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Sample records for sensitive oligonucleotide ligation

  1. Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.

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    Nuttada Panpradist

    Full Text Available Human immunodeficiency virus (HIV is a chronic infection that can be managed by antiretroviral treatment (ART. However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1 lysis and/or nucleic acid extraction, (2 amplification of HIV RNA or DNA, (3 ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4 analysis via oligonucleotide surface capture, denaturation, and detection (CDD. The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.

  2. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

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    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  3. Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array.

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    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2015-01-01

    With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens.

  4. Circular DNA by "Bis-Click" Ligation: Template-Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides.

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    Yang, Haozhe; Seela, Frank

    2016-01-22

    A highly effective and convenient "bis-click" strategy was developed for the template-independent circularization of single-stranded oligonucleotides by employing copper(I)-assisted azide-alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7-deaza-2'-deoxyadenosine and 2'-deoxyuridine residues with different side chains were used in solid-phase synthesis with phosphoramidite chemistry. The bis-click ligation of linear 9- to 36-mer oligonucleotides with 1,4-bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9-mers. Compared with linear duplexes, circular bis-click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis-click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A fast, visible-light-sensitive azobenzene for bioorthogonal ligation.

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    Poloni, Claudia; Szymański, Wiktor; Hou, Lili; Browne, Wesley R; Feringa, Ben L

    2014-01-20

    Azobenzenes have been used as photoresponsive units for the control of numerous biological processes. Primary prerequisites for such applications are site-selective incorporation of photoswitchable units into biomolecules and the possibility of using non-destructive and deep-tissue-penetrating visible light for the photoisomerization. Here we report a push-pull azobenzene that readily undergoes a Staudinger-Bertozzi ligation with azide groups, that can be addressed with visible light (>440 nm) and exhibits the solvato- and acidochromism typical for push-pull systems. The thermal relaxation in aqueous environment proceeds on the low-millisecond timescale, thus enabling control over biological processes on similar timescales. The approach is demonstrated in the modification of a quartz surface and in the incorporation of an azobenzene unit into a functional peptide, the third zinc finger in the mammalian factor Sp1. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Sensitive detection of aggregated prion protein via proximity ligation.

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    Hammond, Maria; Wik, Lotta; Deslys, Jean-Philippe; Comoy, Emmanuel; Linné, Tommy; Landegren, Ulf; Kamali-Moghaddam, Masood

    2014-01-01

    The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.

  7. Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

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    Schoonbroodt, Sonia; Frans, Nicolas; DeSouza, Mark; Eren, Rachel; Priel, Smadar; Brosh, Naama; Ben-Porath, Judith; Zauberman, Arie; Ilan, Ehud; Dagan, Shlomo; Cohen, Edward H.; Hoogenboom, Hennie R.; Ladner, Robert Charles; Hoet, René M.

    2005-01-01

    The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all VH families/segments was observed showing that ONCL did not introduce cloning biases for or against any VH family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 1010 and by selecting five unique Fabs against GAPDH antigen. PMID:15905471

  8. Oligonucleotide-linked gold nanoparticle aggregates for enhanced sensitivity in lateral flow assays.

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    Hu, Jie; Wang, Lin; Li, Fei; Han, Yu Long; Lin, Min; Lu, Tian Jian; Xu, Feng

    2013-11-21

    Lateral flow assays (LFAs) as rapid analytical techniques promise to be widely used in point-of-care (POC) diagnostics because of their affordability and simplicity. However, LFAs still suffer from low sensitivity in detection of various biomarkers, e.g., nucleic acids. In this study, we developed a simple and general one-step signal amplification strategy, which employed oligonucleotide-linked gold nanoparticle (AuNP) aggregates to enhance the sensitivity in nucleic acid lateral flow (NALF) assays. Using a nucleic acid sequence of human immunodeficiency virus type 1 (HIV-1) as a model analyte, we observed that the detection limit of the developed NALF assay was 0.1 nM, which was improved by 2.5-fold compared with that of a non-signal amplification approach. The methodology described here could be used to detect a broad range of nucleic acids, and the general signal amplification approach could be potentially adopted in other types of LFAs.

  9. Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

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    Gustafsdottir Sigrun

    2010-10-01

    Full Text Available Abstract Background Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation. Conclusions The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

  10. A new mixed-backbone oligonucleotide against glucosylceramide synthase sensitizes multidrug-resistant tumors to apoptosis.

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    Gauri A Patwardhan

    2009-09-01

    Full Text Available Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C(18-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.

  11. Oligonucleotide-stabilized fluorescent silver nanoclusters for the specific and sensitive detection of biotin.

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    Xiong, Xiaoli; Tang, Yan; Zhao, Jingjin; Zhao, Shulin

    2016-02-21

    A novel biotin fluorescent probe based on oligonucleotide-stabilized silver nanoclusters (DNA-AgNCs) was synthesized by employing a biotinylated cytosine-rich sequence as a synthesized template. The fluorescence properties of the DNA-AgNCs are related to the modified position of the DNA. When biotin is linked to the middle thymine base of the DNA sequence, the DNA-AgNCs emit the strongest fluorescence. Moreover, the stability of the DNA-AgNCs was affected by avidin through biotin-avidin binding, quenching the fluorescence of the DNA-AgNCs. In contrast, if free biotin is further introduced into this system, the quenching is apparently weakened by competition, leading to the restoration of fluorescence. This phenomenon can be utilized for the detection of biotin. Under the optimal conditions, the fluorescence recovery is linearly proportional to the concentration of biotin in the range of 10 nM-1.0 μM with a detection limit of 6.0 nM. This DNA-AgNCs probe with excellent fluorescent properties is sensitive and selective for the detection of biotin and has been applied for the determination of biotin in wheat flour.

  12. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

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    Peihu Fan

    Full Text Available The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA. Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL. The specificity was 100% and the coefficient of variation (CV was 7.8% at low p24 concentration (1.5 pg/mL with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.

  13. Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

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    Kovaliov, Marina; Segal, Meirav; Kafri, Pinhas; Yavin, Eylon; Shav-Tal, Yaron; Fischer, Bilha

    2014-05-01

    A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Uptake of liposomally entrapped fluorescent antisense oligonucleotides in NG108-15 cells: conventional versus pH-sensitive.

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    Skalko-Basnet, Natasa; Tohda, Michihisa; Watanabe, Hiroshi

    2002-12-01

    Antisense oligodeoxynucleotides (asODN) are novel therapeutic agents designed to alter RNA metabolism, ultimately resulting in decreased production of disease-associated gene products. To investigate internalisation of liposomally delivered asODN in NG108-15 cells, a hybrid cell line of mouse neuroblastoma and rat glioma, and assure that uptake of marker corresponds to that of antisense, we compared the cellular uptake of fluorescently labelled marker (fluorescein isothiocyanate (FITC)-dextran) and antisense oligonucleotide (FITC-asODN), entrapped either in conventional soy phosphatidylcholine (SPC) liposomes or pH-sensitive liposomes (composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate in a molar ratio of 3 : 2). Both SPC and pH-sensitive liposomes were prepared by a modified freeze-thawing method. Entrapment efficiencies (about 20% of the original material) did not depend on the liposome compositions and fluorescent material used. Fluorescence activated cell sorting (FACS) analysis was used to quantify the association of fluorescent material with the NG108-15 cells, whereas confocal microscopy gave insight on the location of cell associated-fluorescence. Conventional liposomes failed to deliver fluorescent material into the cells, but in contrast, pH-sensitive liposomes significantly improved the uptake of both FITC-dextran and FITC-asODN, with the uptake of liposomal FITC-dextran being greater than the uptake of liposomal FITC-asODN. These results suggest that pH-sensitive liposomes can be applied as a carrier system in the delivery of genetic material into the cells.

  15. Hybridization with synthetic oligonucleotides

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    Szostak, J.W.; Stiles, J.I.; Tye, B.K.; Sherman, F.; Wu, R.

    1978-01-01

    Procedures are described for the use of synthetic oligonucleotides for Southern blot experiments and gene bank screening, and the effect of various mismatches on the efficiency of hybridization is demonstrated. The following topics are discussed: sensitivity vs. specificity, hybridization of a 12-mer to the lambda endolysin gene; hybridization of oligonucleotide probes to the E. coli lac operator; hybridization of synthetic probes to the CYC1 gene of yeast; and cloning eucaryotic genes. (HLW)

  16. Tubal Ligation

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    ... details of the procedure Discuss the causes and probability of sterilization failure Share information about tubal ligation ... a not-for-profit organization and proceeds from Web advertising help support our mission. Mayo Clinic does ...

  17. Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids - TINA

    DEFF Research Database (Denmark)

    Schneider, Uffe V; Géci, Imrich; Jøhnk, Nina

    2011-01-01

    -TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch...

  18. Chemoselective ligation

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    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

    2011-05-10

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  19. Chemoselective ligation

    Science.gov (United States)

    Saxon, Eliana; Bertozzi, Carolyn

    2006-10-17

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  20. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn Ruth [Berkeley, CA

    2011-12-13

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  1. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana (Albany, CA); Bertozzi, Carolyn R. (Berkeley, CA)

    2010-02-23

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g. on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  2. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn [Berkeley, CA

    2003-05-27

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  3. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

    2011-04-12

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  4. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana (Albany, CA); Bertozzi, Carolyn Ruth (Berkeley, CA)

    2010-11-23

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  5. A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

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    Stougaard Magnus

    2007-08-01

    Full Text Available Abstract Background The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes. Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules. Results Chemically synthesized oligonucleotides with hairpin structures were acquired from our standard supplier. The stem of the hairpin contained recognition sequences for the Nt. Alw I nicking enzyme and the Mly I restriction enzyme. These double stranded regions were positioned in a way to allow self-templated circularization of the oligonucleotide. Following ligation, tandem repeats of the complementary sequence of the circular oligonucleotide could be produced through rolling circle DNA synthesis. By running successive rounds of ligation, rolling circle DNA synthesis, and nicking, the original oligonucleotide could be amplified as either the (+-strand or the (--strand. Alternatively, the hairpin structure could be removed by cleavage with the Mly I restriction enzyme, thereby releasing the oligonucleotide sequence contained within the hairpin structure from the hairpin. Conclusion We present here a method for the enzymatic production through DNA amplification of oligonucleotides with freely designable 5'-ends and 3'-ends, using hairpin-containing self-templating oligonucleotides. The hairpin comprises recognition sequences for a nicking enzyme and a restriction enzyme. The oligonucleotides are amplified by successive rounds of ligation, rolling circle DNA synthesis and nicking. Furthermore, the

  6. Oligonucleotide conjugates for therapeutic applications.

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    Winkler, Johannes

    2013-07-01

    Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake mechanisms and pharmacokinetic properties.

  7. Highly sensitive electrochemical sensor for mercury(II) ions by using a mercury-specific oligonucleotide probe and gold nanoparticle-based amplification.

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    Zhu, Zhiqiang; Su, Yuanyuan; Li, Jiang; Li, Di; Zhang, Jiong; Song, Shiping; Zhao, Yun; Li, Genxi; Fan, Chunhai

    2009-09-15

    We report a highly sensitive electrochemical sensor for the detection of Hg(2+) ions in aqueous solution by using a thymine (T)-rich, mercury-specific oligonucleotide (MSO) probe and gold nanoparticles (Au NPs)-based signal amplification. The MSO probe contains seven thymine bases at both ends and a "mute" spacer in the middle, which, in the presence of Hg(2+), forms a hairpin structure via the Hg(2+)-mediated coordination of T-Hg(2+)-T base pairs. The thiolated MSO probe is immobilized on Au electrodes to capture free Hg(2+) in aqueous media, and the MSO-bound Hg(2+) can be electrochemically reduced to Hg(+), which provides a readout signal for quantitative detection of Hg(2+). This direct immobilization strategy leads to a detection limit of 1 microM. In order to improve the sensitivity, MSO probe-modified Au NPs are employed to amplify the electrochemical signals. Au NPs are comodified with the MSO probe and a linking probe that is complementary to a capture DNA probe immobilized on gold electrodes. We demonstrated that this Au NPs-based sensing strategy brings about an amplification factor of more than 3 orders of magnitude, leading to a limit of detection of 0.5 nM (100 ppt), which satisfactorily meets the sensitivity requirement of U.S. Environmental Protection Agency (EPA). This Au NPs-based Hg(2+) sensor also exhibits excellent selectivity over a spectrum of interference metal ions. Considering the high sensitivity and selectivity of this sensor, as well as the cost-effective and portable features of electrochemical techniques, we expect this Au NPs amplified electrochemical sensor will be a promising candidate for field detection of environmentally toxic mercury.

  8. Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

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    Townsend Henrik J

    2005-11-01

    Full Text Available Abstract High-density oligonucleotide (oligo arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L. Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

  9. Sensitivity and specificity of in situ proximity ligation for protein interaction analysis in a model of steatohepatitis with Mallory-Denk bodies.

    Directory of Open Access Journals (Sweden)

    Bernhard Zatloukal

    Full Text Available The in situ proximity ligation assay (isPLA is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs. These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC. We investigated the colocalization of all three key MDB components, namely keratin 8 (K8, keratin 18 (K18, and p62 (sequestosome 1 by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8-/- and Krt18-/- mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol.

  10. Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification.

    Science.gov (United States)

    Ehlert, Alexandra; Demmel, Anja; Hupfer, Christine; Busch, Ulrich; Engel, Karl-Heinz

    2009-04-01

    The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg(-1) range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg(-1). The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.

  11. ALIS-FLP: Amplified ligation selected fragment-length polymorphism method for microbial genotyping

    DEFF Research Database (Denmark)

    Brillowska-Dabrowska, A.; Wianecka, M.; Dabrowski, Slawomir

    2008-01-01

    in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the Tsp......A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs...

  12. Tubal Ligation Reversal

    Science.gov (United States)

    ... and other factors. Success rates may be as high as 80 percent or as low as near 40 percent depending on your circumstances. Tubal ligation reversal is abdominal surgery, which carries a risk of infection, bleeding and ...

  13. Click nucleic acid ligation: applications in biology and nanotechnology.

    Science.gov (United States)

    El-Sagheer, Afaf H; Brown, Tom

    2012-08-21

    Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have

  14. Tubal ligation - slideshow

    Science.gov (United States)

    ... GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Tubal ligation - Series—Normal anatomy URL of this page: //medlineplus.gov/ency/presentations/ ...

  15. EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments.

    Science.gov (United States)

    Milewski, Marek C; Kamel, Karol; Kurzynska-Kokorniak, Anna; Chmielewski, Marcin K; Figlerowicz, Marek

    2017-10-01

    Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

  16. "Bis-Click" Ligation of DNA: Template-Controlled Assembly, Circularisation and Functionalisation with Bifunctional and Trifunctional Azides.

    Science.gov (United States)

    Yang, Haozhe; Seela, Frank

    2017-03-08

    Ligation and circularisation of oligonucleotides containing terminal triple bonds was performed with bifunctional or trifunctional azides. Both reactions are high yielding. Template-assisted bis-click ligation of two individual non-complementary oligonucleotide strands was accomplished to yield heterodimers exclusively. In this context, the template fulfils two functions: it accelerates the ligation reaction and controls product assembly (heterodimer vs. homodimer formation). Intermolecular bis-click circularisation of one oligonucleotide strand took place without template assistance. For construction of oligonucleotides with terminal triple bonds in the nucleobase side chain, 7- or 5-functionalised 7-deaza-dA and dU residues were used. These oligonucleotides are directly accessible by solid-phase synthesis. When trifunctional azides were employed instead of bifunctional linkers, functionalisation of the remaining azido group was performed with small molecules such as 1-ethynyl pyrene, biotin propargyl amide or with ethynylated oligonucleotides. By this means, branched DNA was constructed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

    Science.gov (United States)

    Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali

    2017-11-01

    The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

  18. Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification.

    Science.gov (United States)

    Chailleux, Catherine; Aymard, François; Caron, Pierre; Daburon, Virginie; Courilleau, Céline; Canitrot, Yvan; Legube, Gaëlle; Trouche, Didier

    2014-03-01

    Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.

  19. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

    2013-01-01

    Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical...... properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  20. Assessment of the Full Compatibility of Copper(I)-Catalyzed Alkyne-Azide Cycloaddition and Oxime Click Reactions for bis-Labelling of Oligonucleotides.

    Science.gov (United States)

    Estalayo-Adriàn, Sandra; Lartia, Rémy; Meyer, Albert; Vasseur, Jean-Jacques; Morvan, François; Defrancq, Eric

    2015-04-01

    The conjugation of oligonucleotides with reporters is of great interest for improving their intrinsic properties or endowing new ones. In this context, we report herein a new procedure for the bis-labelling of oligonucleotides through oxime ligation (Click-O) and copper(I)-catalyzed alkyne-azide cycloaddition (Click-H). 5'-Azido and 3'-aldehyde precursors were incorporated into oligonucleotides, and subsequent coupling reactions through Click-O and Click-H (or vice versa) were successfully achieved. In particular, we exhaustively investigated the full compatibility of each required step for both tethering strategies. The results demonstrate that click Huisgen and click oxime reactions are fully compatible. However, whilst both approaches can deliver the targeted doubly conjugated oligonucleotide, the route involving click oxime ligation prior to click Huisgen is significantly more successful. Thus the reactions investigated here can be considered to be key elements of the chemical toolbox for the synthesis of highly sophisticated bioconjugates.

  1. Simultaneous detection of DNA from ten food allergens by ligation-dependent probe amplification

    OpenAIRE

    Engel, Karl-Heinz; Demmel, Anja; Ehlert, Alexandra; Hupfer, Christine; Busch, Ulrich

    2009-01-01

    Abstract The simultaneous detection of DNA from different potentially allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows the detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanut, cashew nut, pecan nut, pistachio nut, hazelnut, sesame seeds, macadamia nut, almond, walnut and brazil nut. The specificity of the system was tested with DNA from more than 50...

  2. New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group.

    Science.gov (United States)

    Wu, Zuyan; Lee, Michael D; Carruthers, Thomas J; Szabo, Monika; Dennis, Matthew L; Swarbrick, James D; Graham, Bim; Otting, Gottfried

    2017-06-21

    Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

  3. Oligonucleotide fingerprinting to individualize ungulates.

    Science.gov (United States)

    Schwaiger, F W; Gomolka, M; Geldermann, H; Zischler, H; Buitkamp, J; Epplen, J T; Ammer, H

    1992-01-01

    The optimal combination of restriction enzyme and oligonucleotide probe has been determined for the individualization of hoofed animal species (cattle, pig, goat, sheep, horse and camel). Four different restriction endonucleases were used as well as five synthetic oligonucleotide probes hybridizing to different simple tandem repeats for fingerprint analyses in unrelated cattle (Swiss and German Simmental of unknown relationship and three families): 4 x 10(15) cows and oxes would reveal different banding patterns after HinfI digestion using the probe (CAC)5/(GTG)5. The other species were investigated using HinfI (and HaeIII) and five different oligonucleotide probes specific for simple tandem repeats. Using (CAC)5/(GTG)5 the discrimination potential in sheep was about one order of magnitude lower than in cattle while in goats 6 x 10(10) specimen are easily differentiated with (CA)8/(GT)8. From an evolutionary standpoint it may be of interest that also in all other ungulate species tested, (CAC)5/(GTG)5 and (CA)8/(GT)8 exhibited the highest potential for individualization. Advantages of oligonucleotide fingerprinting are discussed.

  4. Thioctic Acid Derivatives as Building Blocks to Incorporate DNA Oligonucleotides onto Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Sónia Pérez-Rentero

    2014-07-01

    Full Text Available Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid–gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid-L-threoninol derivatives containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.

  5. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    Science.gov (United States)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  6. 4'-C-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine as a sensitive (19)F NMR sensor for the detection of oligonucleotide secondary structures.

    Science.gov (United States)

    Granqvist, Lotta; Virta, Pasi

    2014-04-18

    4'-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine was synthesized and incorporated as a phosphoramidite into oligonucleotide sequences. Its applicability as a sensor for the (19)F NMR spectroscopic detection of DNA and RNA secondary structures was demonstrated. On DNA, the (19)F NMR measurements were focused on monitoring of duplex-triplex conversion, for which this fluorine-labeled 2'-deoxynucleoside proved to be a powerful sensor. This sensor seemingly favors DNA, but its behavior in the RNA environment also turned out to be informative. As a demonstration, invasion of a 2'-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was monitored by (19)F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4'-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.

  7. LNA-modified isothermal oligonucleotide microarray for ...

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of .... Sequences and calculated melting temperatures of DNA oligonucleotide probes and LNA-modified oligonucleotide probes for. Bacillus .... quick chilling on ice for 5 min. 13.5 μL of ...

  8. Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

    Science.gov (United States)

    Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

    2016-04-01

    Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

  9. Formatting and ligating biopolymers using adjustable nanoconfinement

    Science.gov (United States)

    Berard, Daniel J.; Shayegan, Marjan; Michaud, Francois; Henkin, Gil; Scott, Shane; Leslie, Sabrina

    2016-07-01

    Sensitive visualization and conformational control of long, delicate biopolymers present critical challenges to emerging biotechnologies and biophysical studies. Next-generation nanofluidic manipulation platforms strive to maintain the structural integrity of genomic DNA prior to analysis but can face challenges in device clogging, molecular breakage, and single-label detection. We address these challenges by integrating the Convex Lens-induced Confinement (CLiC) technique with a suite of nanotopographies embedded within thin-glass nanofluidic chambers. We gently load DNA polymers into open-face nanogrooves in linear, concentric circular, and ring array formats and perform imaging with single-fluorophore sensitivity. We use ring-shaped nanogrooves to access and visualize confinement-enhanced self-ligation of long DNA polymers. We use concentric circular nanogrooves to enable hour-long observations of polymers at constant confinement in a geometry which eliminates the confinement gradient which causes drift and can alter molecular conformations and interactions. Taken together, this work opens doors to myriad biophysical studies and biotechnologies which operate on the nanoscale.

  10. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    Science.gov (United States)

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2014-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

  11. Gene Assembly from Chip-Synthesized Oligonucleotides

    Science.gov (United States)

    Eroshenko, Nikolai; Kosuri, Sriram; Marblestone, Adam H; Conway, Nicholas; Church, George M.

    2012-01-01

    De novo synthesis of long double-stranded DNA constructs has a myriad of applications in biology and biological engineering. However, its widespread adoption has been hindered by high costs. Cost can be significantly reduced by using oligonucleotides synthesized on high-density DNA chips. However, most methods for using off-chip DNA for gene synthesis have failed to scale due to the high error rates, low yields, and high chemical complexity of the chip-synthesized oligonucleotides. We have recently demonstrated that some commercial DNA chip manufacturers have improved error rates, and that the issues of chemical complexity and low yields can be solved by using barcoded primers to accurately and efficiently amplify subpools of oligonucleotides. This article includes protocols for computationally designing the DNA chip, amplifying the oligonucleotide subpools, and assembling 500-800 basepair (bp) constructs. PMID:25077042

  12. Electronic Structures of LNA Phosphorothioate Oligonucleotides

    DEFF Research Database (Denmark)

    Bohr, Henrik G.; Shim, Irene; Stein, Cy

    2017-01-01

    Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces...... or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical...

  13. Orchidopexy san ligation technique of orchidopexy

    Directory of Open Access Journals (Sweden)

    Jain Vishal

    2011-01-01

    Full Text Available Pediatric hernia surgery is the most common operation done by pediatric general surgeons and it is a core competency for general surgeons in the developing world. Herniotomy is performed for the surgical repair of hernia and along with orchiopexy for the closure of associated patent processus vaginalis. Traditionally, ligation of hernial sac during orchiopexy is considered mandatory to prevent postoperative development of hernia. The present report was designed to study the results of non-ligation of the hernial sac during orchiopexy. It was found that non-ligation has no untoward effect on early complications and recurrence rate on long-term follow-up. It is suggested that it is not necessary to ligate the hernial sac during orchiopexy in children.

  14. Entropy driven chain effects on ligation chemistry

    OpenAIRE

    Pahnke, K.; Brandt, J.; Gryn'ova, G.; Lindner, P.; Schweins, R.; Schmidt, F.G.; Lederer, A; Coote, M.L.; Barner-Kowollik, C

    2015-01-01

    We report the investigation of fundamental entropic chain effects that enable the tuning of modular ligation chemistry – for example dynamic Diels–Alder (DA) reactions in materials applications – not only classically via the chemistry of the applied reaction sites, but also via the physical and steric properties of the molecules that are being joined. Having a substantial impact on the reaction equilibrium of the reversible ligation chemistry, these effects are important when tran...

  15. Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates.

    Science.gov (United States)

    Algar, W Russ; Blanco-Canosa, Juan B; Manthe, Rachel L; Susumu, Kimihiro; Stewart, Michael H; Dawson, Philip E; Medintz, Igor L

    2013-01-01

    Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

  16. Electron detachment dissociation (EDD) pathways in oligonucleotides

    Science.gov (United States)

    Kinet, Catherine; Gabelica, Valérie; Balbeur, Dorothée; de Pauw, Edwin

    2009-06-01

    Electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) are two novel fragmentation methods yielding radicals from negatively charged ions. With the goal of comparing EDD, EPD and the more traditional collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) fragmentation processes in oligonucleotides, we studied here the EDD fragmentation pathways of oligonucleotides of varying length. We chose polythymine oligonucleotides because these are the least prone to secondary structure formation, and found complete sequence coverage by EDD for up to dT20. We also found that the fragmentation pathways change with oligonucleotide length: electron detachment is a mandatory step in the fragmentation of larger sequences, while shorter oligonucleotides can also fragment via direct electronic or vibrational excitation by the electrons. This is supported by (1) the fact that continuous ejection of the charge-reduced species does not totally prevent fragmentation of short oligonucleotides dT5 and dT6, (2) the fact that CID and EDD fragments are more similar for small oligonucleotides (although double resonance experiments show that they are not all issued from the same mechanisms), and (3) the fact that electron-induced dissociation (EID) of singly charged dT3 and dT4 gives similar fragments as EDD of doubly charged dT5 and dT6. Finally, the detachment efficiency as a function of the nature of the nucleobase was studied. The effect of base on electron detachment in EDD (G > T > A > C) is different than in EPD (G > A > C > T), indicating different electron loss mechanisms.

  17. Comparison and transfer testing of multiplex ligation detection methods for GM plants

    Directory of Open Access Journals (Sweden)

    Ujhelyi Gabriella

    2012-01-01

    Full Text Available Abstract Background With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i hybridisation and ligation of specific probes, ii amplification of the ligated probes and iii detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study. Results Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands. Conclusions From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the

  18. Identification of GalNAc-Conjugated Antisense Oligonucleotide Metabolites Using an Untargeted and Generic Approach Based on High Resolution Mass Spectrometry.

    Science.gov (United States)

    Husser, Christophe; Brink, Andreas; Zell, Manfred; Müller, Martina B; Koller, Erich; Schadt, Simone

    2017-06-20

    Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl galactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells. A highly sensitive method was developed to identify metabolites of oligonucleotides using capillary flow liquid chromatography with column switching coupled to a high resolution Orbitrap Fusion mass spectrometer. Detection of GalNAc-conjugated oligonucleotides and their metabolites was achieved by combining full scan MS with two parallel MS2 experiments, one data-dependent scan and an untargeted MS2 experiment (all ion fragmentation) applying high collision energy. In the all ion fragmentation scan, a diagnostic fragment originating from the phosphorothioate backbone (O2PS-: m/z 94.936) was formed efficiently upon collisional activation. Based on this fragment an accurate determination of metabolites of oligonucleotides was achieved, independent of their sequence or conjugation in an untargeted but highly selective manner. The method was effectively applied to investigate uptake and metabolism of GalNAc-conjugated oligonucleotides in incubations of primary rat hepatocytes; the elucidation of expected and unexpected degradation products was achieved in subnanomolar range.

