)) basata su misure dell'angolo di contatto. Successivamente, e' stata realizzata una seconda selezione mediante prove di permeazione in una cella agitata. Infine, con lo scopo di confrontare la stabilita' della SLM con supporto modificato rispetto

  19. CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

    Science.gov (United States)

    Busi, Florent; Cresteil, Thierry

    2005-09-01

    The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

  20. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang; Schwarzer, Dirk

    2016-01-01

    and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity, and target sites

  1. The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA

    DEFF Research Database (Denmark)

    Lartigue, Carole; Lebaudy, Anne; Blanchard, Alain

    2014-01-01

    Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl g...... specifically modifies m5U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.......Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl...... group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses...

  2. Development of a new method to identify aminoacylated RNA

    Directory of Open Access Journals (Sweden)

    Wang Ji

    2014-02-01

    Full Text Available A RT-PCR method is developed to isolate RNA aminoacylated on their 3’ end from large pools of RNA. The method is being applied in two separate projects. We are interested in isolating a new class of ribozymes that could successively catalyze the two chemical reactions leading to their own 3’ aminoacylation (ATP activation of an amino acid followed by 3' esterification of the RNA. The catalysis of each of the two reactions has independently been demonstrated for some RNA isolated with the SELEX methodology [1-2]. However, the coupling of both reactions on a same molecule has not been achieved yet. The identification of these still hypothetical ribozymes may help understand how the former translation system started in the absence of the aminoacyltRNA Synthetase, which catalyzes the above two reactions on tRNA in modern cells. In another project, we would like to identify the whole repertoire of aminoacylated RNA (the “aminoacylome” in cells. There are strong indications that other RNA besides tRNA and tmRNA may be aminoacylated for biological purposes [3-4].

  3. Osteoblast cell response to surface-modified carbon nanotubes

    International Nuclear Information System (INIS)

    Zhang Faming; Weidmann, Arne; Nebe, J. Barbara; Burkel, Eberhard

    2012-01-01

    In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.

  4. Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ1

    International Nuclear Information System (INIS)

    Brooks, Allen F.; Vélez-Martínez, Carol S.; Hollis Showalter, H.D.; Garcia, George A.

    2012-01-01

    Highlights: ► Escherichia coli cells deficient in preQ 1 biosynthesis can utilize exogenous preQ 1 from growth media. ► Exogenous radiolabeled preQ 1 is incorporated into cellular RNA in both wild-type and preQ 1 deficient cells. ► Radiolabeled preQ 1 is incorporated into RNAs of differing sizes larger than tRNA. ► Analysis of isolated ribosomes shows that radiolabeled preQ 1 is incorporated into ribosomal RNA. ► The conclusion of this study is that queuine is incorporated into many other RNAs in addition to the known tRNAs. -- Abstract: There are over 100 modified bases that occur in RNA with the majority found in transfer RNA. It has been widely believed that the queuine modification is limited to four transfer RNA species in vivo. However, given the vast amount of the human genome (60–70%) that is transcribed into non-coding RNA (Mattick ), probing the presence of modified bases in these RNAs is of fundamental importance. The mechanism of incorporation of queuine, via transglycosylation, makes this uniquely poised to probe base modification in RNA. Results of incubations of Escherichia coli cell cultures with [ 3 H] preQ 1 (a queuine precursor in eubacteria) clearly demonstrate preQ 1 incorporation into a number of RNA species of various sizes larger than transfer RNA. Specifically, significant levels of preQ 1 incorporation into ribosomal RNA are observed. The modification of other large RNAs was also observed. These results confirm that non-coding RNAs contain modified bases and lead to the supposition that these modifications are necessary to control non-coding RNA structure and function as has been shown for transfer RNA.

  5. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Wengel, Jesper; Stenvang, Jan

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been a...

  6. The role of microRNAs in the development and progression of chemical-associated cancers

    International Nuclear Information System (INIS)

    Pogribny, Igor P.; Beland, Frederick A.; Rusyn, Ivan

    2016-01-01

    Human exposure to certain natural and man-made chemical carcinogens is one of the major risk factors for cancer development. The effect of chemical carcinogens on genetic and epigenetic alterations and their significance in the development of cancer has been well-established. In contrast, the role of microRNAs (miRNAs) in the etiology of chemical-associated cancers remains relatively unexplored despite extensive reports on changes in miRNA expression upon carcinogen exposure. This review summarizes the current knowledge for the role of miRNAs as drivers of chemical-induced carcinogenesis by bridging the gap between carcinogen exposure and cancer development through functional studies. It also emphasizes the potential for miRNA changes as early indicators of the carcinogenic process, markers for carcinogen exposure, and identification of chemical carcinogenic hazards. - Highlights: • Exposure to chemical carcinogens alters microRNA expression. • MicroRNA alterations may have significance in the development of cancer. • MicroRNAs may be early indicators of the carcinogenic process and carcinogen exposure.

  7. The role of microRNAs in the development and progression of chemical-associated cancers

    Energy Technology Data Exchange (ETDEWEB)

    Pogribny, Igor P., E-mail: igor.pogribny@fda.hhs.gov [Division of Biochemical Toxicology, National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, AR 72079 (United States); Beland, Frederick A. [Division of Biochemical Toxicology, National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, AR 72079 (United States); Rusyn, Ivan [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX 77843 (United States)

    2016-12-01

    Human exposure to certain natural and man-made chemical carcinogens is one of the major risk factors for cancer development. The effect of chemical carcinogens on genetic and epigenetic alterations and their significance in the development of cancer has been well-established. In contrast, the role of microRNAs (miRNAs) in the etiology of chemical-associated cancers remains relatively unexplored despite extensive reports on changes in miRNA expression upon carcinogen exposure. This review summarizes the current knowledge for the role of miRNAs as drivers of chemical-induced carcinogenesis by bridging the gap between carcinogen exposure and cancer development through functional studies. It also emphasizes the potential for miRNA changes as early indicators of the carcinogenic process, markers for carcinogen exposure, and identification of chemical carcinogenic hazards. - Highlights: • Exposure to chemical carcinogens alters microRNA expression. • MicroRNA alterations may have significance in the development of cancer. • MicroRNAs may be early indicators of the carcinogenic process and carcinogen exposure.

  8. Cystoviral polymerase complex protein P7 uses its acidic C-terminal tail to regulate the RNA-directed RNA polymerase P2.

    Science.gov (United States)

    Alphonse, Sébastien; Arnold, Jamie J; Bhattacharya, Shibani; Wang, Hsin; Kloss, Brian; Cameron, Craig E; Ghose, Ranajeet

    2014-07-15

    In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former toward template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in cystoviruses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Bleomycin Can Cleave an Oncogenic Noncoding RNA.

    Science.gov (United States)

    Angelbello, Alicia J; Disney, Matthew D

    2018-01-04

    Noncoding RNAs are pervasive in cells and contribute to diseases such as cancer. A question in biomedical research is whether noncoding RNAs are targets of medicines. Bleomycin is a natural product that cleaves DNA; however, it is known to cleave RNA in vitro. Herein, an in-depth analysis of the RNA cleavage preferences of bleomycin A5 is presented. Bleomycin A5 prefers to cleave RNAs with stretches of AU base pairs. Based on these preferences and bioinformatic analysis, the microRNA-10b hairpin precursor was identified as a potential substrate for bleomycin A5. Both in vitro and cellular experiments demonstrated cleavage. Importantly, chemical cleavage by bleomycin A5 in the microRNA-10b hairpin precursors occurred near the Drosha and Dicer enzymatic processing sites and led to destruction of the microRNA. Evidently, oncogenic noncoding RNAs can be considered targets of cancer medicines and might elicit their pharmacological effects by targeting noncoding RNA. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. RNA interference and Register Machines (extended abstract

    Directory of Open Access Journals (Sweden)

    Masahiro Hamano

    2012-11-01

    Full Text Available RNA interference (RNAi is a mechanism whereby small RNAs (siRNAs directly control gene expression without assistance from proteins. This mechanism consists of interactions between RNAs and small RNAs both of which may be single or double stranded. The target of the mechanism is mRNA to be degraded or aberrated, while the initiator is double stranded RNA (dsRNA to be cleaved into siRNAs. Observing the digital nature of RNAi, we represent RNAi as a Minsky register machine such that (i The two registers hold single and double stranded RNAs respectively, and (ii Machine's instructions are interpreted by interactions of enzyme (Dicer, siRNA (with RISC com- plex and polymerization (RdRp to the appropriate registers. Interpreting RNAi as a computational structure, we can investigate the computational meaning of RNAi, especially its complexity. Initially, the machine is configured as a Chemical Ground Form (CGF, which generates incorrect jumps. To remedy this problem, the system is remodeled as recursive RNAi, in which siRNA targets not only mRNA but also the machine instructional analogues of Dicer and RISC. Finally, probabilistic termination is investigated in the recursive RNAi system.

  11. The Long Non-coding RNA HOTTIP Enhances Pancreatic Cancer Cell Proliferation, Survival and Migration

    Science.gov (United States)

    ABSTRACTHOTTIP is a long non-coding RNA (lncRNA) transcribed from the 5' tip of the HOXA locus and is associated with the polycomb repressor complex 2 (PRC2) and WD repeat containing protein 5 (WDR5)/mixed lineage leukemia 1 (MLL1) chromatin modifying complexes. HOTTIP is expres...

  12. RNA signal amplifier circuit with integrated fluorescence output.

    Science.gov (United States)

    Akter, Farhima; Yokobayashi, Yohei

    2015-05-15

    We designed an in vitro signal amplification circuit that takes a short RNA input that catalytically activates the Spinach RNA aptamer to produce a fluorescent output. The circuit consists of three RNA strands: an internally blocked Spinach aptamer, a fuel strand, and an input strand (catalyst), as well as the Spinach aptamer ligand 3,5-difluoro-4-hydroxylbenzylidene imidazolinone (DFHBI). The input strand initially displaces the internal inhibitory strand to activate the fluorescent aptamer while exposing a toehold to which the fuel strand can bind to further displace and recycle the input strand. Under a favorable condition, one input strand was able to activate up to five molecules of the internally blocked Spinach aptamer in 185 min at 30 °C. The simple RNA circuit reported here serves as a model for catalytic activation of arbitrary RNA effectors by chemical triggers.

  13. REDIdb: an upgraded bioinformatics resource for organellar RNA editing sites.

    Science.gov (United States)

    Picardi, Ernesto; Regina, Teresa M R; Verbitskiy, Daniil; Brennicke, Axel; Quagliariello, Carla

    2011-03-01

    RNA editing is a post-transcriptional molecular process whereby the information in a genetic message is modified from that in the corresponding DNA template by means of nucleotide substitutions, insertions and/or deletions. It occurs mostly in organelles by clade-specific diverse and unrelated biochemical mechanisms. RNA editing events have been annotated in primary databases as GenBank and at more sophisticated level in the specialized databases REDIdb, dbRES and EdRNA. At present, REDIdb is the only freely available database that focuses on the organellar RNA editing process and annotates each editing modification in its biological context. Here we present an updated and upgraded release of REDIdb with a web-interface refurbished with graphical and computational facilities that improve RNA editing investigations. Details of the REDIdb features and novelties are illustrated and compared to other RNA editing databases. REDIdb is freely queried at http://biologia.unical.it/py_script/REDIdb/. Copyright © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  14. BioVLAB-MMIA: a cloud environment for microRNA and mRNA integrated analysis (MMIA) on Amazon EC2.

    Science.gov (United States)

    Lee, Hyungro; Yang, Youngik; Chae, Heejoon; Nam, Seungyoon; Choi, Donghoon; Tangchaisin, Patanachai; Herath, Chathura; Marru, Suresh; Nephew, Kenneth P; Kim, Sun

    2012-09-01

    MicroRNAs, by regulating the expression of hundreds of target genes, play critical roles in developmental biology and the etiology of numerous diseases, including cancer. As a vast amount of microRNA expression profile data are now publicly available, the integration of microRNA expression data sets with gene expression profiles is a key research problem in life science research. However, the ability to conduct genome-wide microRNA-mRNA (gene) integration currently requires sophisticated, high-end informatics tools, significant expertise in bioinformatics and computer science to carry out the complex integration analysis. In addition, increased computing infrastructure capabilities are essential in order to accommodate large data sets. In this study, we have extended the BioVLAB cloud workbench to develop an environment for the integrated analysis of microRNA and mRNA expression data, named BioVLAB-MMIA. The workbench facilitates computations on the Amazon EC2 and S3 resources orchestrated by the XBaya Workflow Suite. The advantages of BioVLAB-MMIA over the web-based MMIA system include: 1) readily expanded as new computational tools become available; 2) easily modifiable by re-configuring graphic icons in the workflow; 3) on-demand cloud computing resources can be used on an "as needed" basis; 4) distributed orchestration supports complex and long running workflows asynchronously. We believe that BioVLAB-MMIA will be an easy-to-use computing environment for researchers who plan to perform genome-wide microRNA-mRNA (gene) integrated analysis tasks.