  19. Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytegenes and Bacillus cereus by Oligonucleotide Microarray

    Directory of Open Access Journals (Sweden)

    Meysam Sarshar

    2015-11-01

    Full Text Available Background: Traditional laboratory methods to detect pathogenic bacteria are time consuming and laborious. Therefore, it is essential to use powerful and reliable molecular methods for quick and simultaneous detection of microbial pathogens. Objectives: The current study aimed to evaluate the capability and efficiency of 23S rDNA sequence for rapid and simultaneous detection of four important food-borne pathogens by an oligonucleotide microarray technique. Materials and Methods: The 23S rDNA sequences of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus were obtained from GenBank databases and used to design the oligonucleotide probes and primers by Vector NTI software. Oligonucleotide probes were placed on a nylon membrane and hybridization was performed between probes and 23S rDNA digoxigenin-labeled polymerase chain reaction (PCR products. Hybridization signals were visualized by NBT/BCIP color development. Results: Positive hybridization color was produced for Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus. The oligonucleotide microarray detected all bacterial strains in a single reaction in less than five hours. The sensitivity of the performed microarray assay was 103 cfu/mL for each species of pathogen. No cross reaction was found between the tested bacterial species. Conclusions: The obtained results indicated that amplification of 23S rDNA gene followed by oligonucleotide microarray hybridization is a rapid and reliable method to identify and discriminate foodborne pathogens tested under the study.

  20. Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

    Directory of Open Access Journals (Sweden)

    Roberta D'Agata

    2017-01-01

    Full Text Available Gold nanoparticles (AuNPs exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays.

  1. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte Stahl; Jensen, Knud Jørgen

    2015-01-01

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our...... results by electronic structure calculations. Functionalized oligonucleotides were prepared in good yields by protein-mediated CuAAC click reactions for the first time with a human copper-binding chaperon. The carbohydrate, peptide, and fluorescent derivatives display high binding affinity and selectivity...

  2. Synthetic oligonucleotides : Useful molecules? A review

    NARCIS (Netherlands)

    Calogero, A; Hospers, GAP; Mulder, NH

    1997-01-01

    Specific inhibition of mammalian genes is possible through the use of antisense oligonucleotides (AS ODNs) or ribozymes. These strategies have led to a better understanding of several cellular and molecular mechanisms, among which cancer development. Recently, these strategies have been applied also

  3. Associating Oligonucleotides with Positively Charged Liposomes

    Czech Academy of Sciences Publication Activity Database

    Jurkiewicz, P.; Okruszek, A.; Hof, Martin; Langner, M.

    2003-01-01

    Roč. 8, č. 1 (2003), s. 77-84 ISSN 1425-8153 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : oligonucleotides * fluorescence correlation spectroscopy * DOTAP Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.455, year: 2003

  4. A comparative analysis of existing oligonucleotides selection ...

    African Journals Online (AJOL)

    SERVER

    2007-07-04

    Jul 4, 2007 ... rence of poly-morphisms in genomic DNA (Schena,. 1996). Two DNA chips format currently in wide use are, the cDNA array format and high density synthetic oligo- nucleotide array format (oligos). The oligonucleotide app- roach has some advantages because it allows the user to design oligos for each ...

  5. Expressed protein ligation for a large dimeric protein

    NARCIS (Netherlands)

    Karagöz, G.E.; Sinnige, T; Hsieh, O.; Rüdiger, S.G.D.

    2011-01-01

    Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic

  6. Artery ligation in the treatment of hemorrhoidal disease

    NARCIS (Netherlands)

    Schuurman, J.P.

    2012-01-01

    The aim of this thesis was to study the working principle in relation to the outcome of the artery ligation procedure; a treatment for hemorrhoidal disease. Hemorrhoidal artery ligation, known as HAL (hemorrhoidal artery ligation) or THD (transanal hemorrhoidal dearterialization) procedure, is a

  7. Prospective Randomized Clinical Trial on Suction Elastic Band Ligator Versus Forceps Ligator in the Treatment of Haemorrhoids

    Directory of Open Access Journals (Sweden)

    A.R. Mohd Ramzisham

    2005-10-01

    Conclusion: Suction band ligation is superior to forceps ligation for the treatment of second- and third-degree haemorrhoids in terms of pain tolerance, amount of analgesia consumed and intra-procedure bleeding.

  8. Nanocrystalline silicon-based oligonucleotide chips.

    Science.gov (United States)

    Zhu, Z Q; Zhu, B; Zhang, J; Zhu, J Z; Fan, C H

    2007-04-15

    A novel oligonucleotide array sensor has been developed with nanocrystalline Si (ncSi) substrates. The ncSi was prepared by electrochemical etching technique. Our study indicated that both the binding capacity and the hybridization efficiency are dependent upon the particle size of ncSi. In contrary, the chips developed with Si substrates exhibit the lower binding capacity and hybridization efficiency. The improved performances of the sensor chips are attributed to the large specific surface area of ncSi compared to the existing conventional techniques. The sensor chips with the ncSi substrate of 13 nm-sized particle can be regenerated and reused for at least 12 times. The oligonucleotide array sensor also shows high stability, which can bear relatively the stringent conditions (e.g. 80 degrees C, 75% of relative humidity and 3.6 klx of irradiation).

  9. Design and validation of DNA libraries for multiplexing proximity ligation assays.

    Directory of Open Access Journals (Sweden)

    Nicolas Gobet

    Full Text Available Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA. PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.

  10. DNA splicing by directed ligation (SDL).

    Science.gov (United States)

    Berlin, Y A

    1999-01-01

    Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.

  11. Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA.

    Science.gov (United States)

    Ihara, T; Maruo, Y; Takenaka, S; Takagi, M

    1996-01-01

    Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5'-aminohexyl-terminated synthetic oligonucleotides. Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436-443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2-3 kcal mol-1 (1-2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent (with a 10-30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and RNA; single- and double strands) and the sequences. PMID:8932383

  12. Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

    Science.gov (United States)

    Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

    2017-08-15

    Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

    Science.gov (United States)

    Roh, Changhyun

    2012-01-01

    Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

  14. Ultrasensitive detection of DNA and RNA based on enzyme-free click chemical ligation chain reaction on dispersed gold nanoparticles.

    Science.gov (United States)

    Kato, Daiki; Oishi, Motoi

    2014-10-28

    An ultrasensitive colorimetric DNA and RNA assay using a combination of enzyme-free click chemical ligation chain reaction (CCLCR) on dispersed gold nanoparticles (GNPs) and a magnetic separation process has been developed. The click chemical ligation between an azide-containing probe DNA-modified GNP and a dibenzocyclooctyne-containing probe biotinyl DNA occurred through hybridization with target DNA (RNA) to form the biotinyl-ligated GNPs (ligated products). Eventually, both the biotinyl-ligated GNPs and target DNA (RNA) were amplified exponentially using thermal cycling. After separation of the biotinyl-ligated GNPs using streptavidin-modified magnetic beads, the change in intensity of the surface plasmon band at 525 nm in the supernatants was observed by UV/vis measurement and was also evident visually. The CCLCR assay provides ultrasensitive detection (50 zM: several copies) of target DNA that is comparable to PCR-based approaches. Note that target RNA could also be detected with similar sensitivity without the need for reverse transcription to the corresponding cDNA. The amplification efficiency of the CCLCR assay was as high as 82% due to the pseudohomogeneous reaction behavior of CCLCR on dispersed GNPs. In addition, the CCLCR assay was able to discriminate differences in single-base mismatches and to specifically detect target DNA and target RNA from the cell lysate.

  15. Comparative analysis of measles virus RNA by oligonucleotide fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Stephenson, J.R.; Meulen, V. ter (Wuerzburg Univ. (Germany, F.R.))

    1982-01-01

    Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T/sub 1/ oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin.

  16. Direct microcontact printing of oligonucleotides for biochip applications

    Directory of Open Access Journals (Sweden)

    Trévisiol E

    2005-07-01

    Full Text Available Abstract Background A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. Results Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. Conclusion The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting

  17. Oligonucleotide probes for DNA fingerprinting in dogs.

    Science.gov (United States)

    Jung, M; Wilke, K; Geldermann, H

    1994-01-12

    Ten different oligonucleotide probes were evaluated for DNA fingerprinting in dogs. Seven probes are able to detect polymorphic bands. Probes (GT)(8) , (GTG)(5) and (GGAT)(4) are most informative for individual identification. The probabilities that two individuals from different breeds have the same DNA fingerprint pattern are 1.7 × 10(-7) , 5.5 × 10(-8) and 4.5 × 10(-6) , respectively. Using a combination of the three probes, paternity tests were performed with exclusion probabilities between 0.006% and 3%. ZUSAMMENFASSUNG: Oligonucleotid Sonden für DNA fingerprinting bei Hunden Zur Darstellung von DNA-Fingerprints beim Hund wurden zehn verschiedene Oligonukleotid-Sonden verglichen. Mit sieben Sonden konnten polymorphe Banden nachgewiesen werden. Die Sonden (GT)(8) , (GTG)(5) und (GGAT)(4) besaßen die größte Informativität für den Indentitätsnachweis. Die Wahrscheinlichkeit, daß zwei Individuen dieselben Fingerprint-Muster aufweisen, liegt bei 1,7 × 10(-7) , 5,5 × 10(-8) bzw. 4,5 × 10(-6) . Bei Verwendung einer Kombination der drei Sonden wurden Vaterschaftskontrollen mit Ausschlußwahrscheinlichkeiten zwischen 0,006% und 3 % erreicht. 1994 Blackwell Verlag GmbH.

  18. The HubBLe trial: haemorrhoidal artery ligation (HAL versus rubber band ligation (RBL for haemorrhoids

    Directory of Open Access Journals (Sweden)

    Tiernan Jim

    2012-10-01

    Full Text Available Abstract Background Haemorrhoids (piles are a very common condition seen in surgical clinics. After exclusion of more sinister causes of haemorrhoidal symptoms (rectal bleeding, perianal irritation and prolapse, the best option for treatment depends upon persistence and severity of the symptoms. Minor symptoms often respond to conservative treatment such as dietary fibre and reassurance. For more severe symptoms treatment such as rubber band ligation may be therapeutic and is a very commonly performed procedure in the surgical outpatient setting. Surgery is usually reserved for those who have more severe symptoms, as well as those who do not respond to non-operative therapy; surgical techniques include haemorrhoidectomy and haemorrhoidopexy. More recently, haemorrhoidal artery ligation has been introduced as a minimally invasive, non destructive surgical option. There are substantial data in the literature concerning efficacy and safety of 'rubber band ligation including multiple comparisons with other interventions, though there are no studies comparing it to haemorrhoidal artery ligation. A recent overview has been carried out by the National Institute for Health and Clinical Excellence which concludes that current evidence shows haemorrhoidal artery ligation to be a safe alternative to haemorrhoidectomy and haemorrhoidopexy though it also highlights the lack of good quality data as evidence for the advantages of the technique. Methods/design The aim of this study is to establish the clinical effectiveness and cost effectiveness of haemorrhoidal artery ligation compared with conventional rubber band ligation in the treatment of people with symptomatic second or third degree (Grade II or Grade III haemorrhoids. Design: A multi-centre, parallel group randomised controlled trial. Outcomes: The primary outcome is patient-reported symptom recurrence twelve months following the intervention. Secondary outcome measures relate to symptoms

  19. The HubBLe trial: haemorrhoidal artery ligation (HAL) versus rubber band ligation (RBL) for haemorrhoids.

    Science.gov (United States)

    Tiernan, Jim; Hind, Daniel; Watson, Angus; Wailoo, Allan J; Bradburn, Michael; Shephard, Neil; Biggs, Katie; Brown, Steven

    2012-10-25

    Haemorrhoids (piles) are a very common condition seen in surgical clinics. After exclusion of more sinister causes of haemorrhoidal symptoms (rectal bleeding, perianal irritation and prolapse), the best option for treatment depends upon persistence and severity of the symptoms. Minor symptoms often respond to conservative treatment such as dietary fibre and reassurance. For more severe symptoms treatment such as rubber band ligation may be therapeutic and is a very commonly performed procedure in the surgical outpatient setting. Surgery is usually reserved for those who have more severe symptoms, as well as those who do not respond to non-operative therapy; surgical techniques include haemorrhoidectomy and haemorrhoidopexy. More recently, haemorrhoidal artery ligation has been introduced as a minimally invasive, non destructive surgical option.There are substantial data in the literature concerning efficacy and safety of 'rubber band ligation including multiple comparisons with other interventions, though there are no studies comparing it to haemorrhoidal artery ligation. A recent overview has been carried out by the National Institute for Health and Clinical Excellence which concludes that current evidence shows haemorrhoidal artery ligation to be a safe alternative to haemorrhoidectomy and haemorrhoidopexy though it also highlights the lack of good quality data as evidence for the advantages of the technique. The aim of this study is to establish the clinical effectiveness and cost effectiveness of haemorrhoidal artery ligation compared with conventional rubber band ligation in the treatment of people with symptomatic second or third degree (Grade II or Grade III) haemorrhoids. A multi-centre, parallel group randomised controlled trial. The primary outcome is patient-reported symptom recurrence twelve months following the intervention. Secondary outcome measures relate to symptoms, complications, health resource use, health related quality of life and cost

  20. Universal ligation-detection-reaction microarray applied for compost microbes

    Directory of Open Access Journals (Sweden)

    Romantschuk Martin

    2008-12-01

    Full Text Available Abstract Background Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones. Results Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array. Conclusion This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.

  1. Investigations of oligonucleotide usage variance within and between prokaryotes

    DEFF Research Database (Denmark)

    Bohlin, J.; Skjerve, E.; Ussery, David

    2008-01-01

    Oligonucleotide usage in archaeal and bacterial genomes can be linked to a number of properties, including codon usage (trinucleotides), DNA base-stacking energy (dinucleotides), and DNA structural conformation (di-to tetranucleotides). We wanted to assess the statistical information potential...... of different DNA 'word-sizes' and explore how oligonucleotide frequencies differ in coding and non-coding regions. In addition, we used oligonucleotide frequencies to investigate DNA composition and how DNA sequence patterns change within and between prokaryotic organisms. Among the results found...... was that prokaryotic chromosomes can be described by hexanucleotide frequencies, suggesting that prokaryotic DNA is predominantly short range correlated, i. e., information in prokaryotic genomes is encoded in short oligonucleotides. Oligonucleotide usage varied more within AT-rich and host-associated genomes than...

  2. Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Olga A. Krasheninina

    2017-11-01

    Full Text Available In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield, ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs, aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed.

  3. Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Gil Tae Hwang

    2018-01-01

    Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

  4. DNA fingerprinting in cattle using oligonucleotide probes.

    Science.gov (United States)

    Buitkamp, J; Zischler, H; Epplen, J T; Geldermann, H

    1991-01-01

    Oligonucleotide probes specific for simple tandem repeat sequences produce individual specific DNA fingerprints in man and all animal species tested so far. Here 11 different synthetic probes were hybridized to bovine genomic DNAs which had been digested with the restriction endonucleases HinfI, AluI and HaeIII. Two of these probes gave DNA fingerprint patterns which were analysed for three German breeds. Different parameters were calculated, such as the average number of bands per individual or the probability of finding identical fingerprints in two unrelated individuals. The number of polymorphic bands varies from 11 to 23 in the different breeds and the probability of finding the same banding pattern in two unrelated individuals ranges from 1.5 x 10(-7) to 2.4 x 10(-7). Hence this DNA fingerprinting procedure allows precise identification of individuals. It is also a useful additional method for paternity testing in cattle.

  5. Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases.

    Science.gov (United States)

    Southwell, Amber L; Skotte, Niels H; Bennett, C Frank; Hayden, Michael R

    2012-11-01

    The rising median age of our population and the age-dependent risk of neurodegeneration translate to exponentially increasing numbers of afflicted individuals in the coming years. Although symptomatic treatments are available for some neurodegenerative diseases, most are only moderately efficacious and are often associated with significant side effects. The development of small molecule, disease-modifying drugs has been hindered by complex pathogenesis and a failure to clearly define the rate-limiting steps in disease progression. An alternative approach is to directly target the mutant gene product or a defined causative protein. Antisense oligonucleotides (ASOs) - with their diverse functionality, high target specificity, and relative ease of central nervous system (CNS) delivery - are uniquely positioned as potential therapies for neurological diseases. Here we review the development of ASOs for the treatment of inherited neurodegenerative diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Laparoscopic-assisted percutaneous internal ring ligation in children

    African Journals Online (AJOL)

    Laparoscopic-assisted percutaneous internal ring ligation in children. Mostafa A. Hamad a,b. , Mohamed A. Osman a,b and Mahmoud Abdelhamed a,b. Aim To evaluate the feasibility and safety of laparoscopic ligation of internal ring in congenital inguinal hernia in children. Patients and methods Laparoscopic ...

  7. Esophageal variceal ligation in the secondary prevention of variceal ...

    African Journals Online (AJOL)

    Introduction: Long-term outcome of patients after band ligation have been poorly defined. Therefore, we conducted a long-term follow-up study to delineate the outcome of ligation in patients with portal hypertension in the Hassan II university hospital, Fes, Morocco. Methods: Over 118 months patients treated by endoscopic ...

  8. Tubal ligation and risk of ovarian cancer subtypes

    DEFF Research Database (Denmark)

    Sieh, Weiva; Salvador, Shannon; McGuire, Valerie

    2013-01-01

    Tubal ligation is a protective factor for ovarian cancer, but it is unknown whether this protection extends to all invasive histological subtypes or borderline tumors. We undertook an international collaborative study to examine the association between tubal ligation and ovarian cancer subtypes....

  9. Laparoscopic-assisted percutaneous internal ring ligation in children

    African Journals Online (AJOL)

    Aim To evaluate the feasibility and safety of laparoscopic ligation of internal ring in congenital inguinal hernia in children. Patients and methods Laparoscopic percutaneous ligation of internal inguinal ring has been performed on 97 children with 133 hernias. The age ranged between 6 months and 11.5 years.

  10. Oxo-ester mediated native chemical ligation: concept and applications.

    Science.gov (United States)

    Wan, Qian; Chen, Jin; Yuan, Yu; Danishefsky, Samuel J

    2008-11-26

    A direct oxo-ester peptide ligation method has been developed. Through the use of an activated C-terminal para nitrophenyl ester (1), it is possible to achieve direct cysteine ligations (1 + 2 --> 4). Peptide substrates incorporating bulky C-terminal amino acids (1) can be accommodated with high reaction efficiency.

  11. An empirical study of choosing efficient discriminative seeds for oligonucleotide design.

    Science.gov (United States)

    Chung, Won-Hyoung; Park, Seong-Bae

    2009-12-03

    Oligonucleotide design is known as a time-consuming work in bioinformatics. In order to accelerate and be efficient the oligonucleotide design process, one of widely used approach is the prescreening unreliable regions using a hashing (or seeding) algorithm. Since the seeding algorithm is originally proposed to increase sensitivity for local alignment, the specificity should be considered as well as the sensitivity for the oligonucleotide design problem. However, a measure of evaluating the seeds regarding how adequate and efficient they are in the oligo design is not yet proposed. Here, we propose novel measures of evaluating the seeding algorithms based on the discriminability and the efficiency. To evaluate the proposed measures, we examine five seeding algorithms in oligonucleotide design. We carried out a series of experiments to compare the seeding algorithms. As the result, the spaced seed is recorded as the most efficient discriminative seed for oligo design. The performance of transition-constrained seed is slightly lower than the spaced seed. Because BLAT seeding algorithm and Vector seeding algorithm give poor scores in specificity and efficiency, we conclude that these algorithms are not adequate to design oligos. Consequently, we recommend spaced seeds or transition-constrained seeds with 15 approximately 18 weight in order to design oligos with the length of 50 mer. The empirical experiments in real biological data reveal that the recommended seeds show consequently good performance. We also propose a software package which enables the users to get the adequate seeds under their own experimental conditions. Our study is valuable to the two points. One is that our study can be applied to the oligo design programs in order to improve the performance by suggesting the experiment-specific seeds. The other is that our study is useful to improve the performance of the mapping assembly in the field of Next-Generation Sequencing. Our proposed measures are

  12. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  13. Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease

    Directory of Open Access Journals (Sweden)

    Valentina Sardone

    2017-04-01

    Full Text Available Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

  14. Antisense oligonucleotide induction of progerin in human myogenic cells.

    Directory of Open Access Journals (Sweden)

    Yue-Bei Luo

    Full Text Available We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA transcript in human myogenic cells. The progerin transcript (LMNA Δ150 lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS. HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model

  15. Oligonucleotides Containing Aminated 2′-Amino-LNA Nucleotides

    DEFF Research Database (Denmark)

    Lou, Chenguang; Samuelsen, Simone V.; Christensen, Niels Johan

    2017-01-01

    Mono- and diaminated 2′-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that stru...... that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2′-amino-LNA monomers and the host oligonucleotide backbone.......Mono- and diaminated 2′-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested...

  16. Modern methods for the synthesis of peptide-oligonucleotide conjugates

    Science.gov (United States)

    Zubin, Evgenii M.; Romanova, Elena A.; Oretskaya, Tat'yana S.

    2002-03-01

    The published data on the methods of chemical solution and solid-phase synthesis of peptide-oligonucleotide conjugates are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to the solution synthesis of peptide-oligonucleotide conjugates are systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates, viz., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 references.

  17. Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    2013-09-01

    Full Text Available UV-VIS spectroscopic analysis of oligonucleotide DNA exposed to different magnetic fields was performed in order to investigate the relationship between DNA extinction coefficients and optical parameters according to magnetic-field strength. The results with the oligonucleotides adenine-thymine 100 mer (AT-100 DNA and cytosine-guanine 100 mer (CG-100 DNA indicate that the magnetic field influences DNA molar extinction coefficients and refractive indexes. The imaginary parts of the refractive index and molar extinction coefficients of the AT-100 and CG-100 DNA decreased after exposure to a magnetic field of 750 mT due to cleavage of the DNA oligonucleotides into smaller segments.

  18. Multiplex, quantitative, ligation-dependent probe amplification for determination of allergens in food.

    Science.gov (United States)

    Mustorp, Stina L; Drømtorp, Signe M; Holck, Askild L

    2011-05-25

    Legislation requires labeling of foods containing allergenic ingredients. Here, we present a robust 10-plex quantitative and sensitive ligation-dependent probe amplification method, the allergen-multiplex ligation-dependent probe amplification (MLPA) method, for specific detection of eight allergens: sesame, soy, hazelnut, peanut, lupine, gluten, mustard, and celery. Ligated probes were amplified by polymerase chain reaction (PCR), and amplicons were detected using capillary electrophoresis. Quantitative results were obtained by comparing signals with an internal positive control. The limit of detection varied from approximately 5 to 400 gene copies, depending on the allergen. The method was tested using different foods spiked with mustard, celery, soy, or lupine flour in the 1-0.001% range. Depending on the allergen, sensitivities were similar or better than those obtained with qPCR. The allergen-MLPA method is modular and can be adapted by adding probe pairs for other allergens. The DNA-based allergen-MLPA method will constitute a complementary method to the traditional protein-based methods.

  19. Metallo-Phthalocyanine Near-IR Fluorophores: Oligonucleotide Conjugates and Their Applications in PCR Assays

    Science.gov (United States)

    Nesterova, Irina V.; Verdree, Vera T.; Pakhomov, Serhii; Strickler, Karen L.; Allen, Michael W.; Hammer, Robert P.; Soper, Steven A.

    2011-01-01

    Water soluble, metallo-pthalocyanine (MPc) near-IR fluorophores were designed, synthesized, and evaluated as highly stable and sensitive reporters for fluorescence assays. Their conjugation to oligonucleotides was achieved via succinimidyl ester-amino coupling chemistry with the conditions for conjugation extensively examined and optimized. In addition, various conjugate purification and isolation techniques were evaluated as well. Results showed that under proper conditions and following purification using reverse-phase ion-pair chromatography, labeling efficiencies near 80% could be achieved using ZnPc (Zn phthalocyanine) as the labeling fluorophore. Absorption and fluorescence spectra accumulated for the conjugates indicated that the intrinsic fluorescence properties of the MPc’s were not significantly altered by covalent attachment to oligonucleotides. As an example of the utility of MPc reporters, we used the MPc–oligonucleotide conjugates as primers for PCR (polymerase chain reaction) amplifications with the products sorted via electrophoresis and detected using near-IR fluorescence (λex = 680 nm). The MPc dyes were found to be more chemically stable under typical thermal cycling conditions used for PCR compared to the carbocyanine-based near-IR reporter systems typically used and produced single and narrow bands in the electrophoretic traces, indicative of producing a single PCR product during amplification. PMID:18030995

  20. Long-range order of organized oligonucleotide monolayers on Au(111) electrodes

    DEFF Research Database (Denmark)

    Wackerbarth, Hainer; Grubb, Mikala; Zhang, Jingdong

    2004-01-01

    , with electrochemical potential control of both the sample electrode and the tip. All the data are based on single-crystal, atomically planar Au(111)-electrode surfaces. The high sensitivity of such surfaces provides accurate HS-10A and HS-10AT electrode coverages on the basis of the reductive desorption of the Au......Oligonucleotides modified by a hexamethylene linker group adsorb on gold electrodes via Au-S bond formation. We have obtained novel data for adsorption of thiol-modified (HS) single-strand HS-10A and double-stranded HS-10AT oligonucleotides and for analogous thiol-free 10A (A = adenine) and 10T (T......-S bond. The coverage is high and in keeping with dense monolayers of adsorbed HS-10A and HS-10AT in an upright or tilted orientation, with the oligonucleotide backbone repelled from the strongly negatively charged electrode surface. Adsorbed thiol-free 10A only gives aAu(111)-reconstruction peak, while...

  1. Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Albretsen, C.; Haukanes, B.I.; Aasland, R.; Kleppe, K.

    1988-04-01

    The authors present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions. Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G+C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11/sup 0/C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5x SSC and found to be 43, 58, and 60/sup 0/C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.

  2. Sequence-dependent theory of oligonucleotide hybridization kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)

    2014-05-07

    A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions.

  3. Oligonucleotide-based theranostic nanoparticles in cancer therapy.

    Science.gov (United States)

    Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

    2016-05-01

    Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering RNA and microRNA, which are powerful therapeutic agents, have been effectively employed in theranostic systems against various cancers. Nanoparticles are used to deliver oligonucleotides into tumors by passive or active targeting while protecting the oligonucleotides from nucleases in the extracellular environment. The use of quantum dots, iron oxide nanoparticles and gold nanoparticles and tagging with contrast agents, like fluorescent dyes, optical or magnetic agents and various radioisotopes, has facilitated early detection of tumors and evaluation of therapeutic efficacy. In this article, we review the advantages of theranostic applications in cancer therapy and imaging, with special attention to oligonucleotide-based therapeutics.

  4. Sequence-dependent theory of oligonucleotide hybridization kinetics.

    Science.gov (United States)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-05-07

    A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions.