  15. Evolutionary dynamics of RNA-like replicators : A bioinformatic approach to the origin of life

    NARCIS (Netherlands)

    Takeuchi, N.

    2010-01-01

    The origin of life has always attracted scientific inquiries. The RNA world hypothesis suggests that, before the evolution of DNAs and proteins, primordial life was based on RNAs both for information storage and chemical catalysis. In its simplest form, an RNA world consists of RNA molecules that

  16. 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.

  17. Silencing VDAC1 Expression by siRNA Inhibits Cancer Cell Proliferation and Tumor Growth In Vivo

    Directory of Open Access Journals (Sweden)

    Tasleem Arif

    2014-01-01

    Full Text Available Alterations in cellular metabolism and bioenergetics are vital for cancer cell growth and motility. Here, the role of the mitochondrial protein voltage-dependent anion channel (VDAC1, a master gatekeeper regulating the flux of metabolites and ions between mitochondria and the cytoplasm, in regulating the growth of several cancer cell lines was investigated by silencing VDAC1 expression using small interfering RNA (siRNA. A single siRNA specific to the human VDAC1 sequence at nanomolar concentrations led to some 90% decrease in VDAC1 levels in the lung A549 and H358, prostate PC-3, colon HCT116, glioblastoma U87, liver HepG2, and pancreas Panc-1 cancer cell lines. VDAC1 silencing persisted 144 hours post-transfection and resulted in profound inhibition of cell growth in cancer but not in noncancerous cells, with up to 90% inhibition being observed over 5 days that was prolonged by a second transfection. Cells expressing low VDAC1 levels showed decreased mitochondrial membrane potential and adenoside triphosphate (ATP levels, suggesting limited metabolite exchange between mitochondria and cytosol. Moreover, cells silenced for VDAC1 expression showed decreased migration, even in the presence of the wound healing accelerator basic fibroblast growth factor (bFGF. VDAC1-siRNA inhibited cancer cell growth in a Matrigel-based assay in host nude mice. Finally, in a xenograft lung cancer mouse model, chemically modified VDAC1-siRNA not only inhibited tumor growth but also resulted in tumor regression. This study thus shows that VDAC1 silencing by means of RNA interference (RNAi dramatically inhibits cancer cell growth and tumor development by disabling the abnormal metabolic behavior of cancer cells, potentially paving the way for a more effective pipeline of anticancer drugs.

  18. Ariadne: a database search engine for identification and chemical analysis of RNA using tandem mass spectrometry data.

    Science.gov (United States)

    Nakayama, Hiroshi; Akiyama, Misaki; Taoka, Masato; Yamauchi, Yoshio; Nobe, Yuko; Ishikawa, Hideaki; Takahashi, Nobuhiro; Isobe, Toshiaki

    2009-04-01

    We present here a method to correlate tandem mass spectra of sample RNA nucleolytic fragments with an RNA nucleotide sequence in a DNA/RNA sequence database, thereby allowing tandem mass spectrometry (MS/MS)-based identification of RNA in biological samples. Ariadne, a unique web-based database search engine, identifies RNA by two probability-based evaluation steps of MS/MS data. In the first step, the software evaluates the matches between the masses of product ions generated by MS/MS of an RNase digest of sample RNA and those calculated from a candidate nucleotide sequence in a DNA/RNA sequence database, which then predicts the nucleotide sequences of these RNase fragments. In the second step, the candidate sequences are mapped for all RNA entries in the database, and each entry is scored for a function of occurrences of the candidate sequences to identify a particular RNA. Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values. The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts. It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

  19. Conference Scene: Advances in RNA nanotechnology promise to transform medicine.

    Science.gov (United States)

    Leontis, Neocles; Sweeney, Blake; Haque, Farzin; Guo, Peixuan

    2013-07-01

    The second International Conference on RNA Nanotechnology and Therapeutics was held on the 3-5 April in Lexington, (KY, USA). The focus of the conference was on leveraging the unique chemical and biological properties of RNA to promote transformative advances in medicine. The conference convened more than 200 researchers from 15 countries and many disciplines, roughly double the participants of the first conference. While many presentations focused on the design, assembly and characterization of RNA nanoparticles and their uses for in vivo and in vitro sensing, diagnosis and therapy, others covered a variety of relevant areas of RNA biology and chemistry.

  20. A ω-mercaptoundecylphosphonic acid chemically modified gold electrode for uranium determination in waters in presence of organic matter.

    Science.gov (United States)

    Merli, Daniele; Protti, Stefano; Labò, Matteo; Pesavento, Maria; Profumo, Antonella

    2016-05-01

    A chemically modified electrode (CME) on a gold surface assembled with a ω-phosphonic acid terminated thiol was investigated for its capability to complex uranyl ions. The electrode, characterized by electrochemical techniques, demonstrated to be effective for the determination of uranyl at sub-μgL(-1) level by differential pulse adsorptive stripping voltammetry (DPAdSV) in environmental waters, also in presence of humic matter and other potential chelating agents. The accuracy of the measurements was investigated employing as model probes ligands of different complexing capability (humic acids and EDTA). Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Thermogravimetric Analysis of Modified Hematite by Methane (CH{sub 4}) for Chemical-Looping Combustion: A Global Kinetics Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Monazam, Esmail R; Breault, Ronald W; Siriwardane, Ranjani; Miller, Duane D

    2013-10-01

    Iron oxide (Fe{sub 2}O{sub 3}) or in its natural form (hematite) is a potential material to capture CO{sub 2} through the chemical-looping combustion (CLC) process. It is known that magnesium (Mg) is an effective methyl cleaving catalyst and as such it has been combined with hematite to assess any possible enhancement to the kinetic rate for the reduction of Fe{sub 2}O{sub 3} with methane. Therefore, in order to evaluate its effectiveness as a hematite additive, the behaviors of Mg-modified hematite samples (hematite –5% Mg(OH){sub 2}) have been analyzed with regard to assessing any enhancement to the kinetic rate process. The Mg-modified hematite was prepared by hydrothermal synthesis. The reactivity experiments were conducted in a thermogravimetric analyzer (TGA) using continuous stream of CH{sub 4} (5, 10, and 20%) at temperatures ranging from 700 to 825 {degrees}C over ten reduction cycles. The mass spectroscopy analysis of product gas indicated the presence of CO{sub 2}, H{sub 2}O, H{sub 2} and CO in the gaseous product. The kinetic data at reduction step obtained by isothermal experiments could be well fitted by two parallel rate equations. The modified hematite samples showed higher reactivity as compared to unmodified hematite samples during reduction at all investigated temperatures.

  2. Modified chemical synthesis of porous α-Sm{sub 2}S{sub 3} thin films

    Energy Technology Data Exchange (ETDEWEB)

    Kumbhar, V.S.; Jagadale, A.D. [Thin Film Physics Laboratory, Department of Physics, Shivaji University, Kolhapur, (M.S.) 416004 (India); Gaikwad, N.S. [Rayat Shikshan Sanstha, Satara, (M.S.) 415 001 (India); Lokhande, C.D., E-mail: l_chandrakant@yahoo.com [Thin Film Physics Laboratory, Department of Physics, Shivaji University, Kolhapur, (M.S.) 416004 (India)

    2014-08-15

    Highlights: • A novel chemical route to prepare α-Sm{sub 2}S{sub 3} thin films. • A porous honeycomb like morphology of the α-Sm{sub 2}S{sub 3} thin film. • An application of α-Sm{sub 2}S{sub 3} thin film toward its supercapacitive behaviour. - Abstract: The paper reports synthesis of porous α-Sm{sub 2}S{sub 3} thin films using modified chemical synthesis, also known as successive ionic layer adsorption and reaction (SILAR) method. The X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), wettability and ultraviolet–visible spectroscopy (UV–vis) techniques are used for the study of structural, elemental, morphological and optical properties of α-Sm{sub 2}S{sub 3} films. An orthorhombic crystal structure of α-Sm{sub 2}S{sub 3} is resulted from XRD study. The SEM and AFM observations showed highly porous α-Sm{sub 2}S{sub 3} film surface. An optical band gap of 2.50 eV is estimated from optical absorption spectrum. The porous α-Sm{sub 2}S{sub 3} thin film tuned for supercapacitive behaviour using cyclic voltammetry and galvanostatic charge discharge showed a specific capacitance and energy density of 294 Fg{sup –1} and 48.9 kW kg{sup –1}, respectively in 1 M LiClO{sub 4}–propylene carbonate electrolyte.

  3. 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

  4. Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

    Science.gov (United States)

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Antimicrobial activity of chemically modified dextran derivatives.

    Science.gov (United States)

    Tuchilus, Cristina G; Nichifor, Marieta; Mocanu, Georgeta; Stanciu, Magdalena C

    2017-04-01

    Cationic amphiphilic dextran derivatives with a long alkyl group attached to the reductive end of the polysaccharide chain and quaternary ammonium groups attached as pendent groups to the main dextran backbone were synthesized and tested for their antimicrobial properties against several bacteria and fungi strains. Dependence of antimicrobial activity on both polymer chemical composition (dextran molar mass, length of end alkyl group and chemical structure of ammonium groups) and type of microbes was highlighted by disc-diffusion method (diameter of inhibition zone) and broth microdilution method (minimum inhibitory concentrations). Polymers had antimicrobial activity for all strains studied, except for Pseudomonas aeruginosa ATCC 27853. The best activity against Staphylococcus aureus (Minimun Inhibitory Concentration 60μg/mL) was provided by polymers obtained from dextran with lower molecular mass (Mn=4500), C 12 H 25 or C 18 H 37 end groups, and N,N-dimethyl-N-benzylammonium pendent groups. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. A reagentless enzymatic fluorescent biosensor for glucose based on upconverting glasses, as excitation source, and chemically modified glucose oxidase.

    Science.gov (United States)

    Del Barrio, Melisa; Cases, Rafael; Cebolla, Vicente; Hirsch, Thomas; de Marcos, Susana; Wilhelm, Stefan; Galbán, Javier

    2016-11-01

    Upon near-infrared excitation Tm(3+)+Yb(3+) doped fluorohafnate glasses present upconversion properties and emit visible light. This property permits to use these glasses (UCG) as excitation sources for fluorescent optical biosensors. Taking this into account, in this work a fluorescent biosensor for glucose determination is designed and evaluated. The biosensor combines the UCG and the fluorescence of the enzyme glucose oxidase chemically modified with a fluorescein derivative (GOx-FS), whose intensity is modified during the enzymatic reaction with glucose. Optical parameters have been optimized and a mathematical model describing the behavior of the analytical signal is suggested. Working in FIA mode, the biosensor responds to glucose concentrations up to, at least, 15mM with a limit of detection of 1.9mM. The biosensor has a minimum lifetime of 9 days and has been applied to glucose determination in drinks. The applicability of the sensor was tested by glucose determination in two fruit juices. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. 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.

  8. Modified Allergens for Immunotherapy.

    Science.gov (United States)

    Satitsuksanoa, Pattraporn; Głobińska, Anna; Jansen, Kirstin; van de Veen, Willem; Akdis, Mübeccel

    2018-02-16

    During the past few decades, modified allergens have been developed for use in allergen-specific immunotherapy (AIT) with the aim to improve efficacy and reduce adverse effects. This review aims to provide an overview of the different types of modified allergens, their mechanism of action and their potential for improving AIT. In-depth research in the field of allergen modifications as well as the advance of recombinant DNA technology have paved the way for improved diagnosis and research on human allergic diseases. A wide range of structurally modified allergens has been generated including allergen peptides, chemically altered allergoids, adjuvant-coupled allergens, and nanoparticle-based allergy vaccines. These modified allergens show promise for the development of AIT regimens with improved safety and long-term efficacy. Certain modifications ensure reduced IgE reactivity and retained T cell reactivity, which facilities induction of immune tolerance to the allergen. To date, multiple clinical trials have been performed using modified allergens. Promising results were obtained for the modified cat, grass and birch pollen, and house dust mite allergens. The use of modified allergens holds promise for improving AIT efficacy and safety. There is however a need for larger clinical studies to reliably assess the added benefit for the patient of using modified allergens for AIT.

  9. Comparação de três protocolos distintos para extração de RNA de amostras fixadas em formalina e emblocadas em parafina Comparison of three different protocols for extracting RNA from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Gisele Rodrigues Gouveia

    2011-12-01

    Full Text Available INTRODUÇÃO: Tecidos fixados em formalina e emblocados em parafina (FFEP são importantes fontes de amostras para estudos retrospectivos. Apesar de sua capacidade de preservação de proteínas e morfologia celular, a formalina interfere negativamente em testes de biologia molecular por fragmentar e modificar quimicamente os ácidos nucleicos, particularmente o ácido ribonucleico (RNA. OBJETIVO: Comparar a eficiência de três diferentes protocolos de extração de RNA para análise de expressão gênica de tecidos FFEP. MATERIAL E MÉTODOS: Amostras de linfonodo humano FEEP foram submetidas à extração de RNA utilizando-se os kits Ambion e Arcturus Bioscience e o método clássico de Trizol. Após a extração, o RNA foi quantificado e testado quanto à sua capacidade de amplificaç��o pela reação em cadeia da polimerase em tempo real (RT-PCR utilizando primers do gene endógeno gliceraldeído-3 fosfato desidrogenase (GAPDH. DISCUSSÃO/CONCLUSÃO: Todos os protocolos testados produziram quantidades adequadas e suficientes de RNA total, entretanto, somente os protocolos com uso dos kits Ambion e Arcturus produziram RNA capaz de ser amplificado pela PCR.INTRODUCTION: Formalin fixed paraffin embedded (FFPE tissues are an important sample source for retrospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders Molecular Biology tests once it fragments and chemically modifies nucleic acids, particularly RNA. OBJECTIVE: To compare the efficiency of three different RNA extraction protocols for gene expression analysis of FFEP tissues. MATERIAL AND METHODS: RNA was extracted from FFPE samples of human lymph by means of Ambion and Arcturus Bioscience kits and the classical Trizol method. After extraction, RNA was quantified and tested for amplification through real time polymerase chain reaction (RT-PCR using glyceraldehydes-3 phosphate dehydrogenase (GAPDH endogenous gene primers. DISCUSSION

  10. Enhancing adsorption capacity of toxic malachite green dye through chemically modified breadnut peel: equilibrium, thermodynamics, kinetics and regeneration studies.

    Science.gov (United States)

    Chieng, Hei Ing; Lim, Linda B L; Priyantha, Namal

    2015-01-01

    Breadnut skin, in both its unmodified (KS) and base-modified (BM-KS) forms, was investigated for its potential use as a low-cost adsorbent for the removal of toxic dye, malachite green (MG). Characterization of the adsorbents was carried out using scanning electron microscope, X-ray fluorescence and Fourier transform infra-red spectroscopy. Batch adsorption experiments, carried out under optimized conditions, for the adsorption of MG were fitted using five isotherm models (Langmuir, Freundlich, Dubinin-Radushkevich, Temkin and Sips) and six error functions to determine the best-fit model. The adsorption capacity was greatly enhanced when breadnut skin was chemically modified with NaOH, leading to an adsorption capacity of 353.0 mg g(-1), that was far superior to most reported adsorbents for the removal of MG. Thermodynamics studies indicated that the adsorption of MG was spontaneous on KS and BM-KS, and the reactions were endothermic and exothermic, respectively. Kinetics studies showed that both followed the pseudo-second order. Regeneration experiments on BM-KS indicated that its adsorption capacity was still maintained at>90% even after five cycles. It can be concluded that NaOH-modified breadfruit skin has great potential to be utilized in real-life application as a low-cost adsorbent for the removal of MG in wastewater treatment.

  11. Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

    Science.gov (United States)

    Clark, Josef P; Rahman, Reazur; Yang, Nachen; Yang, Linda H; Lau, Nelson C

    2017-09-11

    To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Design of a Bioactive Small Molecule that Targets the Myotonic Dystrophy Type 1 RNA Via an RNA Motif-Ligand Database & Chemical Similarity Searching

    Science.gov (United States)

    Parkesh, Raman; Childs-Disney, Jessica L.; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A.; Disney, Matthew D.

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, Sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1), and cardiac troponin T (cTNT). Based on these observations, the development of small molecule ligands that target specifically expanded DM1 repeats could serve as therapeutics. In the present study, computational screening was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of inhibitors of the RNA-protein complex with low micromolar IC50’s, which are >20-fold more potent than the query compounds, were identified. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with virtual screening. PMID:22300544

  13. Photoelectrochemical Properties of CuO Grown by Using a Modified Chemical Bath Deposition Method

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Jin-wook; Ryu, Hyukhyun [Inje University, Gimhae (Korea, Republic of); Lee, Won-Jae [Dong-Eui University, Busan (Korea, Republic of)

    2016-06-15

    In this study, cupric oxide (CuO) nanorods were grown on the fluorine-doped tin oxide (FTO) glass substrate using a modified-chemical bath deposition (M-CBD) method. We investigated the morphology, structural, optical and photoelectrochemical properties of the cupric oxide nanorods with various growth durations by using field-emission scanning-electron microscopy (FE-SEM), X-ray diffraction (XRD), UV-visible spectroscopy (UV-vis) and three-electrode potentiostat, respectively. In this work, we found that the morphologies, thickness, growth rate, crystallinities, grain sizes and optical bandgap were controllable on the growth duration, which affected photocurrent density and photo-stability. The highest growth rate of CuO nanorods was 126 nm/min. From the XRD measurement, we also confirmed that (020) directional growth affected the growth of the CuO nanorods. A maximum photocurrent density of-1.88 mA/cm² at -0.55 V (vs. SCE) and high photo-stability value about 40% was obtained with 10 minutes growth duration.

  14. Phosphonium modified clay/polyimide nanocomposites

    International Nuclear Information System (INIS)

    Ceylan, Hatice; Çakmakçi, Emrah; Beyler-Çiǧil, Asli; Kahraman, Memet Vezir

    2014-01-01

    In this study, octyltriphenylphosphonium bromide [OTPP-Br] was prepared from the reaction of triphenylphosphine and 1 -bromooctane. The modification of clay was done by ion exchange reaction using OTPP-Br in water medium. Poly(amic acid) was prepared from the reaction of 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-Oxydianiline (ODA). Polyimide(PI)/clay hybrids were prepared by blending of poly(amic acid) and organically modified clay as a type of layered clays. The morphology of the Polyimide/ phosphonium modified clay hybrids was characterized by scanning electron microscopy (SEM). Chemical structures of polyimide and Polyimide/ phosphonium modified clay hybrids were characterized by FTIR. SEM and FTIR results showed that the Polyimide/ phosphonium modified clay hybrids were successfully prepared. Thermal properties of the Polyimide/ phosphonium modified clay hybrids were characterized by thermogravimetric analysis (TGA)

  15. Preparation of Small RNA NGS Libraries from Biofluids.

    Science.gov (United States)

    Etheridge, Alton; Wang, Kai; Baxter, David; Galas, David

    2018-01-01

    Next generation sequencing (NGS) is a powerful method for transcriptome analysis. Unlike other gene expression profiling methods, such as microarrays, NGS provides additional information such as splicing variants, sequence polymorphisms, and novel transcripts. For this reason, NGS is well suited for comprehensive profiling of the wide range of extracellular RNAs (exRNAs) in biofluids. ExRNAs are of great interest because of their possible biological role in cell-to-cell communication and for their potential use as biomarkers or for therapeutic purposes. Here, we describe a modified protocol for preparation of small RNA libraries for NGS analysis. This protocol has been optimized for use with low-input exRNA-containing samples, such as plasma or serum, and has modifications designed to reduce the sequence-specific bias typically encountered with commercial small RNA library construction kits.

  16. Radiotracer study of the adsorption of Fe(III), Cr(III) and Cd(II) on natural and chemically modified Slovak zeolite

    International Nuclear Information System (INIS)

    Foeldesova, M.; Dillinger, P.; Lukac, P.

    1998-01-01

    In order to minimize the contamination of environment with metals in ionic form the more types of natural and chemically modified zeolites were examined to their uptake of Fe(III), Cr(III) and Cd(II) from water solutions by batch radio-exchange equilibration method. In this study was used zeolitic tuff from deposit Nizny Hrabovec (content of clinoptilolite 50-70%) with the grain size from 1.2 to 2.2 mm. The granules of zeolite were modified with the following NaOH solutions: ).5, 1, 2 and 4 mol.l -1 at 80 grad C for 4 hours. The sorption of Fe, Cr and Cd ions on all types of zeolites was studied by radio-exchange method and the sorption of Fe and Cr also flame atomic absorption method. From sorption curves the sorption coefficients were calculated. The results obtained in this work show that zeolites modified with NaOH solution are suitable for adsorption of Fe(III), Cr(III) and Cd(II) from underwater, waste water, feed water and coolant water from nuclear plants. The adsorbed zeolites can be solidified by conventional way

  17. Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

    Science.gov (United States)

    Jakobi, Stephan; Nguyen, Tran Xuan Phong; Debaene, François; Metz, Alexander; Sanglier-Cianférani, Sarah; Reuter, Klaus; Klebe, Gerhard

    2014-10-01

    Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. © 2014 Wiley Periodicals, Inc.

  18. Structural Analysis of ‘key’ Interactions in Functional RNA Molecules

    KAUST Repository

    Chawla, Mohit

    2018-04-01

    The main objective of the thesis is to carry out structural bioinformatics study along with usage of advanced quantum chemical methods to look at the structural stability and energetics of RNA building blocks. The main focus of the work described here lies on understanding the reasons behind the intrinsic stability of key interactions in nucleic acids. Crystal structures of RNA molecules exhibit fascinating variety of non-covalent interactions, which play an important role in maintaining the three dimensional structures. An accurate atomic level description of these interactions in the structural building blocks of RNA is a key to understand the structure-function relationship in these molecules. An effort has been made to link the conclusions drawn from quantum chemical computations on RNA base pairs in wide biochemical context of their occurrence in RNA structures. The initial attention was on the impact of natural and non-natural modifications of the nucleic acid bases on the structure and stability of base pairs that they are involved in. In the remaining sections we cover other molecular interactions shaping nucleic acids, as the interaction between ribose and the bases, and the fluoride sensing riboswitch system in order to investigate structure and dynamics of nucleic acids at the atomic level and to gain insight into the physical chemistry behind.

  19. Structural Analysis of ‘key’ Interactions in Functional RNA Molecules

    KAUST Repository

    Chawla, Mohit

    2018-01-01

    The main objective of the thesis is to carry out structural bioinformatics study along with usage of advanced quantum chemical methods to look at the structural stability and energetics of RNA building blocks. The main focus of the work described here lies on understanding the reasons behind the intrinsic stability of key interactions in nucleic acids. Crystal structures of RNA molecules exhibit fascinating variety of non-covalent interactions, which play an important role in maintaining the three dimensional structures. An accurate atomic level description of these interactions in the structural building blocks of RNA is a key to understand the structure-function relationship in these molecules. An effort has been made to link the conclusions drawn from quantum chemical computations on RNA base pairs in wide biochemical context of their occurrence in RNA structures. The initial attention was on the impact of natural and non-natural modifications of the nucleic acid bases on the structure and stability of base pairs that they are involved in. In the remaining sections we cover other molecular interactions shaping nucleic acids, as the interaction between ribose and the bases, and the fluoride sensing riboswitch system in order to investigate structure and dynamics of nucleic acids at the atomic level and to gain insight into the physical chemistry behind.