  5. Oligonucleotide probes for DNA fingerprinting in horses.

    Science.gov (United States)

    Wilke, K; Weimann, M; Jung, M; Geldermann, H

    1993-01-12

    10 different oligonucleotide probes were evaluated for DNA fingerprinting in horses. Five probes were able to detect polymorphic bands. The probes (GT)(8) , (GTG)(5) and (GGAT)(4) are most informative for individual identification and were used to analyze a population of Hannoveranian horses. The probability that two individuals have the same DNA fingerprint pattern is 1.2 × 10(-8) , 5.2 × 10(-10) and 1.5 × 10(-7) respectively. Using a combination of the three probes, paternity tests were performed with exclusion probabilities between 0.08% and 4%. ZUSAMMENFASSUNG: Oligonukleotide-Sonden für DNS-Fingerprints von Pferden Zur Darstellung von DNA-Fingerprints beim Pferd wurden zehn verschiedene Oligonukleotid-Sonden verglichen. Mit fünf Sonden konnten polymorphe Banden nachgewiesen werden. Die Sonden (GT)(8) , (GTG)(5) und (GGAT)(4) besaßen die größte Informativität für den Identitätsnachweis und wurden für die Analyse einer Population von Hannoverschen Pferden benutzt. Die Wahrscheinlichkeit, daß zwei Individuen dieselben Fingerprint-Muster aufweisen, liegt bei 1,2 × 10(-8) , 5,2 × 10(-10) bzw. 1,5 × 10(-7) . Bei Verwendung einer Kombination der drei Sonden wurden Vaterschaftskontrollen mit Ausschlußwahrscheinlichkeiten zwischen 0,08% und 4% erreicht. 1993 Blackwell Verlag GmbH.

  6. Hole hopping rates in single strand oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Borrelli, Raffaele [Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO (Italy); Capobianco, Amedeo [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy); Peluso, Andrea, E-mail: apeluso@unisa.it [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy)

    2014-08-31

    Highlights: • DNA hole transfer rates have been computed. • Delocalized adenine domains significantly affect hole transfer rates in DNA. • Franck–Condon weighted density of state from DFT normal modes. • DNA application in molecular electronics. - Abstract: The rates of hole transfer between guanine and adenine in single strand DNA have been evaluated by using Fermi’s golden rule and Kubo’s generating function approach for the Franck–Condon weighted density of states. The whole sets of the normal modes and vibrational frequencies of the two nucleobases, obtained at DFT/B3LYP level of calculation, have been considered in computations. The results show that in single strand the pyramidalization/planarization mode of the amino groups of both nucleobases plays the major role. At room temperature, the Franck–Condon density of states extends over a wide range of hole site energy difference, 0–1 eV, giving some hints about the design of oligonucleotides of potential technological interest.

  7. Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch.

    Science.gov (United States)

    Hasegawa, Yusuke; Takada, Tadao; Nakamura, Mitsunobu; Yamana, Kazushige

    2017-08-01

    We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Convergent synthesis of proteins by kinetically controlled ligation

    Science.gov (United States)

    Kent, Stephen; Pentelute, Brad; Bang, Duhee; Johnson, Erik; Durek, Thomas

    2010-03-09

    The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.

  9. Spotted cotton oligonucleotide microarrays for gene expression analysis

    Directory of Open Access Journals (Sweden)

    Nettleton Dan

    2007-03-01

    Full Text Available Abstract Background Microarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, two comprehensive assemblies of cotton ESTs were constructed based on three Gossypium species. Using these assemblies as templates, we describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species. Results Synthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of 211,397 cotton ESTs derived from >50 different cDNA libraries representing many different tissue types and tissue treatments. A total of 22,787 oligonucleotide probes are included on the arrays, optimized to target the diversity of the transcriptome and previously studied cotton genes, transcription factors, and genes with homology to Arabidopsis. A small portion of the oligonucleotides target unidentified protein coding sequences, thereby providing an element of gene discovery. Because many oligonucleotides were based on ESTs from fiber-specific cDNA libraries, the microarray has direct application for analysis of the fiber transcriptome. To illustrate the utility of the microarray, we hybridized labeled bud and leaf cDNAs from G. hirsutum and demonstrate technical consistency of results. Conclusion The cotton oligonucleotide microarray provides a reproducible platform for transcription profiling in cotton, and is made publicly available through http://cottonevolution.info.

  10. Protein biomarker validation via proximity ligation assays.

    Science.gov (United States)

    Blokzijl, A; Nong, R; Darmanis, S; Hertz, E; Landegren, U; Kamali-Moghaddam, M

    2014-05-01

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

  11. Esophageal variceal ligation for hemostasis of acute variceal bleeding

    African Journals Online (AJOL)

    Esophageal variceal ligation for hemostasis of acute variceal bleeding: efficacy and safety. Mounia Lahbabi, Mounia Elyousfi, Nouredine Aqodad, Mohammed Elabkari, Ihssane Mellouki, Sidi Adil Ibrahimi, Dafr Allah Benaja ...

  12. Ligation bias in Illumina next-generation DNA libraries

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel

    2013-01-01

    -products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate...... that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting...... of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected...

  13. Elastic band ligation of hemorrhoids using flexible gastroscope

    Directory of Open Access Journals (Sweden)

    Hadi Abd Zaid Al-Khattabi

    2017-03-01

    Conclusion High success rate, cost effectiveness and the simplicity of rubber band ligation as an outpatient procedure promote its use as the frst line of treatment for frst, second and early third degree hemorrhoids.

  14. [Rubber band ligation in treatment of hemorrhoids: our experience].

    Science.gov (United States)

    Gaj, F; Biviano, I; Sportelli, G; Candeloro, L

    2015-01-01

    Hemorrhoids are a very common condition. The treatment depends upon persistence and severity of symptoms. For hemorrhoids of II and III grade the rubber band ligation may be therapeutic. Our aim is to report the outcomes of rubber band ligation of hemorrhoids, with a follow up of 6 months. A total of 50 patients underwent rubber band ligation for symptomatic hemorrhoids (grade II and III) without prolapse, between June 2012 and June 2014. All patients underwent plug test to rule out presence of rectal mucosal prolapse and were classified according to PATE classification (1). Each hemorrhoid was ligated with one rubber band through a ligator. All patients were evaluated immediately at the end of the procedure, after ten days and six months after the treatment. Patient's demographic and operative data were collected and analyzed. The mean patients age was 47.6±12.3 years (range 24-72). All procedures were performed without complications. Before rubber band ligation, 42 patients had rectal bleeding, 38 had perineal discomfort and 27 had itching. Ten days after the treatment, 12 patients presented self-limited rectal bleeding, but 10 of these had more hemorrhoids and underwent a second rubber band ligation. No patients complained perineal discomfort, and 8 patients had itching; 78% and 16% of patients respectively, experienced feeling of a foreign body inside the canal anal and anal pain. After 6 months, only 13 patients were occasionally symptomatic: 4 patients had rectal bleeding, 2 had perineal discomfort and 4 had itching. Three more patients presented both perineal discomfort and hitching. None had the feeling of a foreign body in anal canal or anal pain. Rubber band ligation is an efficacious, cost-effective and simple treatment for the second and third degree hemorrhoids without rectal mucosal prolapsed. In our hands, no severe complications developed and minor complications could be handled with ease.

  15. Polycation Induced Potential Dependent Structural Transitions of Oligonucleotide Monolayers on Au(111)-Surfaces

    DEFF Research Database (Denmark)

    Salvatore, Princia; Karlsen, Kasper Kannegård; Hansen, Allan Glargaard

    2012-01-01

    We have studied self-assembled molecular monolayers (SAMs) of several 3′-C3-SH conjugated single-strand (ss) and double-strand (ds) 20-base oligonucleotides (ONs) immobilized on single-crystal, atomically planar Au(111)-electrode surfaces in the presence of the triply positively charged base...... electrochemical potential control was used. Spd binding was found to increase notably the ds-ON melting temperature. CV displays capacitive features associated with ss- and ds-ON. A robust capacitive peak around −0.35 V versus saturated calomel electrode (SCE), specific to ds-ON and highly sensitive to base pair...

  16. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

    Science.gov (United States)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  17. Axially Ligated Phthalocyanine Conductors with Magnetic Moments

    Directory of Open Access Journals (Sweden)

    Tamotsu Inabe

    2017-04-01

    Full Text Available This mini-review describes electrical conductivity, magnetic properties, and magnetotransport properties of one-dimensional partially oxidized salts composed of axially ligated phthalocyanines, TPP[M(Pc(CN2]2 (TPP = tetraphenylphosphonium, Pc = phthalocyaninato, with M of Fe (d5, S = 1/2 and Cr (d3, S = 3/2. These salts are isomorphous, and π–π interactions in the crystal, that becomes the origin of the charge carriers, are nearly the same. Both the Fe and Cr salts show carrier localization and charge disproportionation which is enhanced by the interaction between local magnetic moments and conduction π-electrons (π–d interaction. However, the magnetic properties are slightly different between them. M = Fe has been found to show unique anisotropic magnetic properties and antiferromagnetic short-range magnetic order between the d-spins. On the other hand, for M = Cr, its magnetic moment is isotropic. Temperature dependence of the magnetic susceptibility shows typical Curie–Weiss behavior with negative Weiss temperature, but the exchange interaction is complicated. Both M = Fe and M = Cr show large negative magnetoresistance, reflecting the difference in the anisotropy. The magnetoresistance ratio (MR is larger in the Fe system than in the Cr system in the low magnetic field range, but MR in the Cr system exceeds that in the Fe system when the magnetic field becomes higher than 15 T. We discuss the mechanism of the giant negative magnetoresistance with reference to the d–d, π–d, and π–π interactions.

  18. Endoscopic variceal ligation-induced ulcer bleeding

    Science.gov (United States)

    Cho, Eunae; Jun, Chung Hwan; Cho, Sung Bum; Park, Chang Hwan; Kim, Hyun Soo; Choi, Sung Kyu; Rew, Jong Sun

    2017-01-01

    Abstract This study was aimed to determine the risk factors of endoscopic variceal ligation-(EVL) induced ulcer bleeding. The prevalence of EVL-induced ulcer bleeding is reported to be 3.6%. However, there are only limited reports of this serious complication, and the risk factors and the treatment methods are not well established. A total of 430 patients who had undergone EVL in Chonnam National University Hospital from January 2014 to October 2016 were studied. EVL was performed for prophylaxis or acute hemorrhage. The patients were classified into 2 groups: a bleeding group (n = 33) and a non-bleeding group (n = 397). The patients who had endoscopically confirmed EVL-induced ulcer bleeding were included in the bleeding group. EVL-induced ulcer bleeding occurred in 7.7% (n = 33) of the patients. In a multivariate analysis, model for end-stage liver disease (MELD) score >10 (odds ratio [OR]: 3.42, 95% confidence interval [CI]: 1.10–10.64), concomitant GV F3 (OR: 14.1, 95% CI: 2.84–71.43), and detachment of o-ring bands on follow-up endoscopy (OR: 8.06, 95% CI: 2.55–25.64) were independent predictive factors of EVL-induced ulcer bleeding. Various endoscopic modalities were attempted for hemostasis (EVL in 8 cases [24.2%], endoscopic variceal obturation [EVO] with cyanoacrylate in 6 cases [18.2%], argon plasma coagulation [APC] in 1 case (3%), Sengstaken–Blakemore (SB) tube in 3 cases [9.1%]), and proton pump inhibitor therapy only in 15 cases (45.5%). MELD score >10, concomitant GV F3, and detachment of o-ring bands on follow-up endoscopy are risk factors for EVL-induced ulcer bleeding. PMID:28614248

  19. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

    2011-01-01

    specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project......A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...

  20. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    Science.gov (United States)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Delivery of RNAi-Based Oligonucleotides by Electropermeabilization

    Directory of Open Access Journals (Sweden)

    Muriel Golzio

    2013-04-01

    Full Text Available For more than a decade, understanding of RNA interference (RNAi has been a growing field of interest. The potent gene silencing ability that small oligonucleotides have offers new perspectives for cancer therapeutics. One of the present limits is that many biological barriers exist for their efficient delivery into target cells or tissues. Electropermeabilization (EP is one of the physical methods successfully used to transfer small oligonucleotides into cells or tissues. EP consists in the direct application of calibrated electric pulses to cells or tissues that transiently permeabilize the plasma membranes, allowing efficient in vitro and in vivo. cytoplasmic delivery of exogenous molecules. The present review reports on the type of therapeutic RNAi-based oligonucleotides that can be electrotransferred, the mechanism(s of their electrotransfer and the technical settings for pre-clinical purposes.

  2. Current progress on aptamer-targeted oligonucleotide therapeutics

    Science.gov (United States)

    Dassie, Justin P; Giangrande, Paloma H

    2014-01-01

    Exploiting the power of the RNAi pathway through the use of therapeutic siRNA drugs has remarkable potential for treating a vast array of human disease conditions. However, difficulties in delivery of these and similar nucleic acid-based pharmacological agents to appropriate organs or tissues, remains a major impediment to their broad clinical application. Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery vehicles for therapeutic oligonucleotides, including siRNAs. In this review, we summarize recent attractive developments in creatively employing cell-internalizing aptamers to deliver therapeutic oligonucleotides (e.g., siRNAs, miRNAs, anti-miRs and antisense oligos) to target cells. We also discuss advancements in aptamer-siRNA chimera technology, as well as, aptamer-functionalized nanoparticles for siRNA delivery. In addition, the challenges and future prospects of aptamer-targeted oligonucleotide drugs for clinical translation are further highlighted. PMID:24304250

  3. Challenges to oligonucleotides-based therapeutics for Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Goyenvalle Aurélie

    2011-02-01

    Full Text Available Abstract Antisense oligonucleotides are short nucleic acids designed to bind to specific messenger RNAs in order to modulate splicing patterns or inhibit protein translation. As such, they represent promising therapeutic tools for many disorders and have been actively developed for more than 20 years as a form of molecular medicine. Although significant progress has been made in developing these agents as drugs, they are yet not recognized as effective therapeutics and several hurdles remain to be overcome. Within the last few years, however, the prospect of successful oligonucleotides-based therapies has moved a step closer, in particular for Duchenne muscular dystrophy. Clinical trials have recently been conducted for this myopathy, where exon skipping is being used to achieve therapeutic outcomes. In this review, the recent developments and clinical trials using antisense oligonucleotides for Duchenne muscular dystrophy are discussed, with emphasis on the challenges ahead for this type of therapy, especially with regards to delivery and regulatory issues.

  4. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Wupeng Liao

    2017-01-01

    Full Text Available Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF. The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO, small interfering RNA (siRNA, and microRNA (miRNA are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.

  5. Immunomodulation of hematological malignancies using oligonucleotides based-nanomedicines.

    Science.gov (United States)

    Hazan-Halevy, Inbal; Landesman-Milo, Dalit; Rosenblum, Daniel; Mizrahy, Shoshy; Ng, Brandon D; Peer, Dan

    2016-12-28

    Hematological malignancies are a group of diseases characterized by clonal proliferation of blood-forming cells. Malignant blood cells are classified as myeloid or lymphoid cells depending on their stem cell origin. Lymphoid malignancies are characterized by lymphocyte accumulation in the blood stream, in the bone marrow, or in lymphatic nodes and organs. Several of these diseases are associated with chromosomal translocations, which cause gene fusion and amplification of expression, while others are characterized with aberrant expression of oncogenes. Overall, these genes play a major role in development and maintenance of malignant clones. The discovery of antisense oligonucleotides and RNA interference (RNAi) mechanisms offer new tools to specifically manipulate gene expression. Systemic delivery of inhibitory oligonucleotides molecules for manipulation of gene expression in lymphocytes holds a great potential for facilitating the development of an oligonucleotides -based therapy platform for lymphoid blood cancer. However, lymphocytes are among the most difficult targets for oligonucleotides delivery, as they are resistant to conventional transfection reagents and are dispersed throughout the body, making it difficult to successfully localize or deliver oligonucleotides payloads via systemic administration. In this review, we will survey the latest progress in the field of oligonucleotides based nanomedicine in the heterogeneous group of hematological malignancies with special emphasis on RNA based strategies. We will describe the most advanced non-viral nanocarriers for RNA delivery to malignant blood cells. We will also discuss targeted strategies for cell specific delivery of RNA molecules using nanoparticles and the therapeutic benefit of manipulating gene function in hematological malignancies. Finally, we will focus on the ex vivo, in vivo, and clinical trial strategies, that are currently under development in hematological malignancies - strategies that

  6. Inhibition of microRNA with antisense oligonucleotides.

    Science.gov (United States)

    Esau, Christine C

    2008-01-01

    Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified AMOs for use in cultured cells and rodent models, and outline important issues to consider when inhibiting miRNAs with antisense oligonucleotides.

  7. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array.

    Science.gov (United States)

    Hung, Wen-Tsung; Su, Shu-Li; Shiu, Lin-Yi; Chang, Tsung C

    2011-04-13

    Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera) that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS) regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies. A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26) and 97.7% (43/44), respectively. Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

  8. Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

    Directory of Open Access Journals (Sweden)

    Palta Priit

    2010-04-01

    Full Text Available Abstract Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

  9. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

    Directory of Open Access Journals (Sweden)

    Shiu Lin-Yi

    2011-04-01

    Full Text Available Abstract Background Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. Methods We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies. Results A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26 and 97.7% (43/44, respectively. Conclusions Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

  10. Treatment of glioblastoma multiforme cells with temozolomide-BioShuttle ligated by the inverse Diels-Alder ligation chemistry

    Directory of Open Access Journals (Sweden)

    Klaus Braun

    2009-01-01

    Full Text Available Klaus Braun1, Manfred Wiessler1, Volker Ehemann2, Ruediger Pipkorn3, Herbert Spring4, Juergen Debus5, Bernd Didinger5, Mario Koch3, Gabriele Muller6, Waldemar Waldeck61German Cancer Research Center, Dept of Imaging and Radiooncology, Heidelberg, Germany; 2University of Heidelberg, Institute of Pathology, Heidelberg, Germany; 3German Cancer Research Center, Central Peptide Synthesis Unit, Heidelberg, Germany; 4German Cancer Research Center, Dept of Structural Analysis of Gene Structure and Function, Heidelberg, Germany; 5University of Heidelberg, Dept of Radiation Oncology, Heidelberg, Germany; 6German Cancer Research Center,Division of Biophysics of Macromolecules, Heidelberg, GermanyAbstract: Recurrent glioblastoma multiforme (GBM, insensitive against most therapeutic interventions, has low response and survival rates. Temozolomide (TMZ was approved for second-line therapy of recurrent anaplastic astrocytoma. However, TMZ therapy in GBM patients reveals properties such as reduced tolerability and inauspicious hemogram. The solution addressed here concerning GBM therapy consolidates and uses the potential of organic and peptide chemistry with molecular medicine. We enhanced the pharmacologic potency with simultaneous reduction of unwanted adverse reactions of the highly efficient chemotherapeutic TMZ. The TMZ connection to transporter molecules (TMZ-BioShuttle was investigated, resulting in a much higher pharmacological effect in glioma cell lines and also with reduced dose rate. From this result we can conclude that a suitable chemistry could realize the ligation of pharmacologically active, but sensitive and highly unstable pharmaceutical ingredients without functional deprivation. The TMZ-BioShuttle dramatically enhanced the potential of TMZ for the treatment of brain tumors and is an attractive drug for combination chemotherapy.Keywords: drug delivery, carrier molecules, facilitated transport, glioblastoma multiforme, temozolomide

  11. Anti sense and sensibility : renal and skin effects of (antisense) oligonucleotides

    NARCIS (Netherlands)

    Meer, van L.

    2017-01-01

    This thesis describes the clinical investigation of a novel treatment strategy for type 2 diabetes mellitus (t2dm) using an antisense oligonucleotide(aon)to inhibit the sglt2 receptor. Furthermore it describes skin effects of oligonucleotides

  12. Ultrasensitive electrochemical biosensor based on the oligonucleotide self-assembled monolayer-mediated immunosensing interface

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Dengyou; Luo, Qimei [Science College of Hunan Agricultural University, Changsha 410128 (China); Deng, Fawen [The Fourth Hospital of Chansha, Changsha 410006 (China); Li, Zhen [Science College of Hunan Agricultural University, Changsha 410128 (China); Li, Benxiang, E-mail: 172170960@qq.com [Science College of Hunan Agricultural University, Changsha 410128 (China); Shen, Zhifa, E-mail: shenzhifa@wmu.edu.cn [Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035 (China)

    2017-06-08

    Highly sensitive and selective quantitation of a variety of proteins over a wide concentration range is highly desirable for increased accuracy of biomarker detection or for multidisease diagnostics. In the present contribution, using human immunoglobulin G (HIgG) as the model target protein, an electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer-mediated (OSAM) sensing interface. For this immunosensor, the “signal-on” signaling mechanism and enzymatic signal amplification effect were integrated into one sensing architecture. Moreover, the thiolated flexible single-stranded DNAs immobilized onto gold electrode surface not only performs the wobbling motion to facilitate the electron transfer between the electrode surface and biosensing layer but also fundamentally prohibiting the direct interaction of proteins with gold substrate. Thus, the electrochemical signal could be efficiently enhanced and the unspecific adsorption or cross-reaction might be eliminated. As a result, utilizing the newly-proposed immunosensor, the HIgG can be detected down to 0.5 ng/mL, and the high detection specificity is offered. The successful design of OSAM and the highly desirable detection capability of new immunosensor are expected to provide a perspective for fabricating new robust immunosensing platform and for promising potential of oligonucleotide probe in biological research and biomedical diagnosis. - Highlights: • An electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer (OASM). • OASM severs as a flexible monolayer to promote electron transfer and prohibits the direct interaction of proteins with gold substrate. • The electrochemical signal is efficiently enhanced and the unspecific adsorption or cross-reaction is eliminated. • Target protein can be detected down to 0.5 ng/mL, and the high detection specificity can be obtained.

  13. Sortase-Mediated Ligation of Purely Artificial Building Blocks

    OpenAIRE

    Xiaolin Dai; Diana M. Mate; Ulrich Glebe; Tayebeh Mirzaei Garakani; Andrea Körner; Ulrich Schwaneberg; Alexander Böker

    2018-01-01

    Sortase A (SrtA) from Staphylococcus aureus has been often used for ligating a protein with other natural or synthetic compounds in recent years. Here we show that SrtA-mediated ligation (SML) is universally applicable for the linkage of two purely artificial building blocks. Silica nanoparticles (NPs), poly(ethylene glycol) and poly(N-isopropyl acrylamide) are chosen as synthetic building blocks. As a proof of concept, NP–polymer, NP–NP, and polymer–polymer structures are formed by SrtA cata...

  14. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2015-06-01

    Full Text Available Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1, DNA ligase 3 (Lig3 and DNA ligase 4 (Lig4. While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER, homologous recombination repair (HRR and nucleotide excision repair (NER. Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ. Lig3 is implicated in a short-patch base excision repair (BER pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche

  15. Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles

    DEFF Research Database (Denmark)

    Cogoi, S; Jakobsen, U; Pedersen, E B

    2016-01-01

    factor essential for KRAS transcription. It is based on the use of palmitoyl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with lipid-modified G4-decoy oligonucleotides and a lipid-modified cell penetrating TAT peptide. The potency of the strategy in pancreatic cancer cells is demonstrated...

  16. Linear model for fast background subtraction in oligonucleotide microarrays

    NARCIS (Netherlands)

    Kroll, K.M.; Barkema, G.T.; Carlon, E.

    2009-01-01

    Background One important preprocessing step in the analysis of microarray data is background subtraction. In high-density oligonucleotide arrays this is recognized as a crucial step for the global performance of the data analysis from raw intensities to expression values. Results We propose here an

  17. Solid-phase-supported synthesis of morpholinoglycine oligonucleotide mimics

    Directory of Open Access Journals (Sweden)

    Tatyana V. Abramova

    2014-05-01

    Full Text Available An efficient solid-phase-supported peptide synthesis (SPPS of morpholinoglycine oligonucleotide (MorGly mimics has been developed. The proposed strategy includes a novel specially designed labile linker group containing the oxalyl residue and the 2-aminomethylmorpholino nucleoside analogues as first subunits.

  18. Chromosome-specific painting in Cucumis species using bulked oligonucleotides

    Science.gov (United States)

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a sing...

  19. Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura

    2017-01-01

    opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

  20. Fragmentation of protonated oligonucleotides by energetic photons and Cq+ ions

    NARCIS (Netherlands)

    Gonzalez-Magana, O.; Tiemens, M.; Reitsma, G.; Boschman, L.; Door, M.; Bari, S.; Lahaie, P. O.; Wagner, J. R.; Huels, M. A.; Hoekstra, R.; Schlathölter, Thomas

    2013-01-01

    The ionization and fragmentation of trapped protonated dGCAT oligonucleotides upon interaction with energetic photons (h nu = 10-570 eV) and keV Cq+ ions was investigated by means of time-of-flight mass spectrometry. The observed fragmentation patterns are dominated by protonated and nonprotonated

  1. 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...

  2. Oligonucleotide-directed mutagenesis for precision gene editing.

    Science.gov (United States)

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes.

    Science.gov (United States)

    Li, Lanying; Wen, Yanli; Xu, Li; Xu, Qin; Song, Shiping; Zuo, Xiaolei; Yan, Juan; Zhang, Weijia; Liu, Gang

    2016-01-15

    Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Oligonucleotide Sensor Based on Selective Capture of Upconversion Nanoparticles Triggered by Target-Induced DNA Interstrand Ligand Reaction.

    Science.gov (United States)

    Mendez-Gonzalez, Diego; Laurenti, Marco; Latorre, Alfonso; Somoza, Alvaro; Vazquez, Ana; Negredo, Ana Isabel; López-Cabarcos, Enrique; Calderón, Oscar G; Melle, Sonia; Rubio-Retama, Jorge

    2017-04-12

    We present a sensor that exploits the phenomenon of upconversion luminescence to detect the presence of specific sequences of small oligonucleotides such as miRNAs among others. The sensor is based on NaYF4:Yb,Er@SiO2 nanoparticles functionalized with ssDNA that contain azide groups on the 3' ends. In the presence of a target sequence, interstrand ligation is possible via the click-reaction between one azide of the upconversion probe and a DBCO-ssDNA-biotin probe present in the solution. As a result of this specific and selective process, biotin is covalently attached to the surface of the upconversion nanoparticles. The presence of biotin on the surface of the nanoparticles allows their selective capture on a streptavidin-coated support, giving a luminescent signal proportional to the amount of target strands present in the test samples. With the aim of studying the analytical properties of the sensor, total RNA samples were extracted from healthy mosquitoes and were spiked-in with a specific target sequence at different concentrations. The result of these experiments revealed that the sensor was able to detect 10-17 moles per well (100 fM) of the target sequence in mixtures containing 100 ng of total RNA per well. A similar limit of detection was found for spiked human serum samples, demonstrating the suitability of the sensor for detecting specific sequences of small oligonucleotides under real conditions. In contrast, in the presence of noncomplementary sequences or sequences having mismatches, the luminescent signal was negligible or conspicuously reduced.

  5. Adrenal function in preterm infants undergoing patent ductus arteriosus ligation.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif

    2013-01-01

    Targeted milrinone treatment for low left ventricular output (LVO) reduces the incidence of acute cardiorespiratory instability following ligation of patent ductus arteriosus (PDA) in preterm infants. Despite this, some infants continue to experience postoperative deterioration. Adrenal insufficiency related to prematurity has been postulated as a possible mechanism.

  6. Tubal ectopic pregnancy after bilateral tubal ligation: A case report ...

    African Journals Online (AJOL)

    Tubal ectopic pregnancy after bilateral tubal ligation: A case report. N Ameh, NH Madugu, US Bawa, MS Adelaiye, M Akpa. Abstract. No Abstract. Nigerian Journal of Medicine Vol. 15 (4) October-December 2006: 453-454. Full Text: EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  7. Experience with rubber band ligation of hemorrhoids in northern ...

    African Journals Online (AJOL)

    Background: Treatment of hemorrhoids in Nigeria is usually done by the traditional open method that requires hospital admission; anesthesia and is associated with high morbidity. Rubber band ligation is a suitable alternative to open hemorrhoidectomy and has the potential to reduce the need for hospital admission.