  20. Removal of phosphorus using chemically modified lignocellulosic materials

    Science.gov (United States)

    James S. Han; N. Hur; B. Choi; Soo-Hong Min

    2003-01-01

    Heavy metals from an acid mine drainage (AMD) site were precipitated on the surface of juniper fiber. The modified fiber was tested in lab-scaled batch and column tests and in the field. Elemental analysis showed that soluble iron species deposited on the fiber act as an inorganic adsorbent for anions. Sorption capacity, determined by fitting results to a Langmuir...

  1. 5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

    Science.gov (United States)

    Ogino, Minako; Ogino, Tomoaki

    2017-03-15

    The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as

  2. HIGHLY EFFECTIVE CHEMICAL MODIFIERS FOR PRODUCTION OF CONCRETES WITH PRE-SET PROPERTIES

    Directory of Open Access Journals (Sweden)

    Tkach Evgeniya Vladimirovna

    2012-10-01

    Full Text Available The paper demonstrates the application of industrial by-products and recycled materials. Waterproofing admixtures improve the structure and the properties of the cement stone. Development and preparation of highly effective waterproofing modifiers of durable effect, as well as development of the process procedure parameters, including mixing, activation, heat treatment, etc. are to be implemented. The composition of waterproofing modifiers is to be fine-tuned to synergize the behaviour of various ingredients of cement systems to assure the substantial improvement of their strength, freeze- and corrosion resistance. Multi-functional waterproofing admixtures were used to produce highly effective modified concretes. The key idea of the new method of modifying cement-based building materials is that the waterproofing admixture concentration is to exceed 10% of the weight of the binding agent within the per-unit weight of the cement stone, given that its strength does not deteriorate. GKM-type modifier coupled with organo-mineral waterproofing admixture concentration agent GT-M may be recommended for mass use in the manufacturing of hydraulic concrete and reinforced concrete products. Overview of their practical implementation has proven that waterproofing modifier GKM-S, if coupled with waterproofing admixture concentration agent GT-M, improves the corrosion control inside the cement stone and makes it possible to manufacture durable concrete and reinforced concrete products that demonstrate pre-set physical and processing behaviour. Comprehensive concrete modification by modifier GKM-S and waterproofing admixture concentration agent GT-M may be regarded as one of the most ambitious methods of production of highly effective waterproof concretes.

  3. Solid-Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages.

    Science.gov (United States)

    Yang, Xianbin

    2017-09-18

    The oligoribonucleotide phosphorodithioate (PS2-RNA) modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2-RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2-aptamers (thioaptamers). Thus, the interest in and promise of PS2-RNAs has drawn attention to synthesizing, isolating, and characterizing these compounds. RNA-thiophosphoramidite monomers are commercially available from AM Biotechnologies and this unit describes an effective methodology for solid-phase synthesis, deprotection, and purification of RNAs having PS2 internucleotide linkages. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  4. Chemically modified Moringa oleifera seed husks as low cost adsorbent for removal of copper from aqueous solution

    Science.gov (United States)

    Ghafar, Faridah; Mohtar, Aminullah; Sapawe, Norzahir; Hadi, Norulakmal Nor; Salleh, Marmy Roshaidah Mohd

    2017-12-01

    Moringa oleifera husks (MOH) are an agricultural byproduct that may have potential as adsorbent for removal of heavy metal ions in wastewater such as copper (Cu2+). The release of Cu2+ to the environment by the mining and electroplating industries cause a major problem because it is toxic and can cause liver and kidney problems. Hence, it is important to remove copper before the wastewater can be discharged to the environment. In order to increase the adsorption capacity, the MOH was chemically modified using citric acid. The raw and modified MOH were analyzed using Fourier Transform Infra-Red (FTIR) for identification of functional groups present at the adsorbent surface. The adsorption study was carried out using the batch technique in water bath shaker investigating different parameters; adsorbent dosage (30 - 70 g/L), initial concentration of copper (30 - 150 mg/L), contact time (2 - 90 min), temperature (27 - 60 °C) at constant agitation of 100 rpm. The concentrations of copper in aqueous solution before and after the adsorption process was analyzed using Atomic Absorption Spectrum (AAS). The highest percentage removal of copper was found at 10g/L of adsorbent dosage with 30 mg/L of initial concentration and temperature 30 °C. It was also observed that the adsorption of copper by MOH was approaching to equilibrium at 60 min of reaction time. From the FTIR analysis, it was found that the MOH contains hydroxyl, carboxyl and amine groups. The high adsorption capacity of modified MOH to remove copper from aqueous solution makes it preferable and attractive alternative to commercial adsorbent.

  5. MicroRNA and dsRNA targeting chitin synthase A reveal a great potential for pest management of the hemipteran insect Nilaparvata lugens.

    Science.gov (United States)

    Li, Tengchao; Chen, Jie; Fan, Xiaobin; Chen, Weiwen; Zhang, Wenqing

    2017-07-01

    Two RNA silencing pathways in insects are known to exist that are mediated by short interfering RNAs (siRNAs) and microRNAs (miRNAs), which have been hypothesised to be promising methods for insect pest control. However, a comparison between miRNA and siRNA in pest control is still unavailable, particularly in targeting chitin synthase gene A (CHSA). The dsRNA for Nilaparvata lugens CHSA (dsNlCHSA) and the microR-2703 (miR-2703) mimic targeting NlCHSA delivered via feeding affected the development of nymphs, reduced their chitin content and led to lethal phenotypes. The protein level of NlCHSA was downregulated after female adults were injected with dsNlCHSA or the miR-2703 mimic, but there were no significant differences in vitellogenin (NlVg) expression or in total oviposition relative to the control group. However, 90.68 and 46.13% of the eggs laid by the females injected with dsNlCHSA and miR-2703 mimic were unable to hatch, respectively. In addition, a second-generation miRNA and RNAi effect on N. lugens was observed. Ingested miR-2703 seems to be a good option for killing N. lugens nymphs, while NlCHSA may be a promising target for RNAi-based pest management. These findings provide important evidence for applications of small non-coding RNAs (snRNAs) in insect pest management. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Nucleic acid helix structure determination from NMR proton chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

    Werf, Ramon M. van der; Tessari, Marco; Wijmenga, Sybren S., E-mail: S.Wijmenga@science.ru.nl [Radboud University Nijmegen, Department of Biophysical Chemistry, Institute of Molecules and Materials (Netherlands)

    2013-06-15

    We present a method for de novo derivation of the three-dimensional helix structure of nucleic acids using non-exchangeable proton chemical shifts as sole source of experimental restraints. The method is called chemical shift de novo structure derivation protocol employing singular value decomposition (CHEOPS) and uses iterative singular value decomposition to optimize the structure in helix parameter space. The correct performance of CHEOPS and its range of application are established via an extensive set of structure derivations using either simulated or experimental chemical shifts as input. The simulated input data are used to assess in a defined manner the effect of errors or limitations in the input data on the derived structures. We find that the RNA helix parameters can be determined with high accuracy. We finally demonstrate via three deposited RNA structures that experimental proton chemical shifts suffice to derive RNA helix structures with high precision and accuracy. CHEOPS provides, subject to further development, new directions for high-resolution NMR structure determination of nucleic acids.

  7. Thin-film chemical sensors based on electron tunneling

    Science.gov (United States)

    Khanna, S. K.; Lambe, J.; Leduc, H. G.; Thakoor, A. P.

    1985-01-01

    The physical mechanisms underlying a novel chemical sensor based on electron tunneling in metal-insulator-metal (MIM) tunnel junctions were studied. Chemical sensors based on electron tunneling were shown to be sensitive to a variety of substances that include iodine, mercury, bismuth, ethylenedibromide, and ethylenedichloride. A sensitivity of 13 parts per billion of iodine dissolved in hexane was demonstrated. The physical mechanisms involved in the chemical sensitivity of these devices were determined to be the chemical alteration of the surface electronic structure of the top metal electrode in the MIM structure. In addition, electroreflectance spectroscopy (ERS) was studied as a complementary surface-sensitive technique. ERS was shown to be sensitive to both iodine and mercury. Electrolyte electroreflectance and solid-state MIM electroreflectance revealed qualitatively the same chemical response. A modified thin-film structure was also studied in which a chemically active layer was introduced at the top Metal-Insulator interface of the MIM devices. Cobalt phthalocyanine was used for the chemically active layer in this study. Devices modified in this way were shown to be sensitive to iodine and nitrogen dioxide. The chemical sensitivity of the modified structure was due to conductance changes in the active layer.

  8. INFLUENCE OF PACKAGING MATERIAL AND STORAGE TIME ON PHYSICAL, CHEMICAL AND MICROBIOLOGICAL PROPERTIES OF SET YOGURT: A COMPARATIVE STUDY BETWEEN MODIFIED BIODEGRADABLE POLY(LACTIC ACID AND POLYPROPYLENE

    Directory of Open Access Journals (Sweden)

    NAWADON PETCHWATTANA

    2016-10-01

    Full Text Available The current paper investigates the influence of storage time and type of polymeric packaging material on the chemical, physical and microbiological properties of set yogurt. Firstly, poly(lactic acid (PLA was modified by using a core-shell rubber (CSR and an acrylic processing aid (PA to produce PLA with high toughness and good processability. Secondly, an appropriate PLA/PA/CSR composition was selected and fabricated to yogurt cup. The yogurt was stored in both modified PLA and polypropylene (PP packages to observe some physical, chemical and biological changes. Finally, the biodegradation test was made on both packages and compared with that cellulose. Experimental results revealed that adding 5wt% CSR gave PLA/PA as tough as PP. Types of packaging material and storage time did not change the color of yogurt. The number of lactic acid bacteria grew significantly after they had been incubated for 6 days. The bacterial viability decreased dramatically due to the increased acidity and the decreased pH. A positive impact on the viability of bacterial growth was found when yogurt was stored in modified PLA package. This made yogurt had more health benefits than stored in PP package. The biodegradation test results indicated that the modified PLA degraded at a rapid rate. It achieved approximately 50% biodegradation within 40 days which was comparable to the time required to degrade the cellulose, whereas PP was non-biodegradable over the period studied. In summary, substitution conventional PP by a novel modified PLA seems to be a better way for both the health and the environment benefits.

  9. Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR.

    Science.gov (United States)

    Bhan, Arunoday; Mandal, Subhrangsu S

    2016-01-01

    HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is transcriptionally regulated by estradiol (E2) in vitro and in vivo. This chapter describes protocols for analysis of the HOTAIR promoter, cloning, transfection and dual luciferase assays, knockdown of protein synthesis by antisense oligonucleotides, and chromatin immunoprecipitation (ChIP) assay. These protocols are useful for studying the estrogen-mediated transcriptional regulation of lncRNA HOTAIR, as well as other protein coding genes and non-coding RNAs.

  10. Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

    Science.gov (United States)

    Saini, Ravi Prakash; Raman, Venkat; Dhandapani, Gurusamy; Malhotra, Era Vaidya; Sreevathsa, Rohini; Kumar, Polumetla Ananda; Sharma, Tilak R; Pattanayak, Debasis

    2018-01-01

    The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

  11. RNA synthesis in primary cultures of adult rat hepatocytes

    International Nuclear Information System (INIS)

    Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

    1983-01-01

    The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

  12. Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

    OpenAIRE

    Kirpekar, F; Douthwaite, S; Roepstorff, P

    2000-01-01

    We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of post...

  13. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    Science.gov (United States)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  14. Surface chemical and biological characterization of flax fabrics modified with silver nanoparticles for biomedical applications

    International Nuclear Information System (INIS)

    Paladini, F.; Picca, R.A.; Sportelli, M.C.; Cioffi, N.; Sannino, A.; Pollini, M.