  8. Esophageal variceal ligation for hemostasis of acute variceal bleeding

    African Journals Online (AJOL)

    Our aim was to evaluate the effectiveness and safety of endoscopic variceal ligation in the management of oesophageal variceal bleeding in cirrhosis in a located population in Morocco. Methods: Via a retrospective study over 118 months (December 2001- October 2011), cirrhotic patients with endoscopically proven ...

  9. The effectiveness of Doppler controlled hemorrhoidal artery ligation ...

    African Journals Online (AJOL)

    In this work, we discuss the preliminary results of the effectiveness of the hemorrhoidal artery ligation under control Doppler as a new technique for the treatment of hemorrhoids. We report the results of patients with hemorrhoids we have followed over a period of one year who were treated with HAL Doppler. The intra-and ...

  10. Synthesis of coumarin or ferrocene labeled nucleosides via Staudinger ligation

    Directory of Open Access Journals (Sweden)

    Kois Pavol

    2006-11-01

    Full Text Available Abstract Background Reaction of azides with triaryl phosphines under mild conditions gives iminophosphoranes which can react with almost any kind of electrophilic reagent, e.g. aldehydes/ketones to form imines or esters to form amides. This so-called Staudinger ligation has been employed in a wide range of applications as a general tool for bioconjugation including specific labeling of nucleic acids. Results A new approach for the preparation of labeled nucleosides via intermolecular Staudinger ligation is described. Reaction of azidonucleosides with triphenylphosphine lead to iminophosphorane intermediates, which react subsequently with derivatives of coumarin or ferrocene to form coumarin or ferrocene labeled nucleosides. Fluorescent properties of coumarin labeled nucleosides are determined. Conclusion New coumarin and ferrocene labeled nucleosides were prepared via intermolecular Staudinger ligation. This reaction joins the fluorescent coumarin and biospecific nucleoside to the new molecule with promising fluorescent and electrochemical properties. The isolated yields of products depend on the structure of azidonucleoside and carboxylic acids. A detailed study of the kinetics of the Staudinger ligation with nucleoside substrates is in progress.

  11. Endoscopic variceal band ligation: a local experience | Jani | East ...

    African Journals Online (AJOL)

    Objective: To evaluate the results of endoscopic variceal band ligation (EVBL) in the local set-up. Design: Retrospective analysis of data of all patients who had EVBL. Setting: Patients having EVBL at the office endoscopy suite. The Nairobi Hospital, the Aga Khan Hospital and M.P Shah Hospital. Methods: The varices were ...

  12. Spontaneous retraction of the ligated hernial sac during herniotomy ...

    African Journals Online (AJOL)

    Inguinal hernias constitute one of the most common surgical conditions for which a child presents to a surgeon 1,2. The treatment for this procedure in children is through an inguinal herniotomy with a high ligation of the hernial sac at the deep inguinal ring or at the level of the preperitoneal fat 3. In boys, it is important to.

  13. Impact of contraception use among women seeking tubal ligation in ...

    African Journals Online (AJOL)

    2007-02-21

    Feb 21, 2007 ... To describe user characteristics and analyse the impact of reversible contraception use among women who underwent tubal ligation ... suggesting a high prevalence of inconsistent or incorrect use of contraception. pg15-18.indd 15. 2/21/07 .... use is associated with smaller family units (Table II). Discussion.

  14. Recent Progress using the Staudinger Ligation for Radiolabeling Applications.

    Science.gov (United States)

    Mamat, Constantin; Gott, Matthew; Steinbach, Jörg

    2017-09-11

    The increasing application of positron emission tomography (PET) and single photon emission computer tomography (SPECT) in radiopharmacy and nuclear medicine has stimulated the development of a multitude of novel and versatile bioorthogonal conjugation techniques. Currently, there is particular interest in radiolabeling biologically active, high molecular weight compounds like peptides, proteins or antibodies, but also for the labeling of small organic compounds. An enormous challenge in radiolabeling these biologically active molecules is that the introduction of radiohalogens like fluorine-18 as well as various radiometals proceeds under harsh conditions, which could destroy the biomolecule. The Staudinger Ligation is one of the most powerful bioorthogonal conjugation techniques. The reaction proceeds over wide temperature and pH ranges; an amide (peptide) bond is formed as the ligation unit, which minimizes distortion of the structure; no isomers are obtained; and the reaction proceeds without any metal catalyst. Due to this adaptability, this robust ligation type is a perfect candidate with a high potential for various applications in the field of radiopharmacy for the labeling of biomolecules under mild conditions. This review summarizes recent research concerning the implementation of the Staudinger Ligation for radiolabeling applications. This article is protected by copyright. All rights reserved.

  15. Assessment and treatment of post patent ductus arteriosus ligation syndrome.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif F

    2014-07-01

    To compare differences in tissue Doppler imaging, global longitudinal strain (GLS), and cardiac troponin T (cTnT) between infants with low (<200 mL\\/kg\\/min) and high (>200 mL\\/kg\\/min) left ventricular (LV) output 1 hour after duct ligation and assess the impact of milrinone treatment on cardiac output and myocardial performance.

  16. Oesophageal variceal band ligation using a Saeed Six-Shooter ...

    African Journals Online (AJOL)

    He had a total of three sessions after which he was maintained on propranolol. Result: The last recheck endoscopy demonstrated obliterated varices after which he was maintained on propranolol. Conclusion: We present a case of successful variceal band ligation of a cirrhotic with extensive oesophageal varices presenting ...

  17. To evaluate the results of endoscopic variceal band ligation (EVBL)

    African Journals Online (AJOL)

    hi-tech

    2004-04-04

    Apr 4, 2004 ... Lay, C.S. Tsai, Y.T. Teg, C.Y. et al. Endoscopic variceal ligation in prophylaxis of first variceal bleeding in cirrhotic patients with high-risk esophageal varices. Hepatology. 1997;. 25:346-350. 24. Stiegmann, G.V. Goff, J.S. Michaletz-Onody, P.A. et al. Endoscopic sclerotherapy as compared with endoscopic.

  18. Impact of contraception use among women seeking tubal ligation in ...

    African Journals Online (AJOL)

    To describe user characteristics and analyse the impact of reversible contraception use among women who underwent tubal ligation in a rural health district of the Democratic Republic of Congo over a 4-year period. Methods. A retrospective analysis of family planning programme registers for 4 years (1990 - 1994). During ...

  19. Thermoresponsive Injectable Hydrogels Cross-Linked by Native Chemical Ligation

    NARCIS (Netherlands)

    Boere, Kristel W M; Soliman, Bram G.; Rijkers, Dirk T S; Hennink, Wim E.; Vermonden, Tina

    2014-01-01

    Temperature-induced physical gelation was combined with native chemical ligation (NCL) as a chemical cross-linking mechanism to yield rapid network formation and mechanically strong hydrogels. To this end, a novel monomer N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys) was synthesized that

  20. Ectopic pregnancy after two times tubal ligation: a case report

    Directory of Open Access Journals (Sweden)

    Farideh Keypour

    2013-06-01

    Full Text Available Background: Tubal sterilization is the permanent and effective contraception method. This can be performed at any time, but at least half are performed in conjunction with cesarean or vaginal delivery and are termed puerperal. The most complication after tubal ligation is ectopic pregnancy. Ectopic pregnancy is the leading cause of maternal death in first trimester.Case presentation: We present a 33 years old woman gravida5, para4, all normal vaginal delivery, presented with complaints of delayed menstrual period, pelvic pain and spotting. She underwent tubal ligation for two times. For the first time she had puerperal Pomeroy tubal sterilization after third child delivery. Intra uterine pregnancy occurred three years later. One day after vaginal delivery of fourth child, she underwent post partum tubal ligation with the Parkland method. Tubal pregnancy occurred nine months later. Physical examination identified acute abdomen. Pelvic ultrasound showed no gestational sac in uterine cavity. The sac with fetal pole was in right adnexa. Beta-HCG was 2840mIU/ml. She underwent laparotomy. Surgical management included salpingectomy with cornual resection in both sides. The surgery identified Ectopic pregnancy.Conclusion: Any symptoms of pregnancy in a woman after tubal ligation must be investigated; an ectopic pregnancy should be excluded. Ectopic pregnancy must be considered, in any woman with lower abdominal pain, missed period and vaginal bleed-ing. Conception after tubal sterilization can be explained by fistula formation and re-canalization of fallopian tube.

  1. Female sterilization by tubal ligation at caesarean section in Makurdi ...

    African Journals Online (AJOL)

    Background: Female sterilization is an important tool in reducing unplanned pregnancy and maternal mortality in our environment. The aim of this study was to determine the incidence, sociodemographic characteristics, technique, effectiveness and complications associated with female sterilization by bilateral tubal ligation ...

  2. Neovascularisation of the ovary post ligation of ovarian vessels in ...

    African Journals Online (AJOL)

    The viable ovarian tissues noticed on histology were evidence of revascularization suggestive of newer invasion of blood vessels from the ovarian attachment from the retroperitoneal tissues and likely from the subcutaneous supplies. Consequently, it is not advisable to neuter a bitch by ligation of the ovarian vessels ...

  3. Kinetics and equilibria for the axial ligation of bromomethyl (aqua ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 121; Issue 6. Kinetics and equilibria for the axial ligation of bromomethyl (aqua)cobaloxime with pyridines - Isolation characterization and DNA binding. Kotha Laxma Reddy K Ashwini Kumar N Ravi Kumar Reddy Penumaka Nagababu A Panasa Reddy S ...

  4. To evaluate the results of endoscopic variceal band ligation (EVBL)

    African Journals Online (AJOL)

    hi-tech

    2004-04-04

    Apr 4, 2004 ... Villanueva, C. Ortiz, J. Minana, J. et al. Somatostatin treatment and risk stratification by continuous portal pressure monitoring during acute variceal bleeding. Gastroenterology. 2001; 121:ll0-117. 5. Nevens, F. and Rutgeerts, P. Variceal band ligation in the management of bleeding oesophageal varices: an ...

  5. Aptamer: A potential oligonucleotide nanomedicine in the diagnosis and treatment of hepatocellular carcinoma

    Science.gov (United States)

    Ladju, Rusdina Bte; Pascut, Devis; Massi, Muhammad Nasrum; Tiribelli, Claudio; Sukowati, Caecilia H.C.

    2018-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers with a high mortality rate. Late diagnosis and poor prognosis are still a major drawback since curative therapies such as liver resection and liver transplantation are effective only for an early stage HCC. Development of novel molecular targeting therapies against HCC may provide new options that will improve the efficiency of the diagnosis and the success of the therapy, thus ameliorating the life expectancy of the patients. The aptamer is an oligonucleotide nanomedicine that has high binding affinity and specificity to small and large target molecules in the intracellular and extracellular environment with agonist or antagonist function. Currently, several aptamers for diagnostic and therapeutic purposes are under development to recognize different molecules of HCC. In in vitro models, the aptamer has been shown to be able to reduce the growth of HCC cells and increase the sensitivity to conventional chemotherapies. In in vivo mouse models, aptamer could induce cell apoptosis with antitumor activity. Overall data had shown that aptamer has limited toxicity and might be safe in clinical application. This review summarizes recent information of aptamer as a potential oligonucleotide nanomedicine tool, in diagnostics, targeted therapy, and as drug delivery nano-vehicles. PMID:29416827

  6. Oligonucleotide microarray-based detection and genotyping of Plum pox virus.

    Science.gov (United States)

    Pasquini, Graziella; Barba, Marina; Hadidi, Ahmed; Faggioli, Francesco; Negri, Rodolfo; Sobol, Iris; Tiberini, Antonio; Caglayan, Kadriye; Mazyad, Hamed; Anfoka, Ghandi; Ghanim, Murad; Zeidan, Mohammad; Czosnek, Henryk

    2008-01-01

    Plum pox virus (PPV) is the most damaging viral pathogen of stone fruits. The detection and identification of its strains are therefore of critical importance to plant quarantine and certification programs. Existing methods to screen strains of PPV suffer from significant limitations such as the simultaneous detection and genotyping of several strains of PPV in samples infected with different isolates of the virus. A genomic strategy for PPV screening based on the viral nucleotide sequence was developed to enable the detection and genotyping of the virus from infected plant tissue or biological samples. The basis of this approach is a long 70-mer oligonucleotide DNA microarray capable of simultaneously detecting and genotyping PPV strains. Several 70-mer oligonucleotide probes were specific for the detection and genotyping of individual PPV isolates to their strains. Other probes were specific for the detection and identification of two or three PPV strains. One probe (universal), derived from the genome highly conserved 3' non-translated region, detected all individual strains of PPV. This universal PPV probe, combined with probes specific for each known strain, could be used for new PPV strain discovery. Finally, indirect fluorescent labeling of cDNA with cyanine after cDNA synthesis enhanced the sensitivity of the virus detection without the use of the PCR amplification step. The PPV microarray detected and identified efficiently the PPV strains in PPV-infected peach, apricot and Nicotiana benthamiana leaves. This PPV detection method is versatile, and enables the simultaneous detection of plant pathogens.

  7. Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo

    Directory of Open Access Journals (Sweden)

    Maria Chiara Munisso

    2014-01-01

    Full Text Available The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.

  8. Synthesis of Oligonucleotides Carrying Thiol Groups Using a Simple Reagent Derived from Threoninol

    Directory of Open Access Journals (Sweden)

    Ramon Eritja

    2012-08-01

    Full Text Available Oligonucleotides carrying thiol groups are useful intermediates for a remarkable number of applications involving nucleic acids. In this study, DNA oligonucleotides carrying tert-butylsulfanyl (t-BuS protected thiol groups have been prepared. A building block derived from threoninol has been developed to introduce a thiol group at any predetemined position of an oligonucleotide. The resulting thiolated oligonucleotides have been used for the preparation of oligonucleotide conjugates and for the functionalization of gold nanoparticles using the reactivity of the thiol groups.

  9. Ligating perforators in abdominoplasty reduces the risk of seroma.

    Science.gov (United States)

    Skillman, J M; Venus, M R; Nightingale, P; Titley, O G; Park, A

    2014-04-01

    Seroma formation, a common complication of abdominoplasty, can cause patient discomfort and inconvenience. This study aimed to compare seroma rates after ligation and diathermy of large abdominal perforating vessels during abdominoplasty. Consecutive patients undergoing abdominoplasty with epigastric undermining between 2004 and 2011 were studied. Body mass index (BMI), age at operation, smoking history, preoperative weight loss, operative details, perioperative fluid infiltration, concomitant abdominal liposuction, ligation of perforators by clips, suture or diathermy, use of quilting sutures, weight of tissue removed, postoperative drainage, inpatient stay, and seroma rates were recorded. Statistical analysis was undertaken using the unpaired t test, Fisher's exact test, the Mann-Whitney U test, and Kendall's tau-b test. The study included 90 patients. The incidence of seroma was significantly lower among the patients who had perforators ligated (4/60, 6.7%) than among those who had diathermy (10/30, 33%) (p=0.002, Fisher's exact test). Seroma formation was significantly associated with a higher BMI, (27.45 vs. 25.16 kg/m2; p=0.025, t test) but not with preoperative weight loss. Postoperative fluid drainage did not differ significantly between ligated and diathermied perforators (p=0.716 Mann-Whitney U test). Use of ligation by clip or suture rather than by diathermy to ablate large abdominal perforators significantly reduced the incidence of seroma among abdominoplasty 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 .

  10. Infrared coagulation versus rubber band ligation in early stage hemorrhoids

    Directory of Open Access Journals (Sweden)

    P.J. Gupta

    2003-10-01

    Full Text Available The ideal therapy for early stages of hemorrhoids is always debated. Some are more effective but are more painful, others are less painful but their efficacy is also lower. Thus, comfort or efficacy is a major concern. In the present randomized study, a comparison is made between infrared coagulation and rubber band ligation in terms of effectiveness and discomfort. One hundred patients with second degree bleeding piles were randomized prospectively to either rubber band ligation (N = 54 or infrared coagulation (N = 46. Parameters measured included postoperative discomfort and pain, time to return to work, relief in incidence of bleeding, and recurrence rate. The mean age was 38 years (range 19-68 years. The mean duration of disease was 17.5 months (range 12 to 34 months. The number of male patients was double that of females. Postoperative pain during the first week was more intense in the band ligation group (2-5 vs 0-3 on a visual analogue scale. Post-defecation pain was more intense with band ligation and so was rectal tenesmus (P = 0.0059. The patients in the infrared coagulation group resumed their duties earlier (2 vs 4 days, P = 0.03, but also had a higher recurrence or failure rate (P = 0.03. Thus, we conclude that band ligation, although more effective in controlling symptoms and obliterating hemorrhoids, is associated with more pain and discomfort to the patient. As infrared coagulation can be conveniently repeated in case of recurrence, it could be considered to be a suitable alternative office procedure for the treatment of early stage hemorrhoids.

  11. Corrosion behavior of self-ligating and conventional metal brackets

    Directory of Open Access Journals (Sweden)

    Lúcio Henrique Esmeraldo Gurgel Maia

    2014-04-01

    Full Text Available Objective: To test the null hypothesis that the aging process in self-ligating brackets is not higher than in conventional brackets. Methods: Twenty-five conventional (GN-3M/Unitek; GE-GAC; VE-Aditek and 25 self-ligating (SCs-3M/Unitek; INs-GAC; ECs-Aditek metal brackets from three manufacturers (n = 150 were submitted to aging process in 0.9% NaCl solution at a constant temperature of 37 ± 1ºC for 21 days. The content of nickel, chromium and iron ions in the solution collected at intervals of 7, 14 and 21 days was quantified by atomic absorption spectrophotometry. After the aging process, the brackets were analyzed by scanning electron microscopy (SEM under 22X and 1,000X magnifications. Results: Comparison of metal release in self-ligating and conventional brackets from the same manufacturer proved that the SCs group released more nickel (p < 0.05 than the GN group after 7 and 14 days, but less chromium (p < 0.05 after 14 days and less iron (p < 0.05 at the three experimental time intervals. The INs group released less iron (p < 0.05 than the GE group after 7 days and less nickel, chromium and iron (p < 0.05 after 14 and 21 days. The ECs group released more nickel, chromium and iron (p < 0.05 than the VE group after 14 days, but released less nickel and chromium (p < 0.05 after 7 days and less chromium and iron (p < 0.05 after 21 days. The SEM analysis revealed alterations on surface topography of conventional and self-ligating brackets. Conclusions: The aging process in self-ligating brackets was not greater than in conventional brackets from the same manufacturer. The null hypothesis was accepted.

  12. Desulfurization of phosphorothioate oligonucleotides via the sulfur-by-oxygen replacement induced by the hydroxyl radical during negative electrospray ionization mass spectrometry.

    Science.gov (United States)

    Wu, Lianming; White, David E; Ye, Connie; Vogt, Frederick G; Terfloth, Gerald J; Matsuhashi, Hayao

    2012-07-01

    While the occurrence of desulfurization of phosphorothioate oligonucleotides in solution is well established, this study represents the first attempt to investigate the basis of the unexpected desulfurization via the net sulfur-by-oxygen (S-O) replacement during negative electrospray ionization (ESI). The current work, facilitated by quantitative mass deconvolution, demonstrates that considerable desulfurization can take place even under common negative ESI operating conditions. The extent of desulfurization is dependent on the molar phosphorothioate oligonucleotide-to-hydroxyl radical ratio, which is consistent with the corona discharge-induced origin of the hydroxyl radical leading to the S-O replacement. This hypothesis is supported by the fact that an increase of the high-performance liquid chromatography (HPLC) flow rate and the on-column concentration of a phosphorothioate oligonucleotide, as well as a decrease of the electrospray voltage reduce the degree of desulfurization. Comparative LC-tandem mass spectrometry (MS/MS) sequencing of a phosphorothioate oligonucleotide and its corresponding desulfurization product revealed evidence that the S-O replacement occurs at multiple phosphorothioate internucleotide linkage sites. In practice, the most convenient and effective strategy for minimizing this P = O artifact is to increase the LC flow rate and the on-column concentration of phosphorothioate oligonucleotides. Another approach to mitigate possible detrimental effects of the undesired desulfurization is to operate the ESI source at a very low electrospray voltage to diminish the corona discharge; however this will significantly compromise sensitivity when analyzing the low-level P = O impurities in phosphorothioate oligonucleotides. Copyright © 2012 John Wiley & Sons, Ltd.

  13. Necrotizing fasciitis following saphenofemoral junction ligation with long saphenous vein stripping: a case report

    Directory of Open Access Journals (Sweden)

    Ferguson Graeme

    2010-05-01

    Full Text Available Abstract Introduction Necrotizing fasciitis is a rare condition with a mortality rate of around 34%. It can be mono- or polymicrobial in origin. Monomicrobial infections are usually due to group A streptococcus and their incidence is on the rise. They normally occur in healthy individuals with a history of trauma, surgery or intravenous drug use. Post-operative necrotizing fasciitis is rare but accounts for 9 to 28% of all necrotizing fasciitis. The incidence of wound infection following saphenofemoral junction ligation and vein stripping is said to be less than 3%, although this complication is probably under-reported. We describe a case of group A streptococcus necrotizing fasciitis following saphenofemoral junction ligation and vein stripping. Case Presentation A 39-year-old woman presented three days following a left sided saphenofemoral junction ligation with long saphenous vein stripping at another institution. She had a three day history of fever, rigors and swelling of the left leg. She was pyrexial and shocked. She had a very tender, swollen left groin and thigh, with a small blister anteriorly and was in acute renal failure. She was prescribed intravenous penicillin and diagnosed with necrotizing fasciitis. She underwent extensive debridement of her left thigh and was commenced on clindamycin and imipenem. Post-operatively, she required ventilatory and inotropic support with continuous veno-venous haemofiltration. An examination 12 hours after surgery showed no requirement for further debridement. A group A streptococcus, sensitive to penicillin, was isolated from the debrided tissue. A vacuum assisted closure device was fitted to the clean thigh wound on day four and split-skin-grafting was performed on day eight. On day 13, a wound inspection revealed that more than 90% of the graft had taken. Antibiotics were stopped on day 20 and she was discharged on day 22. Conclusion Necrotizing fasciitis is a very serious complication for a

  14. Nanomaterial building blocks based on spider silk-oligonucleotide conjugates.

    Science.gov (United States)

    Humenik, Martin; Scheibel, Thomas

    2014-02-25

    Self-assembling protein nanofibrils are promising structures for the "bottom-up" fabrication of bionanomaterials. Here, the recombinant protein eADF4(C16), a variant of Araneus diadematus dragline silk ADF4, which self-assembles into nanofibrils, and short oligonucleotides were modified for site-specific azide-alkyne coupling. Corresponding oligonuleotide-eADF4(C16) "click" conjugates were hybridized in linear or branched fashion according to the designed complementarities of the DNA moieties. Self-assembly properties of higher ordered structures of the spider silk-DNA conjugates were dominated by the silk component. Assembled β-sheet rich conjugate fibrils were similar in appearance to fibrils of unmodified eADF4(C16) but enabled the specific attachment of neutravidin-modified gold nanoparticles on their surface directed by complementary biotin-oligonucleotides, providing the basis for functionalization of such conjugates.

  15. Palladium-Catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Kevin H. Shaughnessy

    2015-05-01

    Full Text Available Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

  16. Typing of enteroviruses by use of microwell oligonucleotide arrays.

    Science.gov (United States)

    Susi, P; Hattara, L; Waris, M; Luoma-Aho, T; Siitari, H; Hyypiä, T; Saviranta, P

    2009-06-01

    We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

  17. Typing of Enteroviruses by Use of Microwell Oligonucleotide Arrays▿ †

    Science.gov (United States)

    Susi, P.; Hattara, L.; Waris, M.; Luoma-aho, T.; Siitari, H.; Hyypiä, T.; Saviranta, P.

    2009-01-01

    We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies. PMID:19357207

  18. Electronic Structure of Ligated CdSe Clusters: Dependence on DFT Methodology

    Energy Technology Data Exchange (ETDEWEB)

    Albert, VV; Ivanov, SA; Tretiak, S; Kilina, SV

    2011-07-07

    Simulations of ligated semiconductor quantum dots (QDs) and their physical properties, such as morphologies, QD-ligand interactions, electronic structures, and optical transitions, are expected to be very sensitive to computational methodology. We utilize Density Functional Theory (DFT) and systematically study how the choice of density functional, atom-localized basis set, and a solvent affects the physical properties of the Cd{sub 33}Se{sub 33} cluster ligated with a trimethyl phosphine oxide ligand. We have found that qualitative performance of all exchange-correlation (XC) functionals is relatively similar in predicting strong QD-ligand binding energy ({approx}1 eV). Additionally, all functionals predict shorter Cd-Se bond lengths on the QD surface than in its core, revealing the nature and degree of QD surface reconstruction. For proper modeling of geometries and QD-ligand interactions, however, augmentation of even a moderately sized basis set with polarization functions (e.g., LANL2DZ* and 6-31G*) is very important. A polar solvent has very significant implications for the ligand binding energy, decreasing it to 0.2-0.5 eV. However, the solvent model has a minor effect on the optoelectronic properties, resulting in persistent blue shifts up to {approx}0.3 eV of the low-energy optical transitions. For obtaining reasonable energy gaps and optical transition energies, hybrid XC functionals augmented by a long-range Hartree-Fock orbital exchange have to be applied.

  19. Oligonucleotide?directed mutagenesis for precision gene editing

    OpenAIRE

    Sauer, Noel J.; Mozoruk, Jerry; Miller, Ryan B.; Warburg, Zachary J.; Walker, Keith A.; Beetham, Peter R.; Sch?pke, Christian R.; Gocal, Greg F. W.

    2015-01-01

    Summary Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide?directed mutagenesis (ODM), one of the many tools of Cibus? Rapid Trait Development System ( RTDS ?) technology, offers a rapid, precise and non?transgenic breeding a...

  20. Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Suriano, Raffaella [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Biella, Serena, E-mail: serena.biella@polimi.it [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Cesura, Federico; Levi, Marinella; Turri, Stefano [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy)

    2013-05-15

    Different thermoplastic polymers were spin-coated to prepare smooth surfaces for the direct deposition of end-group modified oligonucleotides by Dip-Pen Nanolithography. A study of the diffusion process was done in order to investigate the dependence of calibration coefficient and quality of deposited features on environmental parameters (temperature, relative humidity) and ink's molecular weight and functionality. The optimization of the process parameters led to the realization of high quality and density nanoarrays on plastics.

  1. Differentiation of regions with atypical oligonucleotide composition in bacterial genomes

    Directory of Open Access Journals (Sweden)

    Reva Oleg N

    2005-10-01

    Full Text Available Abstract Background Complete sequencing of bacterial genomes has become a common technique of present day microbiology. Thereafter, data mining in the complete sequence is an essential step. New in silico methods are needed that rapidly identify the major features of genome organization and facilitate the prediction of the functional class of ORFs. We tested the usefulness of local oligonucleotide usage (OU patterns to recognize and differentiate types of atypical oligonucleotide composition in DNA sequences of bacterial genomes. Results A total of 163 bacterial genomes of eubacteria and archaea published in the NCBI database were analyzed. Local OU patterns exhibit substantial intrachromosomal variation in bacteria. Loci with alternative OU patterns were parts of horizontally acquired gene islands or ancient regions such as genes for ribosomal proteins and RNAs. OU statistical parameters, such as local pattern deviation (D, pattern skew (PS and OU variance (OUV enabled the detection and visualization of gene islands of different functional classes. Conclusion A set of approaches has been designed for the statistical analysis of nucleotide sequences of bacterial genomes. These methods are useful for the visualization and differentiation of regions with atypical oligonucleotide composition prior to or accompanying gene annotation.