    2015-01-01

    Silver nanophases are increasingly used as effective antibacterial agent for biomedical applications and wound healing. This work aims to investigate the surface chemical composition and biological properties of silver nanoparticle-modified flax substrates. Silver coatings were deposited on textiles through the in situ photo-reduction of a silver solution, by means of a large-scale apparatus. The silver-coated materials were characterized through X-ray Photoelectron Spectroscopy (XPS), to assess the surface elemental composition of the coatings, and the chemical speciation of both the substrate and the antibacterial nanophases. A detailed investigation of XPS high resolution regions outlined that silver is mainly present on nanophases' surface as Ag 2 O. Scanning electron microscopy and energy dispersive X-ray spectroscopy were also carried out, in order to visualize the distribution of silver particles on the fibers. The materials were also characterized from a biological point of view in terms of antibacterial capability and cytotoxicity. Agar diffusion tests and bacterial enumeration tests were performed on Gram positive and Gram negative bacteria, namely Staphylococcus aureus and Escherichia coli. In vitro cytotoxicity tests were performed through the extract method on murine fibroblasts in order to verify if the presence of the silver coating affected the cellular viability and proliferation. Durability of the coating was also assessed, thus confirming the successful scaling up of the process, which will be therefore available for large-scale production. - Highlights: • Silver nanophases are increasingly used as effective antibacterial agent for biomedical applications. • Silver coatings were deposited on textiles through the in situ photo-reduction of a silver solution. • Flax fabrics were characterized from a biological and surface chemical point of view. • Scaling up of the process was confirmed

  15. Direct determination of Cd and Pb in human urine by GFAAS with deuterium-lamp background correction using different chemical modifiers

    International Nuclear Information System (INIS)

    Husakova, L.; Baoinova, M.; Sramkova, J.; Cernohorsky, T.

    2007-01-01

    Several authors have contributed to the elaboration of methodology for direct determination of Cd and Pb in urine by graphite furnace atomic absorption spectrometry (GFAAS). In the proposed approaches, Zeeman background correction systems were predominantly used, without paying much attention to the selection of an appropriate chemical modifier. However, systematic studies on eleven recommended and less commonly used modifiers have resulted in optimization of atomization conditions, so that accurate analysis also with the use of D 2 -lamp background correction became possible. This was confirmed by comparative measurements using both background correction systems. For determination of Cd in urine, NH 4 F has been selected resulting in the lowest limit of detection (LOD): 0.07 μg L -1 . NH 4 F promotes efficient atomization at low temperatures and suppresses chloride interference effect. Pd + Sr (nitrate) has been selected as the most adequate modifier for determination of Pb. Its presence raised the maximum tolerable pyrolysis temperature up to 1200 o C, which resulted in the maximum reduction of the background signal and the lowest LOD of 1.5 mg L -1 for Pb (10 μL aliquots of dispensed urine). Applying the above modifiers to the analysis of standards and samples, direct aqueous calibration for accurate analysis of diluted and acidified urine samples became possible. Accuracy of the analysis was verified by the use of commercially available quality control reference materials. (authors)

  16. Potassium selective chemically modified field effect transistors based on AlGaN/GaN two-dimensional electron gas heterostructures.

    Science.gov (United States)

    Alifragis, Y; Volosirakis, A; Chaniotakis, N A; Konstantinidis, G; Adikimenakis, A; Georgakilas, A

    2007-06-15

    We investigate the use of the AlGaN/GaN high electron mobility transistor (HEMT) as a novel transducer for the development of ion-selective chemically modified HEMT sensors (ChemHEMTs). For this, polyvinyl chloride (PVC) membrane doped with ion-selective ionophores is deposited onto the area of the gate for the chemical recognition step, while the AlGaN/GaN HEMT is used as the transducer. In particular, the use of a valinocycin doped membrane with thickness of 50 microm generates a sensor with excellent analytical characteristics for the monitoring of K(+). The K(+)-ChemHEMT has sensitivity of 52.4 mV/pK(+)in the linear range of 10(-5) to 10(-2)M, while the detection limit is in the order of 3.1 x 10(-6)M. Also, the sensor shows selectivity similar to valinomycin-based ISEs, while the signal stability over time and the measurement to measurement reproducibility are very good.

  17. New route for synthesis of electrocatalytic Ni(OH)2 modified ...

    Indian Academy of Sciences (India)

    Administrator

    potential cycling of modified electrode with the above complex in alkali. (2) By thermal ... Chemically modified electrodes; nickel hydroxide; borohydride oxidation; electrocatalysis. 1. ..... Au, Pt and Ag substrates including bimetallic alloys (Bin.

  18. The fractionation of t-RNA on N,N′-bis(3-aminopropyl)-piperazine substituted-Sepharose

    Science.gov (United States)

    Leberman, Reuben; Giovanelli, Ruth; Acosta, Zenobio

    1974-01-01

    An anion exchange agarose has been prepared by modifying sepharose 6B with N,N′-bis (-3-aminopropyl) piperazine. This material (BAPP-Sepharose) has been used for the fractionation of t-RNA from E.coli by column chromatography. The results obtained with gram quantities of crude t-RNA at pH 4.6 and pH 8.0 as measured by the elution patterns of alanyl, arginyl, aspartyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, tyrosyl, and valyl t-RNA are described. PMID:10793731

  19. Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

    Science.gov (United States)

    Kim, K H; Hemenway, C

    1997-05-26

    The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

  20. A study comparing chemical peeling using modified jessner′s solution and 15% trichloroacetic acid versus 15% trichloroacetic acid in the treatment of melasma

    Directory of Open Access Journals (Sweden)

    Safoury Omar

    2009-01-01

    Full Text Available Background: Melasma is a symmetric progressive hyperpigmentation of the facial skin that occurs in all races but has a predilection for darker skin phenotypes. Depigmenting agents, laser and chemical peeling as classic Jessner′s solution, modified Jessner′s solution and trichloroacetic acid have been used alone and in combination in the treatment of melasma. Objectives: The aim of the study was to compare the therapeutic effect of combined 15% Trichloroacetic acid (TCA and modified Jessner′s solution with 15% TCA on melasma. Materials and Methods: Twenty married females with melasma (epidermal type, with a mean age of 38.25 years, were included in this study. All were of skin type III or IV. Fifteen percent TCA was applied to the whole face, with the exception of the left malar area to which combined TCA 15% and modified Jessner′s solution was applied. Results: Our results revealed statistically highly significant difference between MASI Score (Melasma Area and Severity Index between the right malar area and the left malar area. Conclusion: Modified Jessner′s solution proved to be useful as an adjuvant treatment with TCA in the treatment of melasma, improving the results and minimizing postinflammatory hyperpigmentation.

  1. The Electrochemical Behavior of Carbon Fiber Microelectrodes Modified with Carbon Nanotubes Using a Two-Step Electroless Plating/Chemical Vapor Deposition Process

    Directory of Open Access Journals (Sweden)

    Longsheng Lu

    2017-03-01

    Full Text Available Carbon fiber microelectrode (CFME has been extensively applied in the biosensor and chemical sensor domains. In order to improve the electrochemical activity and sensitivity of the CFME, a new CFME modified with carbon nanotubes (CNTs, denoted as CNTs/CFME, was fabricated and investigated. First, carbon fiber (CF monofilaments grafted with CNTs (simplified as CNTs/CFs were fabricated in two key steps: (i nickel electroless plating, followed by (ii chemical vapor deposition (CVD. Second, a single CNTs/CF monofilament was selected and encapsulated into a CNTs/CFME with a simple packaging method. The morphologies of as-prepared CNTs/CFs were characterized by scanning electron microscopy. The electrochemical properties of CNTs/CFMEs were measured in potassium ferrocyanide solution (K4Fe(CN6, by using a cyclic voltammetry (CV and a chronoamperometry method. Compared with a bare CFME, a CNTs/CFME showed better CV curves with a higher distinguishable redox peak and response current; the higher the CNT content was, the better the CV curves were. Because the as-grown CNTs significantly enhanced the effective electrode area of CNTs/CFME, the contact area between the electrode and reactant was enlarged, further increasing the electrocatalytic active site density. Furthermore, the modified microelectrode displayed almost the same electrochemical behavior after 104 days, exhibiting remarkable stability and outstanding reproducibility.

  2. 5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

    Science.gov (United States)

    Ogino, Minako

    2017-01-01

    ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups

  3. Haiku: New paradigm for the reverse genetics of emerging RNA viruses.

    Directory of Open Access Journals (Sweden)

    Thérèse Atieh

    Full Text Available Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA, respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.

  4. A highly stable and sensitive chemically modified screen-printed electrode for sulfide analysis

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, D.-M. [Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40217, Taiwan (China); Kumar, Annamalai Senthil [Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40217, Taiwan (China); Zen, J.-M. [Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40217, Taiwan (China)]. E-mail: jmzen@dragon.nchu.edu.tw

    2006-01-18

    We report here a highly stable and sensitive chemically modified screen-printed carbon electrode (CMSPE) for sulfide analysis. The CMSPE was prepared by first ion-exchanging ferricyanide into a Tosflex anion-exchange polymer and then sealing with a tetraethyl orthosilicate sol-gel layer. The sol-gel overlayer coating was crucial to stabilize the electron mediator (i.e., Fe(China){sub 6} {sup 3-}) from leaching. The strong interaction between the oxy-hydroxy functional group of sol-gel and the hydrophilic sites of Tosflex makes the composite highly rigid to trap the ferricyanide mediator. An obvious electrocatalytic sulfide oxidation current signal at {approx}0.20 V versus Ag/AgCl in pH 7 phosphate buffer solution was observed at the CMSPE. A linear calibration plot over a wide range of 0.1 {mu}M to 1 mM with a slope of 5.6 nA/{mu}M was obtained by flow injection analysis. The detection limit (S/N = 3) was 8.9 nM (i.e., 25.6 ppt). Practical utility of the system was applied to the determination of sulfide trapped from cigarette smoke and sulfide content in hot spring water.

  5. Uranium adsorption by non-treated and chemically modified cactus fibres in aqueous solutions

    International Nuclear Information System (INIS)

    Melpomeni Prodromou; Ioannis Pashalidis

    2013-01-01

    The adsorption efficiency of Opuntia ficus indica fibres regarding the removal of hexavalent uranium [U(VI)] from aqueous solutions has been investigated prior and after the chemical treatment (e.g. phosphorylation and MnO 2 -coating) of the biomass. The separation/removal efficiency has been studied as a function of pH, uranium concentration, adsorbent mass, ionic strength, temperature and contact time. Evaluation of the experimental data shows that biosorption is strongly pH-depended and that the MnO 2 -coated product presents the highest adsorption capacity followed by the phosphorylated and non-treated material. Experiments with varying ionic strength/salinity don't show any significant effect on the adsorption efficiency, indicating the formation of inner-sphere surface complexes. The adsorption reactions are in all cases exothermic and relatively fast, particularly regarding the adsorption on the MnO 2 -coated product. The results of the present study indicate that adsorption of uranium from waters is very effective by cactus fibres and particularly the modified treated fibres. The increased adsorption efficiency of the cactus fibres is attributed to their primary and secondary fibrillar structure, which result in a relative relative high specific surface available for sorption. (author)

  6. Obtention of chemically modified clays: organovermiculites

    International Nuclear Information System (INIS)

    Santana, Lisiane N.L.; Silva, Andrea L.; Barbosa, Estefane; Neves, Gelmires A.; Menezes, Romualdo Rodrigues

    2012-01-01

    The organovermiculite is obtained by incorporating the quaternary ammonium salt in the clay mineral vermiculite interlayer space. The objective of this work was to prepare organovermiculites for applications in organic contaminants adsorption. The variation of interlayer space was determined when the vermiculite was treated with an ionic salt (Praepagem WB) and a non-ionic salt (Amina Etoxilada TA50) in different concentrations. Before interacting with quaternary ammonium salt, the clay mineral was subjected to cationic change process with Na 2 CO 3 to substitute Mg 2+ by Na + . The results showed enlargement of interlayer space, reaching values up to 60.0 Å. The vermiculite pre-activated with Na 2 CO 3 during 5 days and modified with the Praepagem WB showed the best performance. Amina Etoxilada TA50 salt was not observed significant changes with increasing concentration. The affinity of organovermiculite for organic solvents was confirmed by Foster swelling test and the best results were observed with diesel and petrol as solvents. (author)

  7. Antibiotic Algae by Chemical Surface Engineering.

    Science.gov (United States)

    Kerschgens, Isabel P; Gademann, Karl

    2018-03-02

    Chemical cell-surface engineering is a tool for modifying and altering cellular functions. Herein, we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4-hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability or motility of the cells. The modified cells were used to inhibit bacterial growth of Gram-positive Bacillus subtilis cultures. Delivery of the antibiotic from the microalgae to the bacterial cells was verified by microscopy. Our studies provide compelling evidence that 1) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and 2) living microalgae can serve as new platforms for drug delivery. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Electrochemical properties of the hexacyanoferrate(II)–ruthenium(III) complex immobilized on silica gel surface chemically modified with zirconium(IV) oxide

    International Nuclear Information System (INIS)

    Panice, Lucimara B.; Oliveira, Elisangela A. de; Filho, Ricardo A.D. Molin; Oliveira, Daniela P. de; Lazarin, Angélica M.; Andreotti, Elza I.S.; Sernaglia, Rosana L.; Gushikem, Yoshitaka

    2014-01-01

    Highlights: • The cyano-bridged mixed valence ruthenium composite material was synthesized. • This newly synthesized compound was incorporated into a carbon paste electrode. • The electrode did not show significant changes in response after six months of use. • The modified electrode is very stable and reproducible. • The electrode sensor was successfully applied for ascorbic acid determination. - Abstract: The chemically modified silica gel with zirconium(IV) oxide was used to immobilize the [Fe(CN) 6 ] 4− complex ion initially. The reaction of this material with [Ru(edta)H 2 O] − complex ion formed the immobilized cyano-bridged mixed valence ruthenium complex, (≡Zr) 5 [(edta)RuNCFe(CN) 5 ]. This material was incorporated into a carbon paste electrode and, its electrochemical properties were investigated. However, for an ascorbic acid solution, an enhancement of the anodic peak current was detected due to electrocatalytic oxidation. The electrode presented the same response for at least 150 successive measurements, with a good repeatability. The modified electrode is very stable and reproducible. The sensor was applied for ascorbic acid determination in pharmaceutical preparation with success

  9. RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA?

    Science.gov (United States)

    Subramanya, Sandesh; Kim, Sang-Soo; Manjunath, N; Shankar, Premlata

    2010-02-01

    Despite the clinical benefits of highly active antiretroviral therapy (HAART), the prospect of life-long antiretroviral treatment poses significant problems, which has spurred interest in developing new drugs and strategies to treat HIV infection and eliminate persistent viral reservoirs. RNAi has emerged as a therapeutic possibility for HIV. We discuss progress in overcoming hurdles to translating transient and stable RNAi enabling technologies to clinical application for HIV; covering the past 2 - 3 years. HIV inhibition can be achieved by transfection of chemically or enzymatically synthesized siRNAs or by DNA-based vector systems expressing short hairpin RNAs (shRNAs) that are processed intracellularly into siRNA. We compare these approaches, focusing on technical and safety issues that will guide the choice of strategy for clinical use. Introduction of synthetic siRNA into cells or its stable endogenous production using vector-driven shRNA have been shown to suppress HIV replication in vitro and, in some instances, in vivo. Each method has advantages and limitations in terms of ease of delivery, duration of silencing, emergence of escape mutants and potential toxicity. Both appear to have potential as future therapeutics for HIV, once the technical and safety issues of each approach are overcome.

  10. Applications of Graphene-Modified Electrodes in Microbial Fuel Cells

    Directory of Open Access Journals (Sweden)

    Fei Yu

    2016-09-01

    Full Text Available Graphene-modified materials have captured increasing attention for energy applications due to their superior physical and chemical properties, which can significantly enhance the electricity generation performance of microbial fuel cells (MFC. In this review, several typical synthesis methods of graphene-modified electrodes, such as graphite oxide reduction methods, self-assembly methods, and chemical vapor deposition, are summarized. According to the different functions of the graphene-modified materials in the MFC anode and cathode chambers, a series of design concepts for MFC electrodes are assembled, e.g., enhancing the biocompatibility and improving the extracellular electron transfer efficiency for anode electrodes and increasing the active sites and strengthening the reduction pathway for cathode electrodes. In spite of the challenges of MFC electrodes, graphene-modified electrodes are promising for MFC development to address the reduction in efficiency brought about by organic waste by converting it into electrical energy.

  11. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

    Science.gov (United States)

    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  12. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

    Science.gov (United States)

    Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  13. Understanding the effect of locked nucleic acid and 2'-O-methyl modification on the hybridization thermodynamics of a miRNA-mRNA pair in the presence and absence of AfPiwi protein.

    Science.gov (United States)

    Kumar, Santosh; Mapa, Koyeli; Maiti, Souvik

    2014-03-18

    miRNAs are some of the key epigenetic regulators of gene expression. They act through hybridization with their target mRNA and modulate the level of respective proteins via different mechanisms. Various cancer conditions are known to be associated with up- and downregulation of the oncogenic and tumor suppressor miRNAs, respectively. The levels of aberrantly expressed oncogenic miRNAs can be downregulated in different ways. Similarly, restoration of tumor suppressor miRNAs to their normal levels can be achieved using miRNA mimics. However, the use of miRNA mimics is limited by their reduced biostability and function. We have studied the hybridization thermodynamics of the miRNA 26a (11-mer, including the seed sequence) guide strand with the mRNA (11-mer) target strand in the absence and presence of AfPiwi protein. We have also inserted locked nucleic acids (LNAs) and 2'-O-methyl-modified nucleotides into the guide strand, in a walk-through manner, to assess their effect on the binding efficiency between guide and target RNA. Insertion of LNA and 2'-O-methyl-modified nucleotides into the guide strand helped to strengthen the binding affinity irrespective of the position of insertion. However, in the presence of AfPiwi protein, these modifications reduced the binding affinity to different extents depending on the position of insertion. Insertion of a modification leads to an increase in the enthalpic contribution with an increased unfavorable entropic contribution, which negatively compensates for the higher favorable enthalpy.

  14. A non-heme iron-mediated chemical demethylation in DNA and RNA.

    Science.gov (United States)

    Yi, Chengqi; Yang, Cai-Guang; He, Chuan

    2009-04-21

    DNA methylation is arguably one of the most important chemical signals in biology. However, aberrant DNA methylation can lead to cytotoxic or mutagenic consequences. A DNA repair protein in Escherichia coli, AlkB, corrects some of the unwanted methylations of DNA bases by a unique oxidative demethylation in which the methyl carbon is liberated as formaldehyde. The enzyme also repairs exocyclic DNA lesions--that is, derivatives in which the base is augmented with an additional heterocyclic subunit--by a similar mechanism. Two proteins in humans that are homologous to AlkB, ABH2 and ABH3, repair the same spectrum of lesions; another human homologue of AlkB, FTO, is linked to obesity. In this Account, we describe our studies of AlkB, ABH2, and ABH3, including our development of a general strategy to trap homogeneous protein-DNA complexes through active-site disulfide cross-linking. AlkB uses a non-heme mononuclear iron(II) and the cofactors 2-ketoglutarate (2KG) and dioxygen to effect oxidative demethylation of the DNA base lesions 1-methyladenine (1-meA), 3-methylcytosine (3-meC), 1-methylguanine (1-meG), and 3-methylthymine (3-meT). ABH3, like AlkB, works better on single-stranded DNA (ssDNA) and is capable of repairing damaged bases in RNA. Conversely, ABH2 primarily repairs lesions in double-stranded DNA (dsDNA); it is the main housekeeping enzyme that protects the mammalian genome from 1-meA base damage. The AlkB-family proteins have moderate affinities for their substrates and bind DNA in a non-sequence-specific manner. Knowing that these proteins flip the damaged base out from the duplex DNA and insert it into the active site for further processing, we first engineered a disulfide cross-link in the active site to stabilize the Michaelis complex. Based on the detailed structural information afforded by the active-site cross-linked structures, we can readily install a cross-link away from the active site to obtain the native-like structures of these complexes

  15. Investigation of Physico-Chemical Properties of Sand-Lime Products Modified of Diabase Aggregate and Chalcedonite Meal

    Science.gov (United States)

    Dachowski, Ryszard; Komisarczyk, Katarzyna

    2017-10-01

    In the era of rapid development in the construction industry, particular attention is focused on harmless and natural materials. Some of the best materials for building masonry walls are sand-lime products. Silicates are obtained from a mixture of quartz, sand and a small amount of water. They emerge as a result of the hydrothermal treatment conducted under high pressure and at a temperature of app. 203 °C. Silicates were modified of different kinds of aggregates, glass or plastics, and the content of dry ingredients was changed because of this fact. The paper describes the studies where the combination of diabase aggregate and chalcedonite meal was used. Microstructure of the products was analyzed with the use of mercury intrusion porosimetry, SEM and XRD methods. Variable content of chalcedonite meal changes the internal structure and the physico-chemical properties.

  16. Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with Nitrogen-15

    International Nuclear Information System (INIS)

    Gewirth, D.T.; Abo, S.R.; Leontis, N.B.; Moore, P.B.

    1987-01-01

    A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15 N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a normal chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for the authors understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed

  17. [Construction of RNA-containing virus-like nanoparticles expression vector with cysteine residues on surface and fluorescent decoration].

    Science.gov (United States)

    Cheng, Yang-Jian; Liang, Ji-Xuan; Li, Qing-Ge

    2005-08-01

    Site-directed mutagenesis was performed at the codon 15 of the MS2 bacteriophage coat protein gene,which had been cloned to the virus-like particles expression vector containing non-self RNA fragment. The produced expression vector,termed pARSC, was transformed to E. coli DH5alpha. The positive clones were selected and proliferated. The harvested cells were treated with sonication and the supernatant was then subjected to linear sucrose density gradients centrifugation (15% to 60%) at 32000 r/min for 4 h at 4 degrees C. The virus-like particles, VLP-Cy, were collected at 35% sucrose density. The particles were examined by transmission electron microscopy and the spherical viral particles of approximately 27 nm in diameter were found. The thiolated VLP-Cy was then chemically modified with fluorescein -5'-maleimide. The covalent fluorescent labeling was confirmed by absorption analysis, SDS-PAGE and MALDI-TOF mass spectroscopy. This is the first report of preparation of RNA-containing natural fluorescent nanoparticles. The study highlight the versatility of MS2 bacteriophage capsids as building blocks for functional nanomaterials construction for a variety of application purposes.

  18. An efficient laser vaporization source for chemically modified metal clusters characterized by thermodynamics and kinetics

    Science.gov (United States)

    Masubuchi, Tsugunosuke; Eckhard, Jan F.; Lange, Kathrin; Visser, Bradley; Tschurl, Martin; Heiz, Ulrich

    2018-02-01

    A laser vaporization cluster source that has a room for cluster aggregation and a reactor volume, each equipped with a pulsed valve, is presented for the efficient gas-phase production of chemically modified metal clusters. The performance of the cluster source is evaluated through the production of Ta and Ta oxide cluster cations, TaxOy+ (y ≥ 0). It is demonstrated that the cluster source produces TaxOy+ over a wide mass range, the metal-to-oxygen ratio of which can easily be controlled by changing the pulse duration that influences the amount of reactant O2 introduced into the cluster source. Reaction kinetic modeling shows that the generation of the oxides takes place under thermalized conditions at less than 300 K, whereas metal cluster cores are presumably created with excess heat. These characteristics are also advantageous to yield "reaction intermediates" of interest via reactions between clusters and reactive molecules in the cluster source, which may subsequently be mass selected for their reactivity measurements.

  19. Applicability of Vacuum Impregnation to Modify Physico-Chemical, Sensory and Nutritive Characteristics of Plant Origin Products—A Review

    Directory of Open Access Journals (Sweden)

    Elżbieta Radziejewska-Kubzdela

    2014-09-01

    Full Text Available Vacuum impregnation is a non-destructive method of introducing a solution with a specific composition to the porous matrices of fruit and vegetables. Mass transfer in this process is a result of mechanically induced differences in pressure. Vacuum impregnation makes it possible to fill large volumes of intercellular spaces in tissues of fruit and vegetables, thus modifying physico-chemical properties and sensory attributes of products. This method may be used, e.g., to reduce pH and water activity of the product, change its thermal properties, improve texture, color, taste and aroma. Additionally, bioactive compounds may be introduced together with impregnating solutions, thus improving health-promoting properties of the product or facilitating production of functional food.