  2. Site-directed selection of oligonucleotide antagonists by competitive elution.

    Science.gov (United States)

    Bridonneau, P; Chang, Y F; Buvoli, A V; O'Connell, D; Parma, D

    1999-02-01

    Oligonucleotide ligands that bind a protein or a small molecule of interest are readily isolated by in vitro selection and amplification of rare sequences from combinatorial libraries of sequence-randomized oligonucleotides (Gold et al., 1995). Classic systematic evolution of ligands by exponential enrichment (SELEX) protocols are affinity based (Tuerk and Gold, 1990), but because many problems and applications require antagonists, protocols for selecting inhibitors are both desirable and valuable. A widely applicable approach for isolating inhibitors is competitive elution with a molecule that binds the targeted molecule's active or binding site. We have used this approach to isolate antagonists of wheat germ agglutinin (WGA) from a library of 2'NH2-pyrimidine, 2'OH-purine oligonucleotides by elution with N N' N"-triacetylchitotriose, (GlcNAc)3. The highest affinity aptamers have equilibrium dissociation constants of 1 nM-20 nM for WGA, a 10(3)-10(4)-fold improvement relative to (GlcNAc)3, and unlike the carbohydrate, are highly specific. In addition to competing for binding with (GlcNAc)3, aptamers inhibit WGA-mediated agglutination of sheep erythrocytes, demonstrating that they are able to compete with natural ligands presented on the surfaces of cells. These results illustrate the feasibility of isolating high-affinity, high-specificity antagonists by competitive elution with low molecular weight, relatively low-affinity, and low-specificity small molecules.

  3. Recent developments in reversible photoregulation of oligonucleotide structure and function.

    Science.gov (United States)

    Lubbe, Anouk S; Szymanski, Wiktor; Feringa, Ben L

    2017-02-20

    There is a growing interest in the photoregulation of biological functions, due to the high level of spatiotemporal precision achievable with light. Additionally, light is non-invasive and waste-free. In particular, the photoregulation of oligonucleotide structure and function is a rapidly developing study field with relevance to biological, physical and material sciences. Molecular photoswitches have been incorporated in oligonucleotides for 20 years, and the field has currently grown beyond fundamental studies on photochemistry of the switches and DNA duplex stability, and is moving towards applications in chemical biology, nanotechnology and material science. Moreover, the currently emerging field of photopharmacology indicates the relevance of photocontrol in future medicine. In recent years, a large number of publications has appeared on photoregulation of DNA and RNA structure and function. New strategies are evaluated and novel, exciting applications are shown. In this comprehensive review, the key strategies for photoswitch inclusion in oligonucleotides are presented and illustrated with recent examples. Additionally the applications that have emerged in recent years are discussed, including gene regulation, drug delivery and materials design. Finally, we identify the challenges that the field currently faces and look forward to future applications.

  4. Acute pancreatitis after thoracic duct ligation for iatrogenic chylothorax. A case report.

    Science.gov (United States)

    Bédat, Benoît; Scarpa, Cosimo Riccardo; Sadowski, Samira Mercedes; Triponez, Frédéric; Karenovics, Wolfram

    2017-01-23

    To report the association between thoracic duct ligation and acute pancreatitis. The association between sudden stop of lymphatic flow and pancreatitis has been established in experimental models. A 57-year-old woman operated for thymoma presented a iatrogenic chylothorax. After thoracic duct ligation, she presented an acute pancreatitis which resolved after conservative treatment. The chylothorax disappeared within 4 days of thoracic duct ligation. This is the first report of acute pancreatitis following thoracic duct ligation. The pancreas and digestive tract should be assessed in symptomatic patients after thoracic duct ligation.

  5. Evaluation of dual priming oligonucleotide-based multiplex PCR for detection of HBV YMDD mutants.

    Science.gov (United States)

    Woo, H Y; Park, H; Kim, B I; Jeon, W K; Kim, Y J

    2008-01-01

    We evaluated the usefulness of dual priming oligonucleotide (DPO)-based multiplex PCR, Seeplex HBV Lami-DR assay (Seegene Institute of Life Sciences, Seoul, Korea), to detect lamivudine-resistant HBV mutants in a comparison with the use of TRUGENE HBV genotyping and restriction fragment mass polymorphism (RFMP). Sera from 44 chronic hepatitis B patients were analyzed for the presence of mutations at codons 180 and 204 by performing DPO-based multiplex PCR, RFMP, and TRUGENE. The overall concordance rate among the three assays was 40.9% (18/44). Concordance rates between multiplex PCR and RFMP or multiplex PCR and TRUGENE were 61.4% (27/44) and 50.0% (22/44), respectively. In ten patients, multiplex PCR identified additional mutants not found using the other two methods. DPO-based multiplex PCR is a highly sensitive method to identify minor mutant populations and could be a practical tool in the monitoring of lamivudine resistance.

  6. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    Energy Technology Data Exchange (ETDEWEB)

    Katebi, Samira; Esmaeili, Abolghasem, E-mail: aesmaeili@sci.ui.ac.ir; Ghaedi, Kamran

    2016-03-15

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

  7. Hydrogels Formed by Oxo-ester Mediated Native Chemical Ligation.

    Science.gov (United States)

    Strehin, Iossif; Gourevitch, Dmitri; Zhang, Yong; Heber-Katz, Ellen; Messersmith, Phillip B

    2013-06-01

    Oxo-ester mediated native chemical ligation (OMNCL) is a variation of the more general native chemical ligation (NCL) reaction that is widely employed for chemoselective ligation of peptide fragments. While OMNCL has been used for a variety of peptide ligations and for biomolecular modification of surfaces, it is typically practiced under harsh conditions that are unsuitable for use in a biological context. In this report we describe the use of OMNCL for polymer hydrogel formation, in-vitro cell encapsulation, and in-vivo implantation. Multivalent polymer precursors containing N-hydroxysuccinimide (NHS) activated oxo-esters and N-cysteine (N-Cys) endgroups were chemically synthesized from branched poly(ethylene glycol) (PEG). Hydrogels formed rapidly at physiologic pH upon mixing of aqueous solutions of NHS and N-Cys functionalized PEGs. Quantitative 1H NMR experiments showed that the reaction proceeds through an OMNCL pathway involving thiol capture to form a thioester intermediate, followed by an S-to-N acyl rearrangement to yield an amide cross-link. pH and temperature were found to influence gelation rate, allowing tailoring of gelation times from a few seconds to a few minutes. OMNCL hydrogels initially swelled before contracting to reach an equilibrium increase in relative wet weight of 0%. This unique behavior impacted the gel stiffness and was attributed to latent formation of disulfide cross-links between network-bound Cys residues. OMNCL hydrogels were adhesive to hydrated tissue, generating a lap shear adhesion strength of 46 kPa. Cells encapsulated in OMNCL hydrogels maintained high viability, and in-situ formation of OMNCL hydrogel by subcutaneous injection in mice generated a minimal acute inflammatory response. OMNCL represents a promising strategy for chemical cross-linking of hydrogels in a biological context and is an attractive candidate for in-vivo applications such as wound healing, tissue repair, drug delivery, and tissue engineering.

  8. Feasibility of laparoscopic portal vein ligation prior to major hepatectomy.

    Science.gov (United States)

    Are, C; Iacovitti, S; Prete, F; Crafa, F M

    2008-01-01

    Patients noted to have an inadequate future liver remnant on pre operative volumetric assessment are considered to be candidates for portal vein embolization (PVE). A subset of patients undergo laparoscopic intervention prior to PVE for staging purposes or to address the primary in Stage IV colon cancer. These patients usually undergo PVE as a subsequent additional procedure by the transhepatic route. The aim of this study was to assess the feasibility of portal vein ligation by the laparoscopic approach in suitable patients. A retrospective review of a prospectively maintained database was performed to identify patients that underwent laparoscopic portal vein ligation (LPVL). The demographic, clinical, radiographic, operative and volumetric details were collected to determine the feasibility of portal vein ligation. A total of nine patients underwent LPVL as part of a two stage procedure in preparation for subsequent major hepatectomy. With a median age of 67 yrs, the diagnoses included: colorectal metastasis (five patients), cholangiocarcinoma (three patients) and hepatocellular carcinoma (one patient). The ligation involved the right portal vein in all and was performed with silk ligature (seven patients) and clips (two patients). Volumetric data was available in six patients which showed a mean increase from 209.1 cc+/-97.76 to 495.83 cc+/-310.91 (increase by 181.5%) In two patients, inadequate hypertrophy mandated later embolization by percutaneous technique. Five patients underwent subsequent major hepatic resection as planned. The remaining four patients were noted to have progression of disease that precluded the planned procedure. There were no complications associated with LPVL. LPVL is feasible and can be safely performed. In a select group of patients, it may be considered as an alternative to subsequent embolization and thereby potentially absolve the need for an additional procedure with its attendant complications.

  9. Comparison of Hi-C results using in-solution versus in-nucleus ligation.

    Science.gov (United States)

    Nagano, Takashi; Várnai, Csilla; Schoenfelder, Stefan; Javierre, Biola-Maria; Wingett, Steven W; Fraser, Peter

    2015-08-26

    Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation. We show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries. We conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

  10. Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation.

    Science.gov (United States)

    Gui, Yue; Qiu, Lingqi; Li, Yaohao; Li, Hongxing; Dong, Suwei

    2016-04-13

    Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.

  11. Invader Assisted Enzyme-Linked Immunosorbent Assay for Colorimetric Detection of Disease Biomarkers Using Oligonucleotide Probe-Modified Gold Nanoparticles.

    Science.gov (United States)

    Song, Qinxin; Qi, Xiemin; Jia, Huning; He, Liang; Kumar, Shalen; Pitman, Janet L; Zou, Bingjie; Zhou, Guohua

    2016-04-01

    We successfully developed an invader assisted ELISA assay (iaELISA) for sensitive detection of disease biomarkers. The method includes three key steps as follows; biotinylated detection antibody was at first used to capture targeted antigen by sandwich ELISA. The biotinylated oligonucleotide was then attached to detection antibody via streptavidin. Finally, the cascade invader reactions were employed to amplify the biotinylated oligonucleotide specific to the antigen so that detection of the antigen was transformed into signal amplification of the antigen-specific DNA. To achieve colorimetric detection, oligonucleotide probe and modified gold nanoparticles (AuNPs) were coupled with the invader assay. Utilization of the hairpin probes in the invader reaction brought about free AuNPs, resulting in the positive read-out (red color). On the other hand, aggregation of the AuNPs occurred when the hairpin probes were not utilized in the reaction. This method was successfully used to detect as low as 2.4 x 10(-11) g/mL of HBsAg by both naked eye and spectrophotometer. This sensitivity was about 100 times higher than that of conventional ELISA method. The method was also used to assay 16 serum specimens from HBV-infected patients and 8 serum specimens from HBV-negative donors and results were in good agreement with those obtained from the conventional ELISA. As the invader assay is sensitive to one base sequence, a good specificity was also obtained by detecting other antigens like hepatitis A virus (HAV) and BSA. The method has therefore much potential for ultrasensitive and cost-effective detection of targeted proteins that have clinical importance.

  12. Timing of surgical ligation and morbidities in very low birth weight infants.

    Science.gov (United States)

    Youn, YoungAh; Moon, Cheong-Jun; Lee, Jae-Young; Lee, Cheul; Sung, In Kyung

    2017-04-01

    The present study examined whether early patent ductus arteriosus (PDA) surgical ligation at ≤2 weeks of life was associated with increased morbidities and mortality in very low birth weight infants (VLBWIs) who were diagnosed with hemodynamically significant (hs) PDA. Between December 2013 and December 2015, a total of 407 VLBWIs were admitted, of whom 145 (35.6%) infants were diagnosed with an hs PDA. The clinical data for these infants were retrospectively collected for analysis. Among the 145 VLBWIs with an hs PDA, 58 (40%) infants had surgical ligation for PDA; of these, 29 (50%) infants had early ligation (EL; ligation at ≤2 weeks of life) and 29 (50%) infants had late ligation (LL; ligation at ≥2 weeks of life). The mean gestational age and birth weight were significantly lower in the PDA-ligated group compared with the nonligated group. In addition, pulmonary hypertension at ≤1 week of life and neonatal seizures were significantly more prevalent in the ligated group (P hypertension at ≤1 week of life was significantly associated with LL (P = 0.019), which was consistently a risk factor for hs PDA ligation in our multivariable logistic regression analysis. EL was not significantly associated with increased hospital morbidities and mortality in VLBWIs with hs PDA. Pulmonary hypertension at ≤1 week of life can be a risk factor for the need for surgical ligation of hs PDA.

  13. Production of cis-syn thymine-thymine cyclobutane dimer oligonucleotide in the presence of acetone photosensitizer.

    Science.gov (United States)

    Mu, Wanmeng; Han, Qingkai; Luo, Zhaofeng; Wang, Yuzhen

    2006-06-01

    cis-syn Cyclobutane pyrimidine dimer (CPD) oligonucleotide was produced by UV irradiation in the presence of acetone photosensitizer. Acetone could enhance the productivity but evidently induced the photocleavage of oligonucleotide under a long time irradiation. A statistical approach of orthogonal design was applied to optimize the preparation condition for the production of the modified oligonucleotide. Optimal conditions for maximal cis-syn CPD oligonucleotide productivity were determined based on three factors: acetone concentration, initial oligonucleotide concentration, and irradiation time at several different levels. The optimal modified oligonucleotide that this optimization could produce was 32.7%. Through analysis of 20% polyacrylamide gel electrophoresis, it was found that modified oligonucleotide migrated slightly more slowly than the parent oligonucleotide. The photoreactivation of cis-syn thymine-thymine dimer oligonucleotide displayed the selectivity of the substrate specificity of DNA photolyase with high-performance liquid chromatography (HPLC) analysis.

  14. Oligonucleotide Antiviral Therapeutics: Antisense and RNA Interference for Highly Pathogenic RNA Viruses

    National Research Council Canada - National Science Library

    Spurgers, Kevin B; Sharkey, C. M; Warfield, Kelly L; Bavari, Sina

    2008-01-01

    .... Important advances in the field include the characterization of RNA interference in mammalian cells and chemical modifications that can dramatically increase the in vivo stability of therapeutic oligonucleotides...

  15. Terahertz response of DNA oligonucleotides on the surface of silicon nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Bagraev, N. T., E-mail: bagraev@mail.ioffe.ru [Peter the Great Saint-Petersburg Polytechnic University (Russian Federation); Chernev, A. L. [Russian Academy of Sciences, Saint Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation); Klyachkin, L. E.; Malyarenko, A. M. [Russian Academy of Sciences, Ioffe Physical–Technical Institute (Russian Federation); Emel’yanov, A. K.; Dubina, M. V. [Russian Academy of Sciences, Saint Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation)

    2016-09-15

    The possibility of identifying DNA oligonucleotides deposited onto the region of the edge channels of silicon nanostructures is considered. The role of various THz (terahertz) radiation harmonics of silicon nanostructures in the resonance response of oligonucleotides is analyzed. In particular, this makes it possible to compare single-stranded 100- and 50-mer DNA oligonucleotides. A technique for the rapid identification of different oligonucleotides by measuring changes in the conductance and transverse potential difference of silicon nanostructures with microcavities, embedded in the edge channels for selecting THz radiation characteristics, is proposed.

  16. Fluorous methods for the synthesis of peptides and oligonucleotides.

    Science.gov (United States)

    Miriyala, Bruhaspathy

    2012-01-01

    The non-covalent affinity of a perfluoro chain towards similar has been exploited by many to separate fluorous tagged compounds from non-fluorous compounds by F-SPE or F-LLE. This purification strategy found its application across diverse fields including peptide and oligonucleotide synthesis where even slight inefficient couplings result in deletion sequences that are often difficult to remove from the target sequence. Two commonly employed strategies to address this problem involve end-tagging the target sequence or capping the deletion sequences with fluorous tags. Solution phase syntheses using soluble fluorous supports are easier and quicker. These approaches are reviewed here in detail.

  17. Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Park, Serk In, E-mail: serkin@korea.edu [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); The BK21 Plus Program for Biomedical Sciences, Korea University College of Medicine, Seoul (Korea, Republic of); Department of Medicine and Center for Bone Biology, Vanderbilt University School of Medicine, Nashville, TN (United States); Park, Sung-Jun [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Laboratory of Obesity and Aging Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD (United States); Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Park, Yun Gyu, E-mail: parkyg@korea.ac.kr [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

    2016-01-15

    The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

  18. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery.

    Science.gov (United States)

    Cheng, Xinwei; Lee, Robert J

    2016-04-01

    Lipid nanoparticles (LNPs) have shown promise as delivery vehicles for therapeutic oligonucleotides, including antisense oligos (ONs), siRNA, and microRNA mimics and inhibitors. In addition to a cationic lipid, LNPs are typically composed of helper lipids that contribute to their stability and delivery efficiency. Helper lipids with cone-shape geometry favoring the formation hexagonal II phase, such as dioleoylphosphatidylethanolamine (DOPE), can promote endosomal release of ONs. Meanwhile, cylindrical-shaped lipid phosphatidylcholine can provide greater bilayer stability, which is important for in vivo application of LNPs. Cholesterol is often included as a helper that improves intracellular delivery as well as LNP stability in vivo. Inclusion of a PEGylating lipid can enhance LNP colloidal stability in vitro and circulation time in vivo but may reduce uptake and inhibit endosomal release at the cellular level. This problem can be addressed by choosing reversible PEGylation in which the PEG moiety is gradually released in blood circulation. pH-sensitive anionic helper lipids, such as fatty acids and cholesteryl hemisuccinate (CHEMS), can trigger low-pH-induced changes in LNP surface charge and destabilization that can facilitate endosomal release of ONs. Generally speaking, there is no correlation between LNP activity in vitro and in vivo because of differences in factors limiting the efficiency of delivery. Designing LNPs requires the striking of a proper balance between the need for particle stability, long systemic circulation time, and the need for LNP destabilization inside the target cell to release the oligonucleotide cargo, which requires the proper selection of both the cationic and helper lipids. Customized design and empirical optimization is needed for specific applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

    Science.gov (United States)

    Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

    2014-01-01

    Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

  20. Detection and quantitation of genetically modified maize (Bt-176 transgenic maize) by applying ligation detection reaction and universal array technology.

    Science.gov (United States)

    Bordoni, Roberta; Mezzelani, Alessandra; Consolandi, Clarissa; Frosini, Andrea; Rizzi, Ermanno; Castiglioni, Bianca; Salati, Claudia; Marmiroli, Nelson; Marchelli, Rosangela; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

    2004-03-10

    We have applied the ligation detection reaction (LDR) combined with a universal array approach to the detection and quantitation of the polymerase chain reaction (PCR) amplified cry1A(b) gene from Bt-176 transgenic maize. We demonstrated excellent specificity and high sensitivity. Down to 0.5 fmol (nearly 60 pg) of PCR amplified transgenic material was unequivocally detected with excellent linearity within the 0.1-2.0% range with respect to wild-type maize. We suggest the feasibility of extending the LDR/universal array format to detect in parallel several transgenic sequences that are being developed for food applications.

  1. Menstrual Pattern following Tubal Ligation: A Historical Cohort Study

    Directory of Open Access Journals (Sweden)

    Shahideh Jahanian Sadatmahalleh

    2016-12-01

    Full Text Available Background: Tubal ligation (TL is recommended for women who have completed their family planning. The existence of the menstrual disorders following this procedure has been the subject of debate for decades. This study was conducted to identify the relationship between tubal ligation and menstrual disorders. Materials and Methods: A historical cohort study was carried out on 140 women undergoing tubal ligation (TL group and on 140 women using condom as the main contraceptive method (Non-TL group. They aged between 20 and 40 years and were selected from a health care center in Rudsar, Guilan Province, Iran, during 2013-2014. The two groups were comparable in demographic characteristics, obstetrical features and menstrual bleeding pattern using a routine questionnaire. A validated pictorial blood loss assessment chart (PBLAC was also used to measure the menstrual blood loss. Results: Women with TL had more menstrual irregularity than those without TL (24.3 vs. 10%, P=0.002. Women with TL had more polymenorrhea (9.3 vs. 1.4%, P=0.006, hypermenorrhea (12.1 vs. 2.1%, P=0.002, menorrhagia (62.9 vs. 22.1%, P<0.0001 and menometrorrhagia (15.7 vs. 3.6%, P=0.001 than those without TL. There is a significant difference in the PBLAC score between women with and without TL (P<0.0001. According to logistic regression, age odds ratio [(OR=1.08, confidence interval (CI:1.07-1.17, P=0.03], TL (OR=5.95, CI:3.45-10.26, P<0.0001 and cesarean section (OR=2.72, CI:1.49-4.97, P=0.001 were significantly associated with menorrhagia. Conclusion: We found significant differences in menstrual disorders between women with and without TL. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of TL before the procedures.

  2. Traditional elastic ligatures versus slide ligation system. A morphological evaluation.

    Science.gov (United States)

    Condò, R; Casaglia, A; Armellin, E; Condò, S G; Cerroni, L

    2013-01-01

    Elastomeric materials play an important role in the orthodontic practice, including the retraction force to move teeth into extraction sites, closing diastemas, selective shifting of the midline and generalized space closure. Frictional resistance and ligating strength of archwire-bracket-ligature complex occurs during utilization of elastomeric and metallic ligatures when orthodontic forces are applicated. The aim of this study was to analyze elastic deformation of three types of elastomeric ligatures, after clinical use. ELASTOMERIC LIGATURES: ring-shape, transparent, latex ligatures (Leone® S.p.A.), ring-shape, grey, polyurethane ligatures (Micerium® S.p.A.) and grey, polyurethane, Slide low-friction ligatures (Leone® S.p.A.). A total of 9 orthodontic patients undergoing fixed orthodontic therapy were selected. Three specimens were applied, one for each types of ligature, inside the oral cavity of each subject. Samples were kept in the oral cavity for 28 days, ligating 0.16 X 0.22 inches stainless steel archwires to stainless steel premolars brackets (Leone® S.p.A., Sesto Fiorentino, FI, Italy) for Bidimensional technique. After the pre-established time, the systems of ligature were removed and washed. Control group consisted of 9 unused specimens of each ligation type. Each elastomeric ligature was observed under the scanning electron microscope (SEM) to determine variations in size. The archwire-bracket-ligature complex was also analyzed. Transparent O-ring ligatures showed significant volumetric and structural changes. The external rounded shape was rather maintained, while the internal shape tended to appear square. Both external and internal diameter significantly increased (pO-ring ligatures endure significant volumetric and structural changes, after clinical use, index of a greater degree of friction and early loss in functionality. Grey, polyurethane Slide low-friction ligatures presented limited variation in size after clinical use.

  3. Synthesis of heteroglycoclusters by using orthogonal chemoselective ligations

    Directory of Open Access Journals (Sweden)

    Baptiste Thomas

    2012-03-01

    Full Text Available Synthetic heteroglycoclusters are being subjected to increasing interest due to their potential to serve as selective ligands for carbohydrate-binding proteins. In this paper, we describe an expedient strategy to prepare cyclopeptides displaying well-defined distributions and combinations of carbohydrates. By using both oxime ligation and copper(I-catalyzed alkyne–azide cycloaddition, two series of compounds bearing binary combinations of αMan, αFuc or βLac in an overall tetravalent presentation, and either 2:2 or 3:1 relative proportions, have been prepared.

  4. Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation

    OpenAIRE

    Ulrich Arnold; Hinderaker, Matthew P.; Raines, Ronald T.

    2002-01-01

    The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94) fragment possessed a C-terminal thioester. A pepti...

  5. Self-ligating vs conventional brackets as perceived by orthodontists.

    Science.gov (United States)

    Prettyman, Chase; Best, Al M; Lindauer, Steven J; Tufekci, Eser

    2012-11-01

    To determine if there are significant clinical differences between self-ligating brackets (SLB) and conventional brackets (CB) during orthodontic treatment, as perceived by orthodontists. A survey was developed and distributed to evaluate how SLB compare to CB in terms of orthodontists' perceptions (n  =  430). SLB were preferred during the initial stage of treatment based on the shorter adjustment appointments and faster initial treatment progress they provided (P orthodontists' preference was significantly influenced by (1) the proportion of patients treated with SLB (P < .0001), (2) the number of cases it took them to become accustomed to SLB (P < .0001), and (3) the average appointment intervals associated with SLB (P < .0001).

  6. Ionic strength of electrospray droplets affects charging of DNA oligonucleotides.

    Science.gov (United States)

    Xu, Ning; Chingin, Konstantin; Chen, Huanwen

    2014-01-01

    The fundamental aspects of charging in electrospray ionization (ESI) are hotly debated. In the present study, ESI charging of DNA oligonucleotides was explored in both positive (ESI+) and negative (ESI-) polarity using mass spectrometry detection. Single-stranded 12-mer CCCCAATTCCCC in buffer solution (aqueous NH4Ac, 100 mM) produced similar charge state distribution (CSD) in either ESI+ or ESI-. Similarity of CSD in ESI+ and ESI- was also observed for the double-stranded 12-mer CGCGAATTCGCG. By adding typical low-vapor reagents (e.g. m-nitro benzyl alcohol, m-NBA; sulfolane) into the same buffer solution (sulfolane or m-NBA, the CGCGAATTCGCG duplex dissociated into single strands in ESI-. No SC was observed in both ESI+ and ESI- for thermally denatured CGCGAATTCGCG duplex in NH4 Ac buffer without the reagents. These findings are difficult to reconcile with the earlier model, which attributes SC in aqueous buffer solution to the conformational changes of analytes. Our observations suggest that the ionic strength of ESI droplets strongly affects the CSD of biopolymers such as DNA oligonucleotides and that SC effect is related to the depletion of ionic strength during the ESI process. Copyright © 2014 John Wiley & Sons, Ltd.

  7. RNase H sequence preferences influence antisense oligonucleotide efficiency.

    Science.gov (United States)

    Kielpinski, Lukasz J; Hagedorn, Peter H; Lindow, Morten; Vinther, Jeppe

    2017-11-08

    RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA-DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides. The sequences preferred by HIV-1 RNase H are distributed in the HIV genome in a way suggesting selection for efficient RNA cleavage during replication. Our findings can be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H may have shaped evolution of the viral genome and contributed to the use of tRNA-Lys3 as primer during viral replication. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Multiplex pairwise assembly of array-derived DNA oligonucleotides.

    Science.gov (United States)

    Klein, Jason C; Lajoie, Marc J; Schwartz, Jerrod J; Strauch, Eva-Maria; Nelson, Jorgen; Baker, David; Shendure, Jay

    2016-03-18

    While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Synthesis of peptide-oligonucleotide conjugates for chromium coordination.

    Science.gov (United States)

    Civitello, E R; Leniek, R G; Hossler, K A; Haebe, K; Stearns, D M

    2001-01-01

    The synthesis of the first peptide-oligonucleotide conjugate designed to coordinate chromium(III) is reported. The overall goal of this work is to synthesize di-deoxynucleotides tethered with chromium(III)-coordinating appendages to model chromium-DNA-protein cross-links, which are a type of DNA lesion that may be involved in chromium-induced cancers. The conjugate dGp(NHCH(2)CH(2)S-Ac-Gly-Ser-Gly-OH)G was made by coupling the peptide, ClAc-Gly-Ser-Gly-OH, and dinucleotide, dGp(NHCH(2)CH(2)SH)G, through a thioether moiety. The conjugate was characterized by HPLC and mass spectrometry. Previously reported methods for small-scale solid-phase synthesis of peptides and dinucleotide were unsuitable; therefore, gram-scale solution-phase methods were developed. We also report the gram-scale syntheses of two other serine-containing peptides, ClAc-betaAla-Ser-Gly-OH and ClAc-Ser-Gly-OH, and three histidine-containing peptides, ClAc-Gly-His-Gly-OH, ClAc-betaAla-His-Gly-OH, and ClAc-His-Gly-OH. The synthesis and characterization of chromium-containing peptide-oligonucleotide conjugates will ultimately help us to understand chromium-DNA interactions at a molecular level, which is necessary before we can determine how chromium causes cancer.

  10. The use of oligonucleotide probes for meningococcal serotype characterization

    Directory of Open Access Journals (Sweden)

    SACCHI Claudio Tavares

    1998-01-01

    Full Text Available In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs. Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A and 615U (VR3-B used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

  11. Sense oligonucleotide competition for gene promoter binding and activation.

    Science.gov (United States)

    Cutroneo, Kenneth R; Chiu, Jen-Fu

    2003-01-01

    Considerable evidence has ensued on the importance of growth factors during regeneration both for cell replication and for stimulation of reparative cells to synthesize and secrete extracellular matrix components. During the healing process if the growth factor concentration is too high because of over-expression, abnormal wound healing and tissue fibrosis will occur. The growth factor concentration at the wound site may be controlled by gene therapy and the titration of gene dosage. However, if there is a narrow window between the beneficial effects and adverse effects of gene therapy, oligonucleotide approaches may be used concurrently with gene therapy to control growth factor concentration(s) at the wound site. Antisense oligos offer a method to control the concentration of growth factors at the level of translation. A novel method using sense oligos to the proalpha1 (I) collagen gene to inhibit gene transcription and collagen synthesis has recently been reported. The exogenous modified oligodeoxynucleotide competes with the cis-element (i.e. the transforming growth factor-beta (TGF-beta) element) in the distal 5'-flanking region of the proalpha1 (I) collagen gene for the trans-acting factor (i.e. the TGF-beta activator protein complex), thereby down regulating promoter activity of the proalpha1 (I) collagen gene and inhibiting type I collagen synthesis. The oligonucleotide approaches, both antisense and sense therapies, may be used to regulate over-expression of growth factors and thereby either eliminate or lessen the potential adverse effects of gene therapy.

  12. Adsorption of DNA oligonucleotides by titanium dioxide nanoparticles.

    Science.gov (United States)

    Zhang, Xu; Wang, Feng; Liu, Biwu; Kelly, Erin Y; Servos, Mark R; Liu, Juewen

    2014-01-28

    Titanium dioxide (TiO2) or titania shows great promise in detoxification and drug delivery. To reach its full potential, it is important to interface TiO2 with biomolecules to harness their molecular recognition function. To this end, DNA attachment is an important topic. Previous work has mainly focused on long double-stranded DNA or single nucleotides. For biosensor development and targeted drug delivery, it is more important to use single-stranded oligonucleotides. Herein, the interaction between fluorescently labeled oligonucleotides and TiO2 nanoparticles is reported. The point of zero charge (PZC) of TiO2 is around 6 in water or acetate buffer; therefore, the particles are positively charged at lower pH. However, if in phosphate or citrate buffer, the particles are negatively charged, even at pH ∼2, suggesting strong adsorption of buffer anions. DNA adsorption takes place mainly via the phosphate backbone, although the bases might also have moderate contributions. Peptide nucleic acids (PNAs) with an amide backbone cannot be adsorbed. DNA adsorption is strongly affected by inorganic anions, where phosphate and citrate can strongly inhibit DNA adsorption. DNA adsorption is promoted by adding salt or lowering pH. DNA adsorption is accompanied with fluorescence quenching, and double-stranded DNA showed reduced quenching, allowing for the detection of DNA using TiO2 nanoparticles.

  13. Evaluation of cerebral electrical activity and cardiac output after patent ductus arteriosus ligation in preterm infants.

    LENUS (Irish Health Repository)

    Leslie, A T F S

    2013-11-01

    To characterize and investigate the relationship between systemic blood flow and pre- and postoperative cerebral electrical activity in preterm neonates undergoing patent ductus arteriosus (PDA) ligation.

  14. Identification of rifampin-resistant mycobacterium tuberculosis strains by hybridization, PCR, and ligase detaction reaction on oligonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Mikhailovich, V.; Lapa, S.; Gryadunov, D.; Sobolev, A.; Strizhkov, B.; Chernyh, N.; Skotnikova, O.; Irtuganova, O.; Moroz, A.; Litvinov, V.; Vladimirskii, M.; Perelman, M.; Chernousova, L.; Erokhin, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences; Moscow Antituberculosis Center; Moscow Medical Academy; Russian Academy of Medical Sciences

    2001-07-01

    Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches -- hybridization, PCR, and ligase detection reaction -- were designed to analyze an 81-bp fragment of the gene rpoB encoding the {beta}-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

  15. Detection of human papillomaviruses by polymerase chain reaction and ligation reaction on universal microarray.

    Directory of Open Access Journals (Sweden)

    Jarmo Ritari

    Full Text Available Sensitive and specific detection of human papillomaviruses (HPV in cervical samples is a useful tool for the early diagnosis of epithelial neoplasia and anogenital lesions. Recent studies support the feasibility of HPV DNA testing instead of cytology (Pap smear as a primary test in population screening for cervical cancer. This is likely to be an option in the near future in many countries, and it would increase the efficiency of screening for cervical abnormalities. We present here a microarray test for the detection and typing of 15 most important high-risk HPV types and two low risk types. The method is based on type specific multiplex PCR amplification of the L1 viral genomic region followed by ligation detection reaction where two specific ssDNA probes, one containing a fluorescent label and the other a flanking ZipCode sequence, are joined by enzymatic ligation in the presence of the correct HPV PCR product. Human beta-globin is amplified in the same reaction to control for sample quality and adequacy. The genotyping capacity of our approach was evaluated against Linear Array test using cervical samples collected in transport medium. Altogether 14 out of 15 valid samples (93% gave concordant results between our test and Linear Array. One sample was HPV56 positive in our test and high-risk positive in Hybrid Capture 2 but remained negative in Linear Array. The preliminary results suggest that our test has accurate multiple HPV genotyping capability with the additional advantages of generic detection format, and potential for high-throughput screening.

  16. Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

    Science.gov (United States)

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

    New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

  17. Phosphorothioate oligonucleotides induction into experimental choroidal neovascularization by HVJ-liposome system.

    Science.gov (United States)

    Ogata, N; Otsuji, T; Matsushima, M; Kimoto, T; Yamanaka, R; Takahashi, K; Wada, M; Uyama, M; Kaneda, Y

    1999-04-01

    The purpose of this study was to determine whether the inactivated hemagglutinating virus of Japan (HVJ)-liposome method can induce phosphorothioate oligonucleotides effectively into an experimentally-induced choroidal neovascularization of rats. We also examined whether antisense phosphorothioate oligonucleotides against VEGF could be induced into choroidal neovascularization as a therapeutic agent by the HVJ-liposome method. The experiments were conducted on a rat model of choroidal neovascularization. FITC-labeled phosphorothioate oligonucleotides were coencapsulated in liposomes. The liposomes were coated with the envelope of inactivated HVJ and injected into the vitreous cavity following photocoagulation of pigmented rat eyes. The eyes were removed following injection, fixed, frozen and cut into thin sections. Induction of oligonucleotides was observed under a laser confocal scanning microscope for fluorescence and the development of choroidal neovascularization was evaluated histopathologically. Phosphorothioate oligonucleotides were effectively induced into ganglion cells and into the cells of the choroidal neovascularization induced by laser photocoagulation. Highly effective induction of oligos was observed 3 to 14 days after intravitreal injection of HVJ-liposomes after which the level decreased. Antisense oligonucleotides against VEGF were induced specifically into cells in the choroidal neovascularization, however neovascularization was still observed. Phosphorothioate oligonucleotides can be effectively induced into ganglion cells, and specifically into cells in choroidal neovascularization. Although antisense oligonucleotides against VEGF failed to prevent choroidal neovascularization, the HVJ-liposome method provided a highly effective means of inducing antisense oligos for in vivo antisense therapy.

  18. Sortase-Mediated Ligation of Purely Artificial Building Blocks

    Directory of Open Access Journals (Sweden)

    Xiaolin Dai

    2018-02-01

    Full Text Available Sortase A (SrtA from Staphylococcus aureus has been often used for ligating a protein with other natural or synthetic compounds in recent years. Here we show that SrtA-mediated ligation (SML is universally applicable for the linkage of two purely artificial building blocks. Silica nanoparticles (NPs, poly(ethylene glycol and poly(N-isopropyl acrylamide are chosen as synthetic building blocks. As a proof of concept, NP–polymer, NP–NP, and polymer–polymer structures are formed by SrtA catalysis. Therefore, the building blocks are equipped with the recognition sequence needed for SrtA reaction—the conserved peptide LPETG—and a pentaglycine motif. The successful formation of the reaction products is shown by means of transmission electron microscopy (TEM, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS, and dynamic light scattering (DLS. The sortase catalyzed linkage of artificial building blocks sets the stage for the development of a new approach to link synthetic structures in cases where their synthesis by established chemical methods is complicated.

  19. Longer treatment times with self-ligated orthodontic brackets.

    Science.gov (United States)

    O'Brien, Kevin

    2014-09-01

    The Medline, Cochrane Library, Biomed Central, BBO including LILACS, Ind Med, Sceilo, Clinical trials.gov, Conference paper Index, Digital Dissertations, German National Library of Medicine (ZB MED), Google Scholar, ISI Web of Knowledge, metaRegister of Controlled Trials, OpenSIGLE and Scirus databases were searched. Randomised controlled trials (RCTs) and quasi-RCTs in patients having fixed-appliance orthodontic treatments were considered. Study assessment data extraction and risk of bias assessment was carried out independently by two reviewers. Overall quality of evidence was based on the Grades of Recommendation Assessment, Development and Evaluation (GRADE) approach. Random-effects meta-analyses were performed where data could be pooled. Twenty five trials (1321 patients) were included. The majority (24) compared self-ligated (SL) and conventional brackets (CL). No trials primarily investigated the effect of bracket material and no indirect comparison was possible. Two trials assessed the bracket slot size but found no consistent difference between 0.022'' and 0.018'' brackets. Four studies contributed to a meta-analysis that showed overall duration of the orthodontic treatment be significantly longer in the SL group by 2.01 months (95%CI; 0.45 to 3.57). Based on existing evidence, no clinical recommendation can be made regarding the bracket material or different ligation modules. For Sl brackets, no conclusive benefits could be proven, while their use was associated with longer treatment durations.

  20. Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation

    Directory of Open Access Journals (Sweden)

    Ulrich Arnold

    2002-01-01

    Full Text Available The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94 fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95–124 of RNase A (with residue 95 being cysteine was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1–124–intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.

  1. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

    Science.gov (United States)

    Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

    2015-11-16

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. © 2015 Zambirinis et al.

  2. Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

    Science.gov (United States)

    Kozlowska, Anna Karolina; Florczak, Anna; Smialek, Maciej; Dondajewska, Ewelina; Mackiewicz, Andrzej; Kortylewski, Marcin; Dams-Kozlowska, Hanna

    2017-09-01

    Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel

  3. Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

    DEFF Research Database (Denmark)

    Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

    2017-01-01

    Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

  4. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-01

    of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual...

  5. Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA.

    Science.gov (United States)

    Varizhuk, Anna M; Zatsepin, Timofei S; Golovin, Andrey V; Belyaev, Evgeny S; Kostyukevich, Yury I; Dedkov, Vladimir G; Shipulin, German A; Shpakovski, George V; Aralov, Andrey V

    2017-07-15

    Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry.

    Science.gov (United States)

    Meng, Zhaojing; Limbach, Patrick A

    2005-01-01

    Nozzle-skimmer dissociation in combination with de novo sequencing was investigated as an approach for increasing the throughput of oligonucleotide analysis attainable by electrospray ionization mass spectrometry. An experimental method allowing for the sequential generation of precursor and fragment ion data during direct infusion of sample was developed. These data can then be used with readily available de novo sequencing software to characterize small oligonucleotides. When this approach was applied to mixtures of oligonucleotides, it was found that de novo sequencing becomes limited due to spectral congestion and overlapping oligonucleotide m/z dissociation product values. Self-packed C(18) microspray emitters were investigated as a means of reducing spectral complexity. It was found that such emitters allow for the analysis of oligonucleotide mixtures with minimal component overlap, and these emitters provide additional benefits of pre- concentrating and desalting the sample. These developments can provide a route for the more rapid characterization of ribonucleic acid endonuclease digestion mixtures.

  7. Design of oligonucleotides for microarrays and perspectives for design of multi-transcriptome arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Wernersson, Rasmus; Knudsen, Steen

    2003-01-01

    with an overview of these parameters. We present here a flexible tool named OligoWiz for designing oligonucleotides for multiple purposes. OligoWiz presents a set of parameter scores in a graphical interface to facilitate an overview for the user. Additional custom parameter scores can easily be added......Optimal design of oligonucleotides for microarrays involves tedious and laborious work evaluating potential oligonucleotides relative to a series of parameters. The currently available tools for this purpose are limited in their flexibility and do not present the oligonucleotide designer...... to the program to extend the default parameters: homology, DeltaTm, low-complexity, position and GATC-only. Furthermore we present an analysis of the limitations in designing oligonucleotide sets that can detect transcripts from multiple organisms. OligoWiz is available at www.cbs.dtu.dk/services/OligoWiz/....

  8. Validation of a spectrophotometric method to estimate the adsorption on nanoemulsions of an antimalarial oligonucleotide

    Directory of Open Access Journals (Sweden)

    Fernanda Bruxel

    2011-09-01

    Full Text Available This study describes the validation of a spectrophotometric method to estimate oligonucleotides association with cationic nanoemulsions. Phosphodiester and phosphorothioate oligonucleotides targeting Plasmodium falciparum topoisomerase II were analyzed at 262 nm. Linear response (r > 0.998 was observed from 0.4 to 1.0 nmol/mL, the relative standard deviation values for the intra- and inter-days precision were lower than 2.6% and the recovery ranged from 98.8 to 103.6% for both oligonucleotides. The association efficiency was estimated based on an ultrafiltration/centrifugation method. Oligonucleotides recovery through 30 kDa-membranes was higher than 92%. The extent of oligonucleotides association (42 to 98% varied with the composition of nanoemulsions

  9. Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Michael J Lodes

    Full Text Available Bacterial and viral upper respiratory infections (URI produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

  10. Banding ligation versus beta-blockers as primary prophylaxis in esophageal varices

    DEFF Research Database (Denmark)

    Gluud, Lise L; Klingenberg, Sarah; Nikolova, Dimitrinka

    2007-01-01

    To compare banding ligation versus beta-blockers as primary prophylaxis in patients with esophageal varices and no previous bleeding.......To compare banding ligation versus beta-blockers as primary prophylaxis in patients with esophageal varices and no previous bleeding....

  11. Level of arterial ligation in total mesorectal excision (TME): An anatomical study

    NARCIS (Netherlands)

    M. Buunen (Mark); M.M. Lange (Marilyne); M. Ditzel (Max); G.J. Kleinrensink (Gert Jan); C.J.H. van de Velde (Cornelis); J.F. Lange (Johan)

    2009-01-01

    textabstractIntroduction: High-tie ligation is a common practice in rectal cancer surgery. However, it compromises perfusion of the proximal limb of the anastomosis. This anatomical study was designed to assess the value of low-tie ligation in order to obtain a tension-free anastomosis. Materials

  12. Modulation of collateral artery growth in a porcine hindlimb ligation model using MCP-1

    NARCIS (Netherlands)

    Voskuil, Michiel; van Royen, Niels; Hoefer, Imo E.; Seidler, Randolph; Guth, Brian D.; Bode, Christoph; Schaper, Wolfgang; Piek, Jan J.; Buschmann, Ivo R.

    2003-01-01

    For an appropriate extrapolation to patients with peripheral arterial obstructive disease, we tested the efficacy of monocyte chemoattractant protein 1 (MCP-1) treatment in a porcine hindlimb ligation model. In 40 minipigs, a femoral artery ligation was performed. Control animals were examined

  13. Tubal ligation and risk of ovarian cancer subtypes: a pooled analysis of case-control studies

    NARCIS (Netherlands)

    Sieh, W.; Salvador, S.; McGuire, V.; Weber, R.P.; Terry, K.L.; Rossing, M.A.; Risch, H.; Wu, A.H.; Webb, P.M.; Moysich, K.; Doherty, J.A.; Felberg, A.; Miller, D.; Jordan, S.J.; Goodman, M.T.; Lurie, G.; Chang-Claude, J.; Rudolph, A.; Kjaer, S.K.; Jensen, A.; Hogdall, E.; Bandera, E.V.; Olson, S.H.; King, M.G.; Rodriguez-Rodriguez, L.; Kiemeney, L.A.L.M.; Marees, T.; Massuger, L.F.A.G.; Altena, A.M. van; Ness, R.B.; Cramer, D.W; Pike, M.C.; Pearce, C.L.; Berchuck, A.; Schildkraut, J.M.; Whittemore, A.S.

    2013-01-01

    BACKGROUND: Tubal ligation is a protective factor for ovarian cancer, but it is unknown whether this protection extends to all invasive histological subtypes or borderline tumors. We undertook an international collaborative study to examine the association between tubal ligation and ovarian cancer

  14. En bloc ligation of renal vessels is safe and reduces duration of surgery

    DEFF Research Database (Denmark)

    Azawi, Nessn Htum; Hult, Mariam Annalisa Skibsted; Dahl, Claus

    2016-01-01

    INTRODUCTION: Conventionally, individual ligation of the renal vessels with clips is performed during laparoscopic nephrectomy (LN). Concomitant ligation of the vessels is not a standard procedure due to an expected risk of stapler dysfunction and the development of arteriovenous fistulas (AVF). ...

  15. The Effect of Various Ligation Methods on Friction in Sliding Mechanics

    Directory of Open Access Journals (Sweden)

    Amit Gupta

    2013-01-01

    Conclusion: Slide modules produce least friction followed by loose SS ligation, slick modules, regular modules, tight SS ligation and highest friction was produced by regular modules tied in a ′figure of 8′ pattern. Width of bracket had no influence on friction produced.

  16. Confinement-induced Molecular Templating and Controlled Ligation

    Science.gov (United States)

    Berard, Daniel; Shayegan, Marjan; Michaud, François; Henkin, Gil; Scott, Shane; Leith, Jason; Leslie, Sabrina; Leslie Lab Team

    Loading and manipulating long DNA molecules within sub-50 nm cross-section nanostructures for genomic and biochemical analyses, while retaining their structural integrity, present key technological challenges to the biotechnology sector, such as device clogging and molecular breakage. We overcome these challenges by using Convex Lens-induced Confinement (CLiC) technology to gently load DNA into nanogrooves from above. Here, we demonstrate single-fluorophore visualization of custom DNA barcodes as well as efficient top-loading of DNA into sub-50 nm nanogrooves of variable topographies. We study confinement-enhanced self-ligation of polymers loaded in circular nanogrooves. Further, we use concentric, circular nanogrooves to eliminate confinement gradient-induced drift of stretched DNA.

  17. MANAGEMENT OF INTERNAL HEMORRHOID WITH RUBBER BAND LIGATION PROCEDURE

    Directory of Open Access Journals (Sweden)

    I Made Arya Winangun

    2013-10-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Hemorrhoid is regarded as the cases most seen in population. The prevalence of this cases about 4,4% with the incidence 12 of 1.000 patient. The current management of hemorrhoid is lifestyle modification, conservative management such as farmacology, minimally invasive treatment and more aggressive therapy using surgical procedure. Rubber band ligation was one of the minimally invasive procedures. This procedure was easy, inexpensive, and can be done outpatient using simple tools without complicated procedure like hemorrhoidectomy. Some studies explained rubber band ligation effectively done in internal hemorrhoid grade II and grade III even this procedure still had minimal complication such as bleeding and unpleasentness /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  18. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  19. Development of multiple W/O/W emulsions as dermal carrier system for oligonucleotides: effect of additives on emulsion stability.

    Science.gov (United States)

    Schmidts, T; Dobler, D; Schlupp, P; Nissing, C; Garn, H; Runkel, F

    2010-10-15

    Multiple water-in-oil-in-water (W/O/W) emulsions are of major interest as potential skin delivery systems for water-soluble drugs like oligonucleotides due to their distinct encapsulation properties. However, multiple emulsions are highly sensitive in terms of variations of the individual components. The presence of osmotic active ingredients in the inner water phase is crucial for the generation of stable multiple emulsions. In order to stabilize the emulsions the influence of NaCl, MgSO(4), glucose and glycine and two cellulose derivatives was investigated. Briefly, multiple W/O/W emulsions using Span 80 as a lipophilic emulsifier and different hydrophilic emulsifiers (PEG-40/50 stearate, steareth-20 and polysorbate 80) were prepared. Stability of the emulsions was analyzed over a period of time using rheological measurements, droplet size observations and conductivity analysis. In this study we show that additives strongly influence the properties stability of multiple emulsions. By increasing the concentration of the osmotic active ingredients, smaller multiple droplets are formed and the viscosity is significantly increased. The thickening agents resulted in a slightly improved stability. The most promising emulsions were chosen and further evaluated for their suitability and compatibility to incorporate a DNAzyme oligonucleotide as active pharmaceutical ingredient. Copyright 2010 Elsevier B.V. All rights reserved.

  20. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    Directory of Open Access Journals (Sweden)

    Gbaj A

    2009-01-01

    Full Text Available The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5’-bispyrene and 3’-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5’-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

  1. Selection of long oligonucleotides for gene expression microarrays using weighted rank-sum strategy

    Directory of Open Access Journals (Sweden)

    Preiser Peter

    2007-09-01

    Full Text Available Abstract Background The design of long oligonucleotides for spotted DNA microarrays requires detailed attention to ensure their optimal performance in the hybridization process. The main challenge is to select an optimal oligonucleotide element that represents each genetic locus/gene in the genome and is unique, devoid of internal structures and repetitive sequences and its Tm is uniform with all other elements on the microarray. Currently, all of the publicly available programs for DNA long oligonucleotide microarray selection utilize various combinations of cutoffs in which each parameter (uniqueness, Tm, and secondary structure is evaluated and filtered individually. The use of the cutoffs can, however, lead to information loss and to selection of suboptimal oligonucleotides, especially for genomes with extreme distribution of the GC content, a large proportion of repetitive sequences or the presence of large gene families with highly homologous members. Results Here we present the program OligoRankPick which is using a weighted rank-based strategy to select microarray oligonucleotide elements via an integer weighted linear function. This approach optimizes the selection criteria (weight score for each gene individually, accommodating variable properties of the DNA sequence along the genome. The designed algorithm was tested using three microbial genomes Escherichia coli, Saccharomyces cerevisiae and the human malaria parasite species Plasmodium falciparum. In comparison to other published algorithms OligoRankPick provides significant improvements in oligonucleotide design for all three genomes with the most significant improvements observed in the microarray design for P. falciparum whose genome is characterized by large fluctuations of GC content, and abundant gene duplications. Conclusion OligoRankPick is an efficient tool for the design of long oligonucleotide DNA microarrays which does not rely on direct oligonucleotide exclusion by

  2. Solid-phase synthesis of 2{sup '}-O-methoxyethyl oligonucleotides using dimeric phosphoramidate blocks

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Gi Weon; Kang, Yong Han [Dept. of Applied Chemistry, Hanyang University, Ansan (Korea, Republic of)

    2016-11-15

    This research focused on the method of using dimeric phosphoramidite blocks to synthesize oligonucleotides for development as oligonucleotide drugs. A 16-mer oligonucleotide with the randomly selected sequence of C*C*T*C*G*C *T*C*T*C*G*C*C* C*G*C was synthesized using CC, GC, and TC dimers, a combination of monomers and dimers, or only monomers as building blocks. Using dimer blocks in this synthetic method provided a significant decrease in critical impurities that had similar properties to the main product, which was confirmed by LC-MS and HPLC analysis.

  3. Synthesis and properties of cholesteryl-modified triple-helix forming oligonucleotides containing a triglycyl linker.

    Science.gov (United States)

    Vu, H; Hill, T S; Jayaraman, K

    1994-01-01

    In order to enhance the nuclear uptake of triple-helix forming oligonucleotides (TFOs), a triglycylcholesterol group was attached to the 3' end. The peptide unit was introduced as a "labile" linker with the aim of releasing the oligonucleotide from the endosomes by the action of peptidases after crossing the cell membrane. Cholesteryl-CPG (8) and -TentaGel (9) supports containing 2-[N-(glycylglycylglycyl)amino]propane-1,3-diol (GAP-3) linker were prepared and used for automated oligonucleotide synthesis. The synthesis, characterization, and stability of these compounds are described.

  4. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo

    Science.gov (United States)

    Simons, Michael; Edelman, Elazer R.; Dekeyser, Jean-Luc; Langer, Robert; Rosenberg, Robert D.

    1992-09-01

    SYNTHETIC antisense oligonucleotides have been used to dissect gene function in vitro. Technical difficulties prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense c-myb oligonu-cleotide to suppress intimal accumulation of rat carotid arterial smooth muscle cells. Our results suggest that antisense oligonucleotides can be used to define the in vivo biological role of specific macromolecules in the blood vessel wall and could potentially serve as a new class of therapeutic agents for cardiovascular disorders.

  5. Synthetic Method for Oligonucleotide Block by Using Alkyl-Chain-Soluble Support.

    Science.gov (United States)

    Matsuno, Yuki; Shoji, Takao; Kim, Shokaku; Chiba, Kazuhiro

    2016-02-19

    A straightforward method for the synthesis of oligonucleotide blocks using a Cbz-type alkyl-chain-soluble support (Z-ACSS) attached to the 3'-OH group of 3'-terminal nucleosides was developed. The Z-ACSS allowed for the preparation of fully protected deoxyribo- and ribo-oligonucleotides without chromatographic purification and released dimer- to tetramer-size oligonucleotide blocks via hydrogenation using a Pd/C catalyst without significant loss or migration of protective groups such as 5'-end 4,4'-dimethoxtrityl, 2-cyanoethyl on internucleotide bonds, or 2'-TBS.

  6. An in vivo Study on Bacterial Colonization with Metal, Ceramic and Self-ligating Brackets: A Scanning Electron Microscopy Study

    Directory of Open Access Journals (Sweden)

    Aravind S Raju

    2013-01-01

    Conclusion: This study highlights that higher retention of plaque in ceramic brackets ligated with elastomeric ring followed with metal brackets ligated with steel ligatures and comparatively less plaque retention in self-ligating brackets. Excess composite around the bracket base is the critical site of plaque accumulation associated with fixed appliances due to its rough surface texture.

  7. Application of graphene–pyrenebutyric acid nanocomposite as probe oligonucleotide immobilization platform in a DNA biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xuan; Gao, Feng; Cai, Xili; Zheng, Meixia [Department of Chemistry and Environment Science, Fujian Province University Key Laboratory of Analytical Science, Zhangzhou Normal University, Zhangzhou 363000 (China); Gao, Fei, E-mail: fgao@fjzs.edu.cn [Department of Chemistry and Environment Science, Fujian Province University Key Laboratory of Analytical Science, Zhangzhou Normal University, Zhangzhou 363000 (China); Jiang, Shulian [Zhangzhou Product Quality Supervision and Inspection Institute, Zhangzhou 363000 (China); Wang, Qingxiang, E-mail: axiang236@126.com [Department of Chemistry and Environment Science, Fujian Province University Key Laboratory of Analytical Science, Zhangzhou Normal University, Zhangzhou 363000 (China)

    2013-10-15

    A stable and uniform organic–inorganic nanocomposite that consists of graphene (GR) and pyrenebutyric acid (PBA) was obtained by ultrasonication, which was characterized by scanning electron microscopy (SEM) and UV–vis absorption spectra. The dispersion was dropped onto a gold electrode surface to obtain GR–PBA modified electrode (GR–PBA/Au). Electrochemical behaviors of the modified electrode were characterized by cyclic voltammetry and electrochemical impedance spectroscopy using [Fe(CN){sub 6}]{sup 3−/4−} as the electroactive probe. A novel DNA biosensor was constructed based on the covalent coupling of amino modified oligonucleotides with the carboxylic group on PBA. By using methylene blue (MB) as a redox-active hybridization indicator, the biosensor was applied to electrochemically detect the complementary sequence, and the results suggested that the peak currents of MB showed a good linear relationship with the logarithm values of target DNA concentrations in the range from 1.0 × 10{sup −15} to 5.0 × 10{sup −12} M with a detection limit of 3.8 × 10{sup −16} M. The selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences. - Highlights: • A nanocomposite containing graphene and pyrenebutyric acid was prepared. • The nanocomposite was applied as a function platform for DNA immobilization platform. • The developed biosensor shows excellent selectivity and sensitivity for target DNA detection.