  20. Exploring the impact of the side-chain length on peptide/RNA binding events.

    Science.gov (United States)

    Sbicca, Lola; González, Alejandro López; Gresika, Alexandra; Di Giorgio, Audrey; Closa, Jordi Teixido; Tejedor, Roger Estrada; Andréola, Marie-Line; Azoulay, Stéphane; Patino, Nadia

    2017-07-19

    The impact of the amino-acid side-chain length on peptide-RNA binding events has been investigated using HIV-1 Tat derived peptides as ligands and the HIV-1 TAR RNA element as an RNA model. Our studies demonstrate that increasing the length of all peptide side-chains improves unexpectedly the binding affinity (K D ) but reduces the degree of compactness of the peptide-RNA complex. Overall, the side-chain length appears to modulate in an unpredictable way the ability of the peptide to compete with the cognate TAR RNA partner. Beyond the establishment of non-intuitive fundamental relationships, our results open up new perspectives in the design of effective RNA ligand competitors, since a large number of them have already been identified but few studies report on the modulation of the biological activity by modifying in the same way the length of all chains connecting RNA recognition motives to the central scaffold of a ligand.

  1. Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells

    Science.gov (United States)

    Shu, Yi; Shu, Dan; Haque, Farzin; Guo, Peixuan

    2013-01-01

    RNA nanotechnology is a term that refers to the design, fabrication, and utilization of nanoparticles mainly composed of ribonucleic acids via bottom-up self-assembly. The packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor has been developed into a nano-delivery platform. This protocol describes the synthesis, assembly, and functionalization of pRNA nanoparticles based on three ‘toolkits’ derived from pRNA structural features: interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions, and an RNA three-way junction for branch-extension. siRNAs, ribozymes, aptamers, chemical ligands, fluorophores, and other functionalities can also be fused to the pRNA prior to the assembly of the nanoparticles, so as to ensure the production of homogeneous nanoparticles and the retention of appropriate folding and function of the incorporated modules. The resulting self-assembled multivalent pRNA nanoparticles are thermodynamically and chemically stable, and they remain intact at ultra-low concentrations. Gene silencing effects are progressively enhanced with increasing number of siRNA in each pRNA nanoparticle. Systemic injection of the pRNA nanoparticles into xenograft-bearing mice has revealed strong binding to tumors without accumulation in vital organs or tissues. The pRNA-based nano-delivery scaffold paves a new way towards nanotechnological application of pRNA-based nanoparticles for disease detection and treatment. The time required for completing one round of this protocol is 3–4 weeks, including in vitro functional assays, or 2–3 months including in vivo studies. PMID:23928498

  2. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

  3. Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems.

    Directory of Open Access Journals (Sweden)

    Abdimajid Osman

    Full Text Available Platelet concentrates (PCs are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets' nucleic acids. We previously reported that treatment of stored platelets with the PR system Intercept significantly reduced the level of half of the microRNAs that were monitored, induced platelet activation and compromised the platelet response to physiological agonists. Using genome-wide differential expression (DE RNA sequencing (RNA-Seq, we now report that Intercept markedly perturbs the mRNA transcriptome of human platelets and alters the expression level of >800 mRNAs (P<0.05 compared to other PR systems and control platelets. Of these, 400 genes were deregulated with DE corresponding to fold changes (FC ≥ 2. At the p-value < 0.001, as many as 147 genes were deregulated by ≥ 2-fold in Intercept-treated platelets, compared to none in the other groups. Finally, integrated analysis combining expression data for microRNA (miRNA and mRNA, and involving prediction of miRNA-mRNA interactions, disclosed several positive and inverse correlations between miRNAs and mRNAs in stored platelets. In conclusion, this study demonstrates that Intercept markedly deregulates the platelet mRNA transcriptome, concomitant with reduced levels of mRNA-regulatory miRNAs. These findings should enlighten authorities worldwide when considering the implementation of PR systems, that target nucleic acids and are not specific to pathogens, for the management of blood products.

  4. The origin and effect of small RNA signaling in plants

    Directory of Open Access Journals (Sweden)

    Jean-Sébastien eParent

    2012-08-01

    Full Text Available Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute and other proteins to form the RNA-induced silencing complex (RISC that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the Argonaute protein involved, RISC triggers either DNA methylation and chromatin modification (leading to transcriptional gene silencing, TGS or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS. In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of small RNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

  5. Optimization and characterization of high pressure homogenization produced chemically modified starch nanoparticles.

    Science.gov (United States)

    Ding, Yongbo; Kan, Jianquan

    2017-12-01

    Chemically modified starch (RS4) nanoparticles were synthesized through homogenization and water-in-oil mini-emulsion cross-linking. Homogenization was optimized with regard to z-average diameter by using a three-factor-three-level Box-Behnken design. Homogenization pressure (X 1 ), oil/water ratio (X 2 ), and surfactant (X 3 ) were selected as independent variables, whereas z-average diameter was considered as a dependent variable. The following optimum preparation conditions were obtained to achieve the minimum average size of these nanoparticles: 50 MPa homogenization pressure, 10:1 oil/water ratio, and 2 g surfactant amount, when the predicted z-average diameter was 303.6 nm. The physicochemical properties of these nanoparticles were also determined. Dynamic light scattering experiments revealed that RS4 nanoparticles measuring a PdI of 0.380 and an average size of approximately 300 nm, which was very close to the predicted z-average diameter (303.6 nm). The absolute value of zeta potential of RS4 nanoparticles (39.7 mV) was higher than RS4 (32.4 mV), with strengthened swelling power. X-ray diffraction results revealed that homogenization induced a disruption in crystalline structure of RS4 nanoparticles led to amorphous or low-crystallinity. Results of stability analysis showed that RS4 nanosuspensions (particle size) had good stability at 30 °C over 24 h.

  6. Gene expression differences between PAXgene and Tempus blood RNA tubes are highly reproducible between independent samples and biobanks.

    Science.gov (United States)

    Skogholt, Anne Heidi; Ryeng, Einar; Erlandsen, Sten Even; Skorpen, Frank; Schønberg, Svanhild A; Sætrom, Pål

    2017-03-23

    Gene expression profiling from blood is sensitive to technology choices. For example, the main blood RNA collection systems-the PAXgene and Tempus tubes-differently influence RNA expression signatures. The aim of this study was to establish a common RNA isolation protocol for these two systems and investigate if it could reduce the differences in gene expression between them. We collected identical blood samples on the PAXgene and Tempus systems and retrieved blood samples from two independent biobanks-NOWAC and HUNT3-which are based on PAXgene and Tempus, respectively. High-quality RNA was extracted from both sampling systems by using their original protocols and our common modified protocol, and were profiled on Illumina microarrays. Regardless of the protocol used, we found most of the measured transcripts to be differently affected by the two sampling systems. However, our modified protocol reduced the number of transcripts that were significantly differentially expressed between PAXgene and Tempus by approximately 50%. Expression differences between PAXgene and Tempus were highly reproducible both between protocols and between different independent sample sets (Pearson correlation 0.563-0.854 across 47323 probes). Moreover, the modified protocol increased the microRNA output of the system with lowest microRNA yield, the PAXgene system. Most transcripts are affected by the choice of sampling system, but these effects are highly reproducible between independent samples. We propose that by running a control experiment with samples on both systems in parallel with biologically relevant samples, researchers may adjust for technical differences between the sampling systems.

  7. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination.

    Science.gov (United States)

    Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

    2016-01-01

    Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with

  8. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

    Directory of Open Access Journals (Sweden)

    Dorota Tataruch-Weinert

    2016-06-01

    Full Text Available Background: Urinary extracellular vesicles (UEVs represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods: UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results: The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters and chemical pretreatment (water purification tablet. For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b UEVs were enriched with small RNA, (c Ribosomal RNA peaks were not observed for any RNA extraction method used and (d RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion: Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA

  9. Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

    Directory of Open Access Journals (Sweden)

    Santiago Grijalvo

    2018-02-01

    Full Text Available Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs or restoring the anomalous levels of non-coding RNAs (ncRNAs that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs, carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs, peptide nucleic acids (PNAs as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

  10. Surface chemical reactions probed with scanning force microscopy

    NARCIS (Netherlands)

    Werts, M.P L; van der Vegte, E.W.; Hadziioannou, G

    1997-01-01

    In this letter we report the study of surface chemical reactions with scanning force microscopy (SFM) with chemical specificity. Using chemically modified SFM probes, we can determine the local surface reaction conversion during a chemical surface modification. The adhesion forces between a

  11. Data on cell growth inhibition induced by anti-VEGF siRNA delivered by Stealth liposomes incorporating G2 PAMAM-cholesterol versus Metafectene® as a function of exposure time and siRNA concentration

    Directory of Open Access Journals (Sweden)

    Nasim Golkar

    2016-09-01

    Full Text Available In this data article, carboxyfluorescein-loaded liposomes were prepared and purified from free carboxyfluorescein using gel filtration chromatography in the first part. In the next part, following preparation of anti-VEGF siRNA loaded liposomes incorporating hydrophobically modified G2 PAMAM dendrimer (G2-Chol40% (Golkar et al., 2016 [1], the cell growth inhibition induced by the formulations (siRNA/Metafectene complexes and siRNA loaded liposomes incorporating hydrophobic G2 was evaluated at two exposure times through MTT assay in a breast cancer cell (SKBR-3 and compared by two-way ANOVA. Keywords: Anti-VEGF siRNA, Cell growth inhibition, Polyamidoaminedendrimer, Liposome

  12. Single step production of Cas9 mRNA for zygote injection.

    Science.gov (United States)

    Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

    2018-03-01

    Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

  13. Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility

    DEFF Research Database (Denmark)

    Permuth, Jennifer B; Reid, Brett; Earp, Madalene

    2016-01-01

    RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleo......, including rs1127313 (G/A), a SNP in the 3' untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing.......RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single...... nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1...

  14. How to find the optimal partner--studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine.

    Science.gov (United States)

    Piecyk, Karolina; Niedzwiecka, Anna; Ferenc-Mrozek, Aleksandra; Lukaszewicz, Maciej; Darzynkiewicz, Edward; Jankowska-Anyszka, Marzena

    2015-08-01

    Snurportin 1 is an adaptor protein that mediates the active nuclear import of uridine-rich small nuclear RNAs (U snRNA) by the importin-β receptor pathway. Its cellular activity influences the overall transport yield of small ribonucleoprotein complexes containing N(2),N(2),7-trimethylguanosine (TMG) capped U snRNA. So far little is still known about structural requirements related to molecular recognition of the trimethylguanosine moiety by snurportin in solution. Since these interactions are of a great biomedical importance, we synthesized a series of new 7-methylguanosine cap analogues with extended substituents at the exocyclic 2-amino group to gain a deeper insight into how the TMG-cap is adapted into the snurportin cap-binding pocket. Prepared chemical tools were applied in binding assays using emission spectroscopy. Surprisingly, our results revealed strict selectivity of snurportin towards the TMG-cap structure that relied mainly on its structural stiffness and compactness. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Efficient Inhibition of wear debris-induced inflammation by locally delivered siRNA

    International Nuclear Information System (INIS)

    Peng Xiaochun; Tao Kun; Cheng Tao; Zhu Junfeng; Zhang Xianlong

    2008-01-01

    Aseptic loosening is the most common long-term complication of total joint replacement, which is associated with the generation of wear debris. The purpose of this study was to investigate the inhibitory effect of small interfering RNA (siRNA) targeting tumor necrosis factor-α (TNF-α) on wear debris-induced inflammation. A local delivery of lentivirus-mediated TNF-α siRNA into the modified murine air pouch, which was stimulated by polymethylmethacrylate (PMMA) particles, resulted in significant blockage of TNF-α both in mRNA and protein levels for up to 4 weeks. In addition, significant down-regulation of interleukin-1 (IL-1) and interleukin-6 (IL-6) was observed in TNF-α siRNA-treated pouches. The safety profile of gene therapy was proven by Bioluminescent assay and quantitative fluorescent flux. Histological analysis revealed less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) in TNF-α siRNA-treated pouches. These findings suggest that local delivery of TNF-α siRNA might be an excellent therapeutic candidate to inhibit particle-induced inflammation.