  8. A Statistical Thermodynamic Model for Investigating the Stability of DNA Sequences from Oligonucleotides to Genomes

    Science.gov (United States)

    Khandelwal, Garima; Lee, Rebecca A.; Jayaram, B.; Beveridge, David L.

    2014-01-01

    We describe the development and testing of a simple statistical mechanics methodology for duplex DNA applicable to sequences of any composition and extensible to genomes. The microstates of a DNA sequence are modeled in terms of blocks of basepairs that are assumed to be fully closed (paired) or open. This approach generates an ensemble of bubblelike microstates that are used to calculate the corresponding partition function. The energies of the microstates are calculated as additive contributions from hydrogen bonding, basepair stacking, and solvation terms parameterized from a comprehensive series of molecular dynamics simulations including solvent and ions. Thermodynamic properties and nucleotide stability constants for DNA sequences follow directly from the partition function. The methodology was tested by comparing computed free energies per basepair with the experimental melting temperatures of 60 oligonucleotides, yielding a correlation coefficient of −0.96. The thermodynamic stability of genic/nongenic regions was tested in terms of nucleotide stability constants versus sequence for the Escherichia coli K-12 genome. It showed clear differentiation of the genes from promoters and captures genic regions with a sensitivity of 0.94. The statistical thermodynamic model presented here provides a seemingly new handle on the challenging problem of interpreting genomic sequences. PMID:24896126

  9. Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

    Science.gov (United States)

    Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

    2015-09-01

    We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

  10. A statistical thermodynamic model for investigating the stability of DNA sequences from oligonucleotides to genomes.

    Science.gov (United States)

    Khandelwal, Garima; Lee, Rebecca A; Jayaram, B; Beveridge, David L

    2014-06-03

    We describe the development and testing of a simple statistical mechanics methodology for duplex DNA applicable to sequences of any composition and extensible to genomes. The microstates of a DNA sequence are modeled in terms of blocks of basepairs that are assumed to be fully closed (paired) or open. This approach generates an ensemble of bubblelike microstates that are used to calculate the corresponding partition function. The energies of the microstates are calculated as additive contributions from hydrogen bonding, basepair stacking, and solvation terms parameterized from a comprehensive series of molecular dynamics simulations including solvent and ions. Thermodynamic properties and nucleotide stability constants for DNA sequences follow directly from the partition function. The methodology was tested by comparing computed free energies per basepair with the experimental melting temperatures of 60 oligonucleotides, yielding a correlation coefficient of -0.96. The thermodynamic stability of genic/nongenic regions was tested in terms of nucleotide stability constants versus sequence for the Escherichia coli K-12 genome. It showed clear differentiation of the genes from promoters and captures genic regions with a sensitivity of 0.94. The statistical thermodynamic model presented here provides a seemingly new handle on the challenging problem of interpreting genomic sequences. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  12. Practical biophysics: Sensors for rapid detection of biological targets utilizing target-induced oligonucleotide annealing.

    Science.gov (United States)

    Heyduk, Tomasz

    2010-10-01

    Detection and quantitation of biomolecules is one of the most commonly performed measurements in biomedical research and clinical diagnostics. There is high demand for convenient, rapid and sensitive biomolecule detection methodologies. In this review we discuss a family of sensors that have been developed in our laboratory that share a common simple biophysical mechanism of action and that are capable of rapid detection of a diverse range of biological targets. The sensors generate fluorescence signal in the presence of the target molecule through target-induced association of short fluorochrome-labeled complementary oligonucleotides that are attached to target recognition elements of the sensors (antibodies, aptamers, etc.) via nanometer scale flexible linkers. This sensor design can be used for detecting proteins, antibodies, nucleic acids and whole cells. The assays using these sensors require only adding a sample to the sensor mix followed by simple fluorescence intensity readout. The simplicity, the speed of detection and the potential for miniaturization are the main assets of these sensors. 2010 Elsevier B.V. All rights reserved.

  13. Persistent postpartum haemorrhage after failed arterial ligation: value of pelvic embolisation

    Energy Technology Data Exchange (ETDEWEB)

    Fargeaudou, Yann; Soyer, Philippe; Sirol, Marc; Boudiaf, Mourad; Dahan, Henri; Dref, Olivier le [Hopital Lariboisiere AP-HP et Universite Diderot-Paris 7, Department of Abdominal and Interventional Imaging, Paris (France); Morel, Olivier; Barranger, Emmanuel [Hopital Lariboisiere AP-HP, Department of Obstetrics and Gynecology, Paris (France); Gayat, Etienne; Mebazaa, Alexandre [Hopital Lariboisiere AP-HP, Department of Anesthesiology and Intensive Care Medicine, Paris (France)

    2010-07-15

    To evaluate the role and efficacy of pelvic embolisation in the treatment of persistent postpartum haemorrhage after failed arterial ligation and to identify the complications of this procedure in this specific population. The clinical files and angiographic examinations of 12 consecutive women (mean age 32 years) who were treated with pelvic embolisation because of persistent, severe postpartum haemorrhage after failed arterial ligation were reviewed. Angiography revealed that persistent bleeding was due to incomplete arterial ligation (n = 4) or the presence of newly developed anastomotic routes (n = 8). In 11 women, pelvic embolisation stopped the bleeding. Hysterectomy was needed in one woman with retained placenta. Two complications due to pelvic embolisation, including leg ischaemia and transient sciatic nerve ischaemia, were identified, both after internal iliac artery ligation. In women with persistent postpartum haemorrhage after failed arterial ligation, pelvic embolisation is an effective treatment in most cases. However, embolisation of the anastomotic routes that contribute to persistent bleeding may result in ischaemic complications. These potential complications reaffirm that arterial ligation should not be the favoured option for postpartum haemorrhage and that special care must be given during pelvic embolisation after failed arterial ligation. (orig.)

  14. Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids.

    Science.gov (United States)

    Paris, Clément; Brun, Omar; Pedroso, Enrique; Grandas, Anna

    2015-04-10

    This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  15. Antiproliferative effects of antisense oligonucleotides directed to the RNA of c-myc oncogene.

    Science.gov (United States)

    Degols, G; Leonetti, J P; Mechti, N; Lebleu, B

    1991-01-01

    Several groups have reported the use of antisense oligonucleotides to inhibit c-myc gene expression and study its biological role. However high concentrations of free oligonucleotides were generally needed. To lower their concentration and stabilize the antisense effect against c-myc, oligonucleotides were covalently linked to poly(L-lysine) and administered in ternary complexes formed with heparin (100 micrograms/ml). A sequence specific growth inhibition was observed at concentrations lower than 1 microM, while oligonucleotide-poly(L-lysine) conjugates alone were inefficient. Similar results occurred with other polyanionic compounds. Inhibition of proliferation was correlated to a reduction of c-myc protein and to a transient decrease in c-myc mRNA level. However, implication of RNase H in this process could not be demonstrated. Images PMID:1708128

  16. Exploiting Protected Maleimides to Modify Oligonucleotides, Peptides and Peptide Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Clément Paris

    2015-04-01

    Full Text Available This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  17. Terminalia Chebula provides protection against dual modes of necroptotic and apoptotic cell death upon death receptor ligation

    Science.gov (United States)

    Lee, Yoonjung; Byun, Hee Sun; Seok, Jeong Ho; Park, Kyeong Ah; Won, Minho; Seo, Wonhyoung; Lee, So-Ra; Kang, Kidong; Sohn, Kyung-Cheol; Lee, Ill Young; Kim, Hyeong-Geug; Son, Chang Gue; Shen, Han-Ming; Hur, Gang Min

    2016-01-01

    Death receptor (DR) ligation elicits two different modes of cell death (necroptosis and apoptosis) depending on the cellular context. By screening a plant extract library from cells undergoing necroptosis or apoptosis, we identified a water extract of Terminalia chebula (WETC) as a novel and potent dual inhibitor of DR-mediated cell death. Investigation of the underlying mechanisms of its anti-necroptotic and anti-apoptotic action revealed that WETC or its constituents (e.g., gallic acid) protected against tumor necrosis factor-induced necroptosis via the suppression of TNF-induced ROS without affecting the upstream signaling events. Surprisingly, WETC also provided protection against DR-mediated apoptosis by inhibition of the caspase cascade. Furthermore, it activated the autophagy pathway via suppression of mTOR. Of the WETC constituents, punicalagin and geraniin appeared to possess the most potent anti-apoptotic and autophagy activation effect. Importantly, blockage of autophagy with pharmacological inhibitors or genetic silencing of Atg5 selectively abolished the anti-apoptotic function of WETC. These results suggest that WETC protects against dual modes of cell death upon DR ligation. Therefore, WETC might serve as a potential treatment for diseases characterized by aberrantly sensitized apoptotic or non-apoptotic signaling cascades. PMID:27117478

  18. Postpartum Permanent Sterilization: Could Bilateral Salpingectomy Replace Bilateral Tubal Ligation?

    Science.gov (United States)

    Danis, Rachel B; Della Badia, Carl R; Richard, Scott D

    2016-01-01

    There has recently been an expansion in the use of bilateral salpingectomy at the time of sterilization to theoretically decrease ovarian cancer risk. We sought to determine if postpartum salpingectomy is equivalent to postpartum bilateral tubal ligation (BTL) in terms of duration, estimated blood loss (EBL), and complication rate. A retrospective case series (Canadian Task Force Classification II-2). An academic inner-city hospital. All patients admitted for delivery of full-term intrauterine pregnancy desiring permanent sterilization between March 2014 and March 2015 were included. Excluded patients included those who had sterilization at the time of the cesarean section or other surgical procedure. Two cohorts were identified, those who had a planned postpartum tubal ligation and those having a postpartum salpingectomy. Postpartum sterilization. Researchers of this study recorded demographics, medical histories, and abdominal surgical histories for all patients who met the inclusion criteria. Surgical times, EBL, and complication rates were reviewed. Unpaired t test calculations were used to identify differences between age, body mass index, parity, and surgical time between the 2 cohorts. Chi-square tests were used to determine the statistical significance between complication rates, history of abdominal surgery, and past medical history of tubal disease between the 2 cohorts. Eighty women were identified, 64 in the BTL group and 16 in the salpingectomy cohort. The demographics of each cohort were equivocal. The average surgical time was 59.13 and 71.44 minutes in the BTL and salpingectomy cohorts, respectively. Of the 80 patients, only 1 had an EBL greater than 50 mL; this patient was in the BTL group. Four complications were noted in the BTL cohort, but none were evident in the salpingectomy group. There were no documented sterilization failures in the follow-up period (median = 9 months). Postpartum salpingectomy is slightly longer in duration but with

  19. Direct in situ hybridization with oligonucleotide functionalized quantum dot probes.

    Science.gov (United States)

    Bentolila, Laurent A

    2010-01-01

    Coming from the material sciences, fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have emerged as powerful fluorescent probes for a wide range of biological imaging applications. QDs have several advantages over organic dyes which include higher brightness, better resistance to photobleaching, and simplified multicolor target detection. In this chapter, we describe a rapid assay for the direct imaging of multiple repetitive subnuclear genetic sequences using QD-based FISH probes. Streptavidin-coated QDs (SAvQDs) are functionalized with short biotinylated oligonucleotides and used in a single hybridization/detection step. These QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and show good targeting of dense chromatin domains. Importantly, the broad absorption spectra of QDs allows two sequence specific QD-FISH probes of different colors to be simultaneously imaged with a single laser excitation wavelength. This method, which requires minimal custom conjugation, is easily expandable and offers the experimentalist a new alternative to increase flexibility in multicolor cytogenetic FISH applications of repetitive DNAs.

  20. Linear model for fast background subtraction in oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Barkema Gerard T

    2009-11-01

    Full Text Available Abstract Background One important preprocessing step in the analysis of microarray data is background subtraction. In high-density oligonucleotide arrays this is recognized as a crucial step for the global performance of the data analysis from raw intensities to expression values. Results We propose here an algorithm for background estimation based on a model in which the cost function is quadratic in a set of fitting parameters such that minimization can be performed through linear algebra. The model incorporates two effects: 1 Correlated intensities between neighboring features in the chip and 2 sequence-dependent affinities for non-specific hybridization fitted by an extended nearest-neighbor model. Conclusion The algorithm has been tested on 360 GeneChips from publicly available data of recent expression experiments. The algorithm is fast and accurate. Strong correlations between the fitted values for different experiments as well as between the free-energy parameters and their counterparts in aqueous solution indicate that the model captures a significant part of the underlying physical chemistry.

  1. Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

    Directory of Open Access Journals (Sweden)

    Tomoko Nishina

    2015-01-01

    Full Text Available We developed an efficient system for delivering short interfering RNA (siRNA to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid-DNA gapmer antisense oligonucleotide (ASO was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS molecules are bound to ASO with UNA sequences.

  2. Alternative interpretations of the oligonucleotide transport literature: insights from nature.

    Science.gov (United States)

    Wu-Pong, S

    2000-10-31

    Elucidation of the mechanism of oligonucleotide (ON) cellular internalization has met an impasse at the lipid penetration stage. ON internalization is commonly regarded to involve endocytosis, yet the method by which the ON penetrates the endosome membrane remains a mystery despite more than 10 years of research by multiple laboratories. In addition, the literature regarding this topic is fraught with discrepancies and inconsistencies. Therefore, the goal of this review is to propose and illustrate the feasibility of the notion that the literature discrepancies are perhaps an indication of a complex transport mechanism involving more than one uptake pathway. Accordingly, ON- and cell-differences in uptake may be attributed to differences in the relative importance of these pathways for different cell types and ONs. An example of one such pathway is reviewed and critiqued in this communication with respect to its hypothetical role in ON uptake. Other innovative mechanisms should similarly be considered to stimulate new ideas, discussion and research in this unique and interesting field.

  3. Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin

    Directory of Open Access Journals (Sweden)

    Christof M. Niemeyer

    2011-08-01

    Full Text Available Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV with phosphorothioate oligonucleotides (psDNA containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.

  4. EvoOligo: oligonucleotide probe design with multiobjective evolutionary algorithms.

    Science.gov (United States)

    Shin, Soo-Yong; Lee, In-Hee; Cho, Young-Min; Yang, Kyung-Ae; Zhang, Byoung-Tak

    2009-12-01

    Probe design is one of the most important tasks in successful deoxyribonucleic acid microarray experiments. We propose a multiobjective evolutionary optimization method for oligonucleotide probe design based on the multiobjective nature of the probe design problem. The proposed multiobjective evolutionary approach has several distinguished features, compared with previous methods. First, the evolutionary approach can find better probe sets than existing simple filtering methods with fixed threshold values. Second, the multiobjective approach can easily incorporate the user's custom criteria or change the existing criteria. Third, our approach tries to optimize the combination of probes for the given set of genes, in contrast to other tools that independently search each gene for qualifying probes. Lastly, the multiobjective optimization method provides various sets of probe combinations, among which the user can choose, depending on the target application. The proposed method is implemented as a platform called EvoOligo and is available for service on the web. We test the performance of EvoOligo by designing probe sets for 19 types of Human Papillomavirus and 52 genes in the Arabidopsis Calmodulin multigene family. The design results from EvoOligo are proven to be superior to those from well-known existing probe design tools, such as OligoArray and OligoWiz.

  5. Stable oligonucleotide-functionalized gold nanosensors for environmental biocontaminant monitoring.

    Science.gov (United States)

    Riquelme, Maria V; Leng, Weinan; Carzolio, Marcos; Pruden, Amy; Vikesland, Peter

    2017-12-01

    The global propagation of environmental biocontaminants such as antibiotic resistant pathogens and their antibiotic resistance genes (ARGs) is a public health concern that highlights the need for improved monitoring strategies. Here, we demonstrate the environmental stability and applicability of an oligonucleotide-functionalized gold nanosensor. The mecA ARG was targeted as model biocontaminant due to its presence in clinically-relevant pathogens and to its emergence as an environmental contaminant. mecA-specific nanosensors were tested for antibiotic resistance gene (ARG) detection in ARG-spiked effluent from four wastewater treatment plants (WWTPs). The mecA-specific nanosensors showed stability in environmental conditions and in high ionic strength ([MgCl2]<50mM), and high selectivity against mismatched targets. Spectrophotometric detection was reproducible with an LOD of 70pM (≈4×107genes/μL), even in the presence of interferences associated with non-target genomic DNA and complex WWTP effluent. This contribution supports the environmental applicability of a new line of cost-effective, field-deployable tools needed for wide-scale biocontaminant monitoring. Copyright © 2017. Published by Elsevier B.V.

  6. Oligonucleotide fishing for STAT6: cross-talk between IL-4 and chemokines

    DEFF Research Database (Denmark)

    Eriksen, K W; Nielsen, M; Kaltoft, K

    2001-01-01

    -stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline epsilon transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover...... activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level....

  7. Complexes of carbon nanotubes with oligonucleotides in thin Langmuir-Blodgett films to detect electrochemically hybridization

    Science.gov (United States)

    Egorov, A. S.; Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Orekhovskaya, T. I.; Veligura, A. A.; Govorov, M. I.; Shulitsky, B. G.

    2014-10-01

    Self-assembled complexes consisting of thin multi-walled carbon nanotubes (MWCNTs) and DNA-oligonucleotides which are able to a cooperative binding to complementary oligonucleotides have been investigated. It was establised a high-performance charge transport in nanostructured Langmuir-Blodgett complexes thin MWCNTs/DNA. A method to electrochemically detect DNA hybridization on the self-organized structures has been proposed.

  8. Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2'-Amino-LNA Functionalized with Galactose Units

    DEFF Research Database (Denmark)

    Kumar, Rajesh; Ries, Annika; Wengel, Jesper

    2017-01-01

    A convenient method for the preparation of oligonucleotides containing internally-attached galactose and triantennary galactose units has been developed based on click chemistry between 2'-N-alkyne 2'-amino-LNA nucleosides and azido-functionalized galactosyl building blocks. The synthesized oligo...... oligonucleotides show excellent binding affinity and selectivity towards complementary DNA/RNA strands with an increase in the melting temperature of up to +23.5 °C for triply-modified variants....

  9. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

    Science.gov (United States)

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes

    2016-10-04

    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. Efficient Bioconjugation of Protein Capture Agents to Biosensor Surfaces Using Aniline-Catalyzed Hydrazone Ligation

    Science.gov (United States)

    Byeon, Ji-Yeon; Limpoco, F. T.; Bailey, Ryan C.

    2010-01-01

    Aniline-catalyzed hydrazone ligation between surface immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method for attaching protein capture agents to model oxide-coated biosensor substrates. Silicon photonic microring resonators are used to directly evaluate the efficiency of this surface bioconjugate reaction at various pHs and in the presence or absence of aniline as a nucleophilic catalyst. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, allowing derivatization of substrates with reduced incubation time and sample consumption. This increase in antibody loading directly results in more sensitive antigen detection when functionalized microrings are employed in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH dependent non-covalent binding trend that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent. PMID:20809595

  11. Cross-Linked Collagen Triple Helices by Oxime Ligation.

    Science.gov (United States)

    Hentzen, Nina B; Smeenk, Linde E J; Witek, Jagna; Riniker, Sereina; Wennemers, Helma

    2017-09-13

    Covalent cross-links are crucial for the folding and stability of triple-helical collagen, the most abundant protein in nature. Cross-linking is also an attractive strategy for the development of synthetic collagen-based biocompatible materials. Nature uses interchain disulfide bridges to stabilize collagen trimers. However, their implementation into synthetic collagen is difficult and requires the replacement of the canonical amino acids (4R)-hydroxyproline and proline by cysteine or homocysteine, which reduces the preorganization and thereby stability of collagen triple helices. We therefore explored alternative covalent cross-links that allow for connecting triple-helical collagen via proline residues. Here, we present collagen model peptides that are cross-linked by oxime bonds between 4-aminooxyproline (Aop) and 4-oxoacetamidoproline placed in coplanar Xaa and Yaa positions of neighboring strands. The covalently connected strands folded into hyperstable collagen triple helices (Tm ≈ 80 °C). The design of the cross-links was guided by an analysis of the conformational properties of Aop, studies on the stability and functionalization of Aop-containing collagen triple helices, and molecular dynamics simulations. The studies also show that the aminooxy group exerts a stereoelectronic effect comparable to fluorine and introduce oxime ligation as a tool for the functionalization of synthetic collagen.

  12. Colonoscopic band ligation for internal hemorrhoids - A tertiary care experience

    Directory of Open Access Journals (Sweden)

    Umesh Jalihal

    2013-01-01

    Full Text Available Background and objectives: Rubber band ligation (BL is the most widely used technique for treatment of symptomatic internal haemorrhoids (IH that are refractory to conservative treatment. The aim of this study is to assess the efficacy of colonoscopic BL as therapy for symptomatic IH. Methods: Patients seen at our center with symptomatic IH who underwent BL between January 2006 and December 2011 were included in this prospective study. The clinical and colonoscopic details were entered in uniform structured data forms. Results: Two hundred and eighteen consecutive patients with symptomatic IH were enrolled in the study. The presentations were rectal bleeding in 150 (69% and prolapse in remaining 68 (31% patients. Twenty-four patients (11% had chronic liver disease (child B-C. Same operator treated all the patients. The severity of the IH was classified by using Goligher grading system. The mean age of patients was 48.3 + 15 years with range of 22 - 85 years. The mean follow up was 3months (range 1 month - 36 months. In 209 patients (96% there was at least 1 grade reduction in hemorrhoids as well the symptoms were controlled. Two patients required surgery and another 7 patients required repeat session of banding. After banding session 32 (15% patients had perianal pain and 13 (6% had mild bleeding. Conclusions: Colonoscopic BL is a safe, and effective outpatient therapeutic procedure for symptomatic internal hemorrhoids. Furthermore, the BL is safe and effective in patients of coagulopathy associated with chronic liver disease.

  13. A novel endoscopic fluorescent band ligation method for tumor localization.

    Science.gov (United States)

    Hyun, Jong Hee; Kim, Seok-Ki; Kim, Kwang Gi; Kim, Hong Rae; Lee, Hyun Min; Park, Sunup; Kim, Sung Chun; Choi, Yongdoo; Sohn, Dae Kyung

    2016-10-01

    Accurate tumor localization is essential for minimally invasive surgery. This study describes the development of a novel endoscopic fluorescent band ligation method for the rapid and accurate identification of tumor sites during surgery. The method utilized a fluorescent rubber band, made of indocyanine green (ICG) and a liquid rubber solution mixture, as well as a near-infrared fluorescence laparoscopic system with a dual light source using a high-powered light-emitting diode (LED) and a 785-nm laser diode. The fluorescent rubber bands were endoscopically placed on the mucosae of porcine stomachs and colons. During subsequent conventional laparoscopic stomach and colon surgery, the fluorescent bands were assayed using the near-infrared fluorescence laparoscopy system. The locations of the fluorescent clips were clearly identified on the fluorescence images in real time. The system was able to distinguish the two or three bands marked on the mucosal surfaces of the stomach and colon. Resection margins around the fluorescent bands were sufficient in the resected specimens obtained during stomach and colon surgery. These novel endoscopic fluorescent bands could be rapidly and accurately localized during stomach and colon surgery. Use of these bands may make possible the excision of exact target sites during minimally invasive gastrointestinal surgery.

  14. Reverse thioether ligation route to multimeric peptide antigens.

    Science.gov (United States)

    Monsó, Marta; Kowalczyk, Wioleta; Andreu, David; de la Torre, Beatriz G

    2012-04-21

    Multimeric presentation, a rather effective way of enhancing peptide immunogenicity, is best exemplified by MAP (multiple antigenic peptide) dendrimers consisting of a branched Lys core on which several copies of the peptide epitope are displayed. While accessible by solid-phase synthesis, MAPs can also be conveniently made in solution, e.g., by linking the epitope (N-acetylated and with a C-terminal Cys) through a thioether bond onto the α and ε (haloacetyl-activated) positions of the Lys core. We now report the reverse version of this approach, whereby a chloroacetyl-derivatised epitope is tethered to a thiol-functionalised form of a Lys dendron core. This convergent approach can be carried out either in solution or in the solid phase and is advantageous because (i) in situ tris(2-carboxyethyl)phosphine (TCEP)-mediated reduction of disulfide bonds maintains the thiol platform reactive throughout the ligation process; (ii) the low amounts of TCEP used pose minimal risk to chloroacetyl groups in the peptide, resulting in (iii) significantly reduced byproduct formation, hence cleaner products. For the solid phase version of the method, an optimised procedure has been devised to convert the Lys core into a tetrathiol dendron. This journal is © The Royal Society of Chemistry 2012

  15. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    Directory of Open Access Journals (Sweden)

    Lifeng Liang

    Full Text Available A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH to evaluate accuracy of the results. We found that most (58.1% of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s, partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal

  16. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation.

    Science.gov (United States)

    Kudirka, Romas; Barfield, Robyn M; McFarland, Jesse; Albers, Aaron E; de Hart, Gregory W; Drake, Penelope M; Holder, Patrick G; Banas, Stefanie; Jones, Lesley C; Garofalo, Albert W; Rabuka, David

    2015-02-19

    There is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation. The speed and stability of the trapped-Knoevenagel ligation further advances the repertoire of aldehyde-based bioconjugations and expands the toolbox for site-specific protein modifications. The trapped-Knoevenagel ligation reaction can be run at near neutral pH in the absence of catalysts to produce conjugates that are stable under physiological conditions. Using this new ligation, we generated an antibody-drug conjugate that demonstrates excellent efficacy in vitro and in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Cross-catalytic peptide nucleic acid (PNA) replication based on templated ligation

    DEFF Research Database (Denmark)

    Singhal, Abhishek; Nielsen, Peter E

    2014-01-01

    We report the first PNA self-replicating system based on template directed cross-catalytic ligation, a process analogous to biological replication. Using two template PNAs and four pentameric precursor PNAs, all four possible carbodiimide assisted amide ligation products were detected and identif......We report the first PNA self-replicating system based on template directed cross-catalytic ligation, a process analogous to biological replication. Using two template PNAs and four pentameric precursor PNAs, all four possible carbodiimide assisted amide ligation products were detected...... and identified by HPLC and MALDI-TOF analysis. We conclude that the two template complementary reaction products are generated via cross-catalysis, while the other two self-complementary (and in principle auto-catalytic) products are formed via intra-complex coupling between the two sets of complementary PNA...

  18. Controllable nanoreactor confined to atomic force microscopy tips and its application in low copy DNA ligation.

    Science.gov (United States)

    Zhou, Hualan; Shi, Wenjian

    2010-11-01

    Less molecules reaction, especially at the single molecule level, plays an important role in biochemical or chemical research. It is also significant to achieve low copy or single molecule DNA ligation during the whole genome project. In this paper, a new type of nanoreactor was constructed around atomic force microscopy (AFM) tips under certain humidity, where DNA molecules can be limited to a special space through water meniscus, so the probability of molecules collision was increased and the efficiency of DNA ligation was greatly enhanced. Combined with the nanomanipulation based on AFM, controllable nanoreactor may provide a new tool to single molecule reaction. Low copy DNA ligation was successfully achieved by this method. Results showed the number of DNA molecules involved in the nanoreactor can not be more than sixty. This method will found a base for the ultimate realization of single-molecule DNA ligation.