  16. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2009-08-01

    Full Text Available Abstract Background The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction. Results With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution. Conclusion It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

  17. Smart Inulin-Based Polycationic Nanodevices for siRNA Delivery.

    Science.gov (United States)

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

    2017-01-01

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

  18. The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

    Science.gov (United States)

    Gaston, Kirk W; Limbach, Patrick A

    2014-01-01

    The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems. PMID:25616408

  19. The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry.

    Science.gov (United States)

    Gaston, Kirk W; Limbach, Patrick A

    2014-01-01

    The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

  20. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Kèvin Knoops

    2008-09-01

    Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

  1. Inorganic-organic hybrid polyoxometalate containing supramolecular helical chains: Preparation, characterization and application in chemically bulk-modified electrode

    International Nuclear Information System (INIS)

    Han Zhangang; Zhao Yulong; Peng Jun; Liu Qun; Wang Enbo

    2005-01-01

    An inorganic-organic hybrid polyoxometalate (POM) (Hbpy) 4 [SiMo 12 O 40 ] (1) (bpy = 2,4-bipyridine), has been prepared and characterized. X-ray diffraction study reveals that compound 1 contains interesting organic double helical chains. The hybrid nanoparticles was used as a solid bulkmodifier to fabricate a three-dimensional chemically modified carbon paste electrode (1-CPE) by direct mixing. The electrochemical behavior and electrocatalysis of 1-CPE has been studied in detail. The results indicate that 1-CPE has a good electrocatalytic activity toward the reduction of nitrite in 1 M H 2 SO 4 aqueous solution. 1-CPE shows remarkable stability that can be ascribed to the interactions existed between POM anions and organic double helical bpy chains, which are very important for practical applications in electrode modification

  2. Temperature Dependence of Logarithmic-like Relaxational Dynamics of Hydrated tRNA.

    Science.gov (United States)

    Chu, Xiang-Qiang; Mamontov, Eugene; O'Neill, Hugh; Zhang, Qiu

    2013-03-21

    The dynamics of RNA within the β-relaxation region of 10 ps to 1 ns is crucial to its biological function. Because of its simpler chemical building blocks and the lack of the side methyl groups, faster relaxational dynamics of RNA compared to proteins can be expected. However, the situation is actually opposite. In this work, the relaxational dynamics of tRNA is measured by quasielastic neutron scattering and analyzed using the mode coupling theory, originally developed for glass-forming liquids. Our results reveal that the dynamics of tRNA follows a log-decay within the β-relaxation region, which is an important trait demonstrated by the dynamics of proteins. The dynamics of hydrated tRNA and lysozyme compared in the time domain further demonstrate that the slower dynamics of tRNA relative to proteins originates from the difference in the folded states of tRNA and proteins, as well as the influence of their hydration water.

  3. High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

    Science.gov (United States)

    Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

    2015-06-01

    Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  4. An Assay of RNA Synthesis in Hepatic Nuclei from Control and Streptococcus pneumoniae-Infected Rats

    Science.gov (United States)

    1982-02-22

    26) as modified by pended in 2.0 ml of 1.0 N KOH and incu- McNamara et al. (27). Nuclei (about 0.25 mg bated for 20 hr at 370 to hydrolyze RNA (28...containing hydrolyzed RNA was ml yeast RNA, 18 units pyruvate kinase, I counted in Scintisol. The pellet was solubi- mM cytidine triphosphate (CTP), I mM...WC. Lecithin biosynthesis in liver mi- 16. Blobel G, Potter VR. Nuclei from rat liver, isolation tochondrial fractions. Biochem Biophys Res Coin

  5. Phylogenetic distribution of plant snoRNA families.

    Science.gov (United States)

    Patra Bhattacharya, Deblina; Canzler, Sebastian; Kehr, Stephanie; Hertel, Jana; Grosse, Ivo; Stadler, Peter F

    2016-11-24

    Small nucleolar RNAs (snoRNAs) are one of the most ancient families amongst non-protein-coding RNAs. They are ubiquitous in Archaea and Eukarya but absent in bacteria. Their main function is to target chemical modifications of ribosomal RNAs. They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of chemical modification that they govern. Similarly to microRNAs, snoRNAs appear in distinct families of homologs that affect homologous targets. In animals, snoRNAs and their evolution have been studied in much detail. In plants, however, their evolution has attracted comparably little attention. In order to chart the phylogenetic distribution of individual snoRNA families in plants, we applied a sophisticated approach for identifying homologs of known plant snoRNAs across the plant kingdom. In response to the relatively fast evolution of snoRNAs, information on conserved sequence boxes, target sequences, and secondary structure is combined to identify additional snoRNAs. We identified 296 families of snoRNAs in 24 species and traced their evolution throughout the plant kingdom. Many of the plant snoRNA families comprise paralogs. We also found that targets are well-conserved for most snoRNA families. The sequence conservation of snoRNAs is sufficient to establish homologies between phyla. The degree of this conservation tapers off, however, between land plants and algae. Plant snoRNAs are frequently organized in highly conserved spatial clusters. As a resource for further investigations we provide carefully curated and annotated alignments for each snoRNA family under investigation.

  6. Synthesis, Improved Antisense Activity and Structural Rationale for the Divergent RNA Affinities of 3;#8242;-Fluoro Hexitol Nucleic Acid (FHNA and Ara-FHNA) Modified Oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Egli, Martin; Pallan, Pradeep S.; Allerson, Charles R.; Prakash, Thazha P.; Berdeja, Andres; Yu, Jinghua; Lee, Sam; Watt, Andrew; Gaus, Hans; Bhat, Balkrishen; Swayze, Eric E.; Seth, Punit P. (Isis Pharm.); (Vanderbilt)

    2012-03-16

    The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F {hor_ellipsis} H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py) {hor_ellipsis} H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

  7. Electron Transfer Reactivity Patterns at Chemically Modified Electrodes: Fundamentals and Application to the Optimization of Redox Recycling Amplification Systems

    Energy Technology Data Exchange (ETDEWEB)

    Bergren, Adam Johan [Iowa State Univ., Ames, IA (United States)

    2006-01-01

    Electroanalytical chemistry is often utilized in chemical analysis and Fundamental studies. Important advances have been made in these areas since the advent of chemically modified electrodes: the coating of an electrode with a chemical film in order to impart desirable, and ideally, predictable properties. These procedures enable the exploitation of unique reactivity patterns. This dissertation presents studies that investigate novel reaction mechanisms at self-assembled monolayers on gold. In particular, a unique electrochemical current amplification scheme is detailed that relies on a selective electrode to enable a reactivity pattern that results in regeneration of the analyte (redox recycling). This regenerating reaction can occur up to 250 times for each analyte molecule, leading to a notable enhancement in the observed current. The requirements of electrode selectivity and the resulting amplification and detection limit improvements are described with respect to the heterogeneous and homogeneous electron transfer rates that characterize the system. These studies revealed that the heterogeneous electrolysis of the analyte should ideally be electrochemically reversible, while that for the regenerating agent should be held to a low level. Moreover, the homogeneous reaction that recycles the analyte should occur at a rapid rate. The physical selectivity mechanism is also detailed with respect to the properties of the electrode and redox probes utilized. It is shown that partitioning of the analyte into/onto the adlayer leads to the extraordinary selectivity of the alkanethiolate monolayer modified electrode. Collectively, these studies enable a thorough understanding of the complex electrode mechanism required for successful redox recycling amplification systems, Finally, in a separate (but related) study, the effect of the akyl chain length on the heterogeneous electron transfer behavior of solution-based redox probes is reported, where an odd-even oscillation

  8. Chemical reagent and process for refuse disposal

    International Nuclear Information System (INIS)

    Somerville, R.B.; Fan, L.T.

    1989-01-01

    A process for treating refuse by mixing them with a reactive chemical and a puzzolana-type material. Said chemical includes a retarding agent which modifies the viscosity and an accelerating agent. (author)

  9. Immunoprecipitation of Tri-methylated Capped RNA.

    Science.gov (United States)

    Hayes, Karen E; Barr, Jamie A; Xie, Mingyi; Steitz, Joan A; Martinez, Ivan

    2018-02-05

    Cellular quiescence (also known as G 0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27 Kip1 . Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm by the Exportin-1 (XPO1) protein. We used an antibody against (TMG)-caps (which does not cross-react with the (m 7 G)-caps that most pri-miRNAs or mRNAs contain [Luhrmann et al ., 1982]) to perform RNA immunoprecipitations from total RNA extracts of proliferating or quiescent HFFs. The novelty of this assay is the specific isolation of pri-miRNAs as well as other non-coding RNAs containing a TMG-cap modification.

  10. Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

    Directory of Open Access Journals (Sweden)

    Lui Wing-bong

    2006-02-01

    Full Text Available Abstract Background We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR. In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. Methods An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen for quantitative performance, analytical sensitivity and precision. Results The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. Conclusion As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

  11. Construction of RNA-Quantum Dot Chimera for Nanoscale Resistive Biomemory Application.

    Science.gov (United States)

    Lee, Taek; Yagati, Ajay Kumar; Pi, Fengmei; Sharma, Ashwani; Choi, Jeong-Woo; Guo, Peixuan

    2015-07-28

    RNA nanotechnology offers advantages to construct thermally and chemically stable nanoparticles with well-defined shape and structure. Here we report the development of an RNA-QD (quantum dot) chimera for resistive biomolecular memory application. Each QD holds two copies of the pRNA three-way junction (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor. The fixed quantity of two RNAs per QD was achieved by immobilizing the pRNA-3WJ with a Sephadex aptamer for resin binding. Two thiolated pRNA-3WJ serve as two feet of the chimera that stand on the gold plate. The RNA nanostructure served as both an insulator and a mediator to provide defined distance between the QD and gold. Immobilization of the chimera nanoparticle was confirmed with scanning tunneling microscopy. As revealed by scanning tunneling spectroscopy, the conjugated pRNA-3WJ-QD chimera exhibited an excellent electrical bistability signal for biomolecular memory function, demonstrating great potential for the development of resistive biomolecular memory and a nano-bio-inspired electronic device for information processing and computing.

  12. The bioavailability of chemicals in soil for earthworms

    Science.gov (United States)

    Lanno, R.; Wells, J.; Conder, Jason M.; Bradham, K.; Basta, N.

    2004-01-01

    The bioavailability of chemicals to earthworms can be modified dramatically by soil physical/chemical characteristics, yet expressing exposure as total chemical concentrations does not address this problem. In order to understand the effects of modifying factors on bioavailability, one must measure and express chemical bioavailability to earthworms in a consistent, logical manner. This can be accomplished by direct biological measures of bioavailability (e.g., bioaccumulation, critical body residues), indirect biological measures of bioavailability (e.g., biomarkers, reproduction), or indirect chemical measures of bioavailability (e.g., chemical or solid-phase extracts of soil). If indirect chemical measures of bioavailability are to be used, they must be correlated with some biological response. Bioavailability can be incorporated into ecological risk assessment during risk analysis, primarily in the estimation of exposure. However, in order to be used in the site-specific ecological risk assessment of chemicals, effects concentrations must be developed from laboratory toxicity tests based on exposure estimates utilizing techniques that measure the bioavailable fraction of chemicals in soil, not total chemical concentrations. ?? 2003 Elsevier Inc. All rights reserved.

  13. Negative-strand RNA viruses: the plant-infecting counterparts.

    Science.gov (United States)

    Kormelink, Richard; Garcia, Maria Laura; Goodin, Michael; Sasaya, Takahide; Haenni, Anne-Lise

    2011-12-01

    While a large number of negative-strand (-)RNA viruses infect animals and humans, a relative small number have plants as their primary host. Some of these have been classified within families together with animal/human infecting viruses due to similarities in particle morphology and genome organization, while others have just recently been/or are still classified in floating genera. In most cases, at least two striking differences can still be discerned between the animal/human-infecting viruses and their plant-infecting counterparts which for the latter relate to their adaptation to plants as hosts. The first one is the capacity to modify plasmodesmata to facilitate systemic spread of infectious viral entities throughout the plant host. The second one is the capacity to counteract RNA interference (RNAi, also referred to as RNA silencing), the innate antiviral defence system of plants and insects. In this review an overview will be presented on the negative-strand RNA plant viruses classified within the families Bunyaviridae, Rhabdoviridae, Ophioviridae and floating genera Tenuivirus and Varicosavirus. Genetic differences with the animal-infecting counterparts and their evolutionary descendants will be described in light of the above processes. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. 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.

  15. Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

    Science.gov (United States)

    Watters, Kyle E; Choudhary, Krishna; Aviran, Sharon; Perry, Keith L

    2018-01-01

    Abstract In single stranded (+)-sense RNA viruses, <