  19. Proximity ligation in situ assay for monitoring the global DNA methylation in cells

    National Research Council Canada - National Science Library

    Hervouet, Eric; Hulin, Philippe; Vallette, François M; Cartron, Pierre-François

    2011-01-01

    .... We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since...

  20. Esophageal variceal ligation for hemostasis of acute variceal bleeding: efficacy and safety

    OpenAIRE

    Lahbabi, Mounia; Elyousfi, Mounia; Aqodad, Nouredine; Elabkari, Mohammed; Mellouki, Ihssane; Ibrahimi, Sidi Adil; Benajah, Dafr Allah

    2013-01-01

    Introduction Endoscopic variceal ligation is widely accepted as the optimum endoscopic treatment for esophageal variceal hemorrhage. In Morocco, there are no data regarding the efficacy of this technique. Our aim was to evaluate the effectiveness and safety of endoscopic variceal ligation in the management of oesophageal variceal bleeding in cirrhosis in a located population in Morocco. Methods Via a retrospective study over 118 months (December 2001- October 2011), cirrhotic patients with en...

  1. Realizing Serine/Threonine Ligation: Scope and Limitations and Mechanistic Implication Thereof

    Directory of Open Access Journals (Sweden)

    Clarence T. T. Wong

    2014-05-01

    Full Text Available Serine/Threonine ligation (STL has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the serine/threonine ligation strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky β-branched amino acid (Thr, Val and Ile is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.

  2. Prospective comparison of ligation and bipolar cautery technique in non-scalpel vasectomy

    Directory of Open Access Journals (Sweden)

    Muammer Altok

    2015-12-01

    Full Text Available Objectives: There is no trial comparing bipolar cautery and ligation for occlusion of vas in non-scalpel vasectomy. This study aimed to compare the effectiveness of these vasectomy occlusion techniques. Materials and Methods: Between January 2002-June 2009, patients were allocated in alternate order. We recruited 100 cases in cautery group and 100 cases in ligation group. Non-scalpel approach was performed during vasectomy and fascial interposition was performed in all cases. First semen analysis was done 3 months after vasectomy. Vasectomy success was defined as azoospermia or non-motile sperm lower than 100.000/mL. Results: Four patients from the cautery group were switched to the ligation group due to technical problem of cautery device. Thus, data of 96 patients as cautery group and 104 patients as ligation group were evaluated. After vasectomy, semen analyses were obtained from 59 of 96 (61.5% patients in cautery group and to 66 of 104 (63.5% patients in ligation group. There was no statistical significant difference between the two groups in terms of the success of vasectomy (p=0.863. Conclusion: Although bipolar cautery technique is safe, effective and feasible in non-scalpel vasectomy, it has no superiority to ligation. There was no statistically significant difference in terms of the success and complications between the two groups.

  3. Retroperitoneal approach for laparoscopic nephroureterectomy with stripping technique: extracorporeal ligation of ureter and ureteral catheter.

    Science.gov (United States)

    Nakamura, K; Nagata, D; Kajikawa, K; Kobayashi, I; Zennami, K; Nishikawa, G; Yoshizawa, T; Tobiume, M; Aoki, S; Yamada, Y; Sumitomo, M

    2012-02-01

    The pluck and stripping techniques are used for lower ureter management in renal pelvic cancer patients. Herein, we report our experience of extracorporeal ligation of the ureter and the ureteral catheter through the trocar port, which differs from conventional laparoscopic ligation in the retroperitoneal space. This technique was selected to reduce the time needed for ureter management using the stripping technique and to provide secure ligation. We performed this stripping technique in patients with T1 and T2 stage renal pelvic cancer without imaging-evident lymph node metastasis. After transurethrally placing a ureteral catheter, we resected the circumference of the ureteral orifice. After laparoscopic nephrectomy via a retroperitoneal approach, the ureteral catheter and distal ureter were ligated extracorporeally. The catheter was pulled to invaginate the ureter so it could then be pulled through the external urethral orifice. This technique of extracorporeal ligation ensures more a secure ligation of the ureter and ureteral catheter. This modified stripping technique does not require lower ureter management with laparotomy, and it is also useful in shortening the operative time. This method is effective for relatively early stage renal pelvic cancer. © 2012 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and Blackwell Publishing Asia Pty Ltd.

  4. Apricot DNA as an indicator for persipan: detection and quantitation in marzipan using ligation-dependent probe amplification.

    Science.gov (United States)

    Luber, Florian; Demmel, Anja; Hosken, Anne; Busch, Ulrich; Engel, Karl-Heinz

    2012-06-13

    The confectionery ingredient marzipan is exclusively prepared from almond kernels and sugar. The potential use of apricot kernels, so-called persipan, is an important issue for the quality assessment of marzipan. Therefore, a ligation-dependent probe amplification (LPA) assay was developed that enables a specific and sensitive detection of apricot DNA, as an indicator for the presence of persipan. The limit of detection was determined to be 0.1% persipan in marzipan. The suitability of the method was confirmed by the analysis of 20 commercially available food samples. The integration of a Prunus -specific probe in the LPA assay as a reference allowed for the relative quantitation of persipan in marzipan. The limit of quantitation was determined to be 0.5% persipan in marzipan. The analysis of two self-prepared mixtures of marzipan and persipan demonstrated the applicability of the quantitation method at concentration levels of practical relevance for quality control.

  5. Trolox mitigates fibrosis in a bile duct ligation model.

    Science.gov (United States)

    Galicia-Moreno, Marina; Favari, Liliana; Muriel, Pablo

    2013-06-01

    Several studies suggest that free radicals may play a role in cholestatic liver injury. The aim of this work was to evaluate the role of trolox in chronic bile duct ligation (BDL). Liver injury was induced by 28-day BDL to male Wistar rats. Animals were divided in four groups of six rats. Trolox was administered daily (50 mg/kg, p.o.). Alanine aminotransferase (ALT) was quantified in serum. Fibrosis was assessed measuring liver hydroxyproline content. Reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, catalase (CAT), and glutathione peroxidase (GPx) activities were measured in liver. Transforming growth factor-β (TGF-β), interleukin-6 (IL-6), and interleukin-10 (IL-10) were determined by western blot and quantified densitometrically. Our results show that trolox treatment in BDL rats prevented the increase in ALT. Collagen was increased by chronic BDL, but trolox administration preserved the normal collagen concentration. BDL produced high levels of the cytokine TGF-β1, IL-6, and IL-10 levels. Trolox administration was effective to partially prevent the increase of TGF-β1 and IL-6, and it was able to further augment the levels of IL-10. Oxidative stress (assessed by lipid peroxidation and liver glutathione content) was increased by BDL; this process was normalized by trolox. The activities of CAT and GPx were altered by BDL, and trolox prevented these events. We found that there is a close relationship between cholestatic liver damage and oxidative stress generation, and this was effectively prevented by trolox. Our study shows that the beneficial effects of trolox are because of its important antioxidant and immunomodulatory properties. © 2011 The Authors Fundamental and Clinical Pharmacology © 2011 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.

  6. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    OpenAIRE

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  7. [Effect of bile duct ligation and recanalization on rat hepatocyte epithelial-mesenchymal phenotype and NOX4 protein expression].

    Science.gov (United States)

    Lou, An-Ni; Pan, Chun-Qiu; Li, Yang; Yang, Ren-Qiang; Li, Xu

    2015-10-01

    To observe epithelial-mesenchymal phenotypes and oxidative stress related protein expressions of the liver cells in a rat model of liver fibrosis induced by bile duct ligation and recanalization. Twenty-four male Wistar rats were randomized into 4 groups, including a sham-operated group, two bile duct ligation groups with ligation for 2 and 4 weeks, and a bile duct ligation group with a 2-week ligation followed by a 2-week recanalization. HE staining and Masson staining were used to assess liver fibrosis in the rats, and immunohistochemistry and Western blotting were employed to detect expressions of the epithelial and mesenchymal marker proteins and oxidative stress-related proteins. Compared with the sham-operated group, the rats with bile duct ligation showed obvious liver fibrosis, which worsened as the ligation time extended, accompanied by significantly increased expression of α-SMA, collagen I, NOX(4) and vimetin and reduced E-cadherin expression. Compared with the rats with bile duct ligation for 4 weeks, the rats in bile duct ligation-recanalization group showed obviously lessened liver fibrosis, significantly lowered expressions of NOX(4) and mesenchymal cell maker proteins, and enhanced expressions of epithelial cell marker proteins. Bile duct ligation up-regulates mesenchymal phenotype-related proteins and NOX(4) protein expression and down-regulates the expression of epithelial phenotype-related proteins, and these changes can be reversed by subsequent bile duct recanalization.

  8. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides.

    Science.gov (United States)

    Muñoz-Alarcón, Andrés; Eriksson, Jonas; Langel, Ülo

    2015-12-01

    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2'-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

  9. Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

    Science.gov (United States)

    Weber, D G; Sahm, K; Polen, T; Wendisch, V F; Antranikian, G

    2008-10-01

    The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

  10. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

    Science.gov (United States)

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (psunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  11. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Chen Feng

    2010-10-01

    Full Text Available Abstract Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals and Plasmodium falciparum (a related parasite responsible for severe human malaria, we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at

  12. Efficient oligonucleotide probe selection for pan-genomic tiling arrays

    Directory of Open Access Journals (Sweden)

    Zhang Wei

    2009-09-01

    Full Text Available Abstract Background Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results This paper presents a new probe selection algorithm (PanArray that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on

  13. Use of oligonucleotide array for identification of six foodborne pathogens and Pseudomonas aeruginosa grown on selective media.

    Science.gov (United States)

    Lin, Miao Chu; Huang, Ay Huey; Tsen, Hau Yang; Wong, Hin-Chung; Chang, Tsung Chain

    2005-11-01

    Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (> 500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.

  14. Leveraging two-way probe-level block design for identifying differential gene expression with high-density oligonucleotide arrays.

    Science.gov (United States)

    Barrera, Leah; Benner, Chris; Tao, Yong-Chuan; Winzeler, Elizabeth; Zhou, Yingyao

    2004-04-20

    To identify differentially expressed genes across experimental conditions in oligonucleotide microarray experiments, existing statistical methods commonly use a summary of probe-level expression data for each probe set and compare replicates of these values across conditions using a form of the t-test or rank sum test. Here we propose the use of a statistical method that takes advantage of the built-in redundancy architecture of high-density oligonucleotide arrays. We employ parametric and nonparametric variants of two-way analysis of variance (ANOVA) on probe-level data to account for probe-level variation, and use the false-discovery rate (FDR) to account for simultaneous testing on thousands of genes (multiple testing problem). Using publicly available data sets, we systematically compared the performance of parametric two-way ANOVA and the nonparametric Mack-Skillings test to the t-test and Wilcoxon rank-sum test for detecting differentially expressed genes at varying levels of fold change, concentration, and sample size. Using receiver operating characteristic (ROC) curve comparisons, we observed that two-way methods with FDR control on sample sizes with 2-3 replicates exhibits the same high sensitivity and specificity as a t-test with FDR control on sample sizes with 6-9 replicates in detecting at least two-fold change. Our results suggest that the two-way ANOVA methods using probe-level data are substantially more powerful tests for detecting differential gene expression than corresponding methods for probe-set level data.

  15. Leveraging two-way probe-level block design for identifying differential gene expression with high-density oligonucleotide arrays

    Directory of Open Access Journals (Sweden)

    Tao Yong-Chuan

    2004-04-01

    Full Text Available Abstract Background To identify differentially expressed genes across experimental conditions in oligonucleotide microarray experiments, existing statistical methods commonly use a summary of probe-level expression data for each probe set and compare replicates of these values across conditions using a form of the t-test or rank sum test. Here we propose the use of a statistical method that takes advantage of the built-in redundancy architecture of high-density oligonucleotide arrays. Results We employ parametric and nonparametric variants of two-way analysis of variance (ANOVA on probe-level data to account for probe-level variation, and use the false-discovery rate (FDR to account for simultaneous testing on thousands of genes (multiple testing problem. Using publicly available data sets, we systematically compared the performance of parametric two-way ANOVA and the nonparametric Mack-Skillings test to the t-test and Wilcoxon rank-sum test for detecting differentially expressed genes at varying levels of fold change, concentration, and sample size. Using receiver operating characteristic (ROC curve comparisons, we observed that two-way methods with FDR control on sample sizes with 2–3 replicates exhibits the same high sensitivity and specificity as a t-test with FDR control on sample sizes with 6–9 replicates in detecting at least two-fold change. Conclusions Our results suggest that the two-way ANOVA methods using probe-level data are substantially more powerful tests for detecting differential gene expression than corresponding methods for probe-set level data.

  16. Effects of ligation of lateral intermuscular septum perforating vessels on blood supply to the femur.

    Science.gov (United States)

    Grob, K; Manestar, M; Lang, A; Ackland, T; Gilbey, H; Kuster, M S

    2015-12-01

    With a subvastus approach to the femur, the vessels that perforate the lateral intermuscular septum (LISP-vessels) must be ligated. The effect on the blood supply to the femur remains unclear. The purpose of the current study was to investigate the effect of ligation of the LISP-Vessels on the blood supply and to examine the anatomy of the LISP-vessels and the anastomoses around the femur. In six human cadavers the LISP vessels were ligated by a lateral subvastus approach on one side. The contralateral side served as control group. After bilateral injection of different coloured silicon dyes into the lateral and medial circumflex femoral artery (green), deep femoral artery (red) and the superficial femoral artery (blue) dissection was performed bilaterally. The arterial perfusion on both sides was compared and the anatomy of the LISP vessels studied. The medullary perfusion of the femur was not altered by the ligation of the LISP vessels. It did also not lead to a decrease in periosteal vessel filling. The LISP vessels were shown to be a part of a complex and rich anastomotic network and play an important role in the perfusion of the femur and quadriceps muscle group. The ligature could be compensated for by this anastomotic network. Branches to the periosteum separate from the LISP vessels immediately after perforating the lateral intermuscular septum. The linea aspera turned out to be an important area for the femoral blood supply. Exposure of the femur through a lateral subvastus approach with ligation of LISP vessels causes a certain degree of soft tissue trauma. However, by using a gentle surgical technique the periostal perfusion of the femur can be preserved by a potent anastomotic network after ligation of the LISP vessels if they are not ligated to close to the lateral intermuscular septum and the linea aspera is not unnecessarily exposed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Multiple ligation of the proximal greater saphenous vein in the CHIVA treatment of primary varicose veins

    Directory of Open Access Journals (Sweden)

    Roberto Delfrate

    2014-06-01

    Full Text Available Saphenous femoral disconnection is the key point of most surgical techniques in the treatment of primary varicose vein surgery. The aim of this study is to compare and analyze different techniques for conservative saphenousfemoral ligation or disconnection. These techniques can be to perform mini invasive open surgery and are suitable for implementation of the conservative hemodynamic correction of venous insufficiency (CHIVA method. The aim was to present the follow-up by retrospective analysis of three different ligation-disconnection techniques of the proximal great saphenous vein (GSV according to the CHIVA method at the GSV end, i.e. between the very end of the GSV and the first arch tributary, according to the CHIVA method. The first thecnique consisted of a surgical division (crossotomy. The other two consisted of triple superposed ligation with No. 2 non-absorbable braided coated suture without division labeled TSFL (triple saphenous flush ligation and No. 0 polypropylenene ligation TPL (triple polypropylene ligation. The difference between TSFL and TPL was in the thickness and type of material of the thread, though both were non-absorbable. The follow up of 56 TPL procedures, 61 crossotomy procedures, and 82 TSFL procedures was analysed. The follow-up consisted of checking the sapheno-femoral junction occlusion with Duplex color ultra sound. The incidence rates of neovascularization (new vessels in the ligation or surgical disconnection site with saphenous-femoral reflux during the Valsalva maneuver were: 4.9% for the crossotomy group, 6.1% for the TSFL group and 37.5% for the TPL group. The data analysed show satisfactory results with both crossotomy and TSFL. Crossotomy has proven to be an effective technique for performing saphenous-femoral disconnection, but TSFL could also be a reliable, safe and low-cost varicose mini-invasive surgery in outpatients. TPL appeared to be less reliable.

  18. Vocal cord paralysis post patent ductus arteriosus ligation surgery: risks and co-morbidities.

    Science.gov (United States)

    Rukholm, Gavin; Farrokhyar, Forough; Reid, Diane

    2012-11-01

    1. To determine the prevalence of left vocal cord paralysis (LVCP) post patent ductus arteriosus (PDA) ligation at a Tertiary Care Centre. 2. To identify risk factors associated with LVCP. 3. To identify co-morbidities associated with LVCP. 4. To determine the frequency of pre- and post-operative nasopharyngolaryngoscopic (NPL) examination in this patient population. Retrospective chart review of all infants who underwent PDA ligation surgery at a tertiary care academic hospital between July 2003 and July 2010. Data on patient age, gender, weight, method of PDA ligation, and results of NPL scoping were collected, as well as patient co-morbidities post PDA ligation. One hundred and fifteen patients underwent PDA ligation surgery. Four patients were excluded due to bilateral vocal cord paralysis. Of the remaining 111 patients, nineteen patients (17.1%) were found to have LVCP. Low birth weight was identified as a significant risk factor for LVCP (p=0.002). Gastroesophageal reflux was identified as a significant co-morbidity associated with LVCP post PDA ligation (p=0.002). Only 0.9% of patients were scoped pre-operatively, and 27.9% were scoped postoperatively. LVCP is associated with multiple morbidities. The authors strongly recommend routine post-operative scoping of all patients post PDA ligation surgery, and preoperative scoping when possible. A prospective study is warranted, in order to confirm the prevalence of LVCP as well as risk factors and associated co-morbidities. Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

  19. Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance.

    Science.gov (United States)

    Merritt, Kristen K; Bradley, Kevin M; Hutter, Daniel; Matsuura, Mariko F; Rowold, Diane J; Benner, Steven A

    2014-01-01

    Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide "letters", the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson-Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson-Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2'-deoxyisocytidine and 2'-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides

  20. Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide.

    Science.gov (United States)

    Hwang, Byungjin; Bang, Duhee

    2016-11-23

    All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials.

  1. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

    Science.gov (United States)

    Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A.

    2015-07-01

    The dynamics of the human oligonucleotide AG3(T2AG3)3 has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  2. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

    Energy Technology Data Exchange (ETDEWEB)

    Sebastiani, F.; Comez, L.; Sacchetti, F. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); CNR, Istituto Officina dei Materiali, Unità di Perugia, c/o Dipartimento di Fisica e Geologia, Università di Perugia, 06123 Perugia (Italy); Longo, M. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); Elettra—Sincrotrone Trieste, 34149 Basovizza, Trieste (Italy); Orecchini, A.; Petrillo, C.; Paciaroni, A., E-mail: alessandro.paciaroni@fisica.unipg.it [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); De Francesco, A. [CNR-IOM OGG c/o Institut Laue-Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France); Muthmann, M. [Jülich Centre for Neutron Science, Forschungszentrum Jülich GmbH, Outstation at Heinz Maier-Leibnitz Zentrum, Lichtenbergstrasse 1, 85747 Garching (Germany); Teixeira, S. C. M. [EPSAM, Keele University, Staffordshire ST5 5BG (United Kingdom); Institut Laue–Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France)

    2015-07-07

    The dynamics of the human oligonucleotide AG{sub 3}(T{sub 2}AG{sub 3}){sub 3} has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  3. Esophageal variceal ligation in the secondary prevention of variceal bleeding: Result of long term follow-up

    OpenAIRE

    Lahbabi, Mounia; Mellouki, Ihssane; Aqodad, Nouredine; Elabkari, Mohammed; Elyousfi, Mounia; Ibrahimi, Sidi Adil; Benajah, Dafr Allah

    2013-01-01

    Introduction Long-term outcome of patients after band ligation have been poorly defined. Therefore, we conducted a long-term follow-up study to delineate the outcome of ligation in patients with portal hypertension in the Hassan II university hospital, Fes, Morocco. Methods Over 118 months patients treated by endoscopic variceal ligation were received regular follow- up and detailed clinical assessment of at least 24 months. Results One hundred twenty five patients were followed up for a mean...

  4. Post-Patent Ductus Arteriosus ligation syndrome with hypertension and masking of renal artery stenosis in an infant.

    Science.gov (United States)

    ElSeed Peterson, Erica E; Mauriello, Daniel

    2018-02-07

    Post-patent ductus arteriosus ligation syndrome is common, but rarely has hypertension been described following ductal ligation with an unclear mechanism. We report a case of an infant who exhibited features of post-patent ductus arteriosus ligation syndrome and hypertension, but was found to have bilateral renal artery stenosis. Increased systemic vascular resistance can be masked by the parallel circuit physiology of a patent ductus arteriosus.

  5. Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation.

    Science.gov (United States)

    Krivos, Kady L; Limbach, Patrick A

    2010-08-01

    Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links. Copyright 2010. Published by Elsevier Inc.

  6. Characterization of mRNA-Cytoskeleton Interactions In Situ Using FMTRIP and Proximity Ligation

    Science.gov (United States)

    Jung, Jeenah; Lifland, Aaron W.; Alonas, Eric J.; Zurla, Chiara; Santangelo, Philip J.

    2013-01-01

    Many studies have demonstrated an association between the cytoskeleton and mRNA, as well as the asymmetric distribution of mRNA granules within the cell in response to various signaling events. It is likely that the extensive cytoskeletal network directs mRNA transport and localization, with different cytoskeletal elements having their own specific roles. In order to understand the spatiotemporal changes in the interactions between the mRNA and the cytoskeleton as a response to a stimulus, a technique that can visualize and quantify these changes across a population of cells while capturing cell-to-cell variations is required. Here, we demonstrate a method for imaging and quantifying mRNA-cytoskeleton interactions on a per cell basis with single-interaction sensitivity. Using a proximity ligation assay with flag-tagged multiply-labeled tetravalent RNA imaging probes (FMTRIP), we quantified interactions between mRNAs and β-tubulin, vimentin, or filamentous actin (F-actin) for two different mRNAs, poly(A) + and β-actin mRNA, in two different cell types, A549 cells and human dermal fibroblasts (HDF). We found that the mRNAs interacted predominantly with F-actin (>50% in HDF, >20% in A549 cells), compared to β-tubulin (cytoskeleton itself and mRNA localization. Both perturbations led to a decrease in poly(A) + mRNA interactions with F-actin and an increase in the interactions with microtubules, in a time dependent manner. PMID:24040294

  7. Hemothorax following Uncomplicated Endoscopic Variceal Sclerotherapy and Ligation for Esophageal Varices

    Directory of Open Access Journals (Sweden)

    Tomoko Ochiai

    2017-09-01

    Full Text Available Endoscopic variceal sclerotherapy and ligation are standard treatment modalities used for the management of esophageal varices. Reportedly, sclerotherapy and ligation are associated with complications such as hematuria, pulmonary thrombus formation, pleural effusion, renal dysfunction, and esophageal stenosis. However, hemothorax following sclerotherapy and ligation has not yet been reported. We treated a patient who presented with liver cirrhosis and polycythemia vera and later developed hemothorax following the above-mentioned procedures. An 86-year-old man diagnosed with liver cirrhosis due to chronic hepatitis type B and alcohol abuse underwent variceal sclerotherapy using ethanolamine oleate to treat his esophageal varices. Oozing from the esophageal varices continued even after the sclerotherapy procedure; therefore, we performed endoscopic variceal ligation. The patient developed left-sided hemothorax within 24 h after treatment of his varices, and an emergency thoracotomy was performed. A pulmonary ligament of the left lung was bulging and ripping because of mediastinal hematoma, and oozing was noted. Cessation of bleeding was noted after the laceration of the left pulmonary ligament had been sutured. Ours is the first case of hemothorax reported in a patient following an uncomplicated procedure of sclerotherapy and ligation.

  8. Esophageal variceal ligation for hemostasis of acute variceal bleeding: efficacy and safety.

    Science.gov (United States)

    Lahbabi, Mounia; Elyousfi, Mounia; Aqodad, Nouredine; Elabkari, Mohammed; Mellouki, Ihssane; Ibrahimi, Sidi Adil; Benajah, Dafr Allah

    2013-01-01

    Endoscopic variceal ligation is widely accepted as the optimum endoscopic treatment for esophageal variceal hemorrhage. In Morocco, there are no data regarding the efficacy of this technique. Our aim was to evaluate the effectiveness and safety of endoscopic variceal ligation in the management of oesophageal variceal bleeding in cirrhosis in a located population in Morocco. Via a retrospective study over 118 months (December 2001- October 2011), cirrhotic patients with endoscopically proven esophageal variceal hemorrhage were treated by endoscopic variceal ligation. We studied the rate of haemostasis, rebleeding, complications and mortality. 360 cirrhotic patients were included and 378 haemostatic variceal ligations were performed. Primary haemostasis was obtained in 96.5 % (N=365) of cases. Thirty three patients (8.7%) bled during follow-up. The rate of minor complications was 15.3 % (N=58). Retrosternal pain, fever, dysphagia and Overtube's migration developed in 8.4 % (N=32); 2.6 % (N=10); 3,7 % (N=14) and 0.5 % (N=2) of the patients respectively. Severity of these complications was mild and transient. The rate of oesophageal ulcers was 5 % (N=19), while the mortality rate by haemorrhage was 5 % (N=18). Our data showed that band ligation is an effective and safe treatment modality of esophageal variceal bleeding with low rates of rebleeding and complications.

  9. Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

    DEFF Research Database (Denmark)

    Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

    2015-01-01

    Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...... insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.......Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale...

  10. Synthesis and properties of cationic 2'-O-[N-(4-aminobutyl)carbamoyl] modified oligonucleotides.

    Science.gov (United States)

    Seio, Kohji; Tokugawa, Munefumi; Kanamori, Takashi; Tsunoda, Hirosuke; Ohkubo, Akihiro; Sekine, Mitsuo

    2012-04-01

    2'-O-[N-(4-Aminobutylcarbamoyl)]uridine (U(abcm)) was synthesized and incorporated into oligonucleotides. The oligonucleotides incorporating U(abcm) formed more stable duplexes with their complementary and mismatched RNAs than those containing 2'-O-carbamoyluridine (U(cm)). The stability of duplex with a U(abcm)-rG base pair showed higher thermostability than the duplex having unmodified U-rG base pair. The U(abcm) residue showed enhanced resistance to snake venome phosphodiesterase. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. The thermal stability of oligonucleotide duplexes is sequence independent in tetraalkylammonium salt solutions: application to identifying recombinant DNA clones.

    OpenAIRE

    Jacobs, K. A.; Rudersdorf, R; Neill, S D; Dougherty, J P; Brown, E L; Fritsch, E F

    1988-01-01

    In solutions of tetraalkylammonium salts the melting temperature of oligonucleotide duplexes is independent of nucleotide sequence and thus GC content. Data quantitating the destabilizing effects of various mismatches in these solvents are also presented. The results are in accord with theories on DNA melting and establish conditions under which oligonucleotides can be used as hybridization probes with predictable and controllable specificity.

  12. Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA and 2'-Amino-LNA Monomers

    DEFF Research Database (Denmark)

    Ries, Annika; Kumar, Rajesh; Lou, Chenguang

    2016-01-01

    Galactose-modified thymidine, LNA-T and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'...

  13. Antisense oligonucleotide inhibition of Heat Shock Protein (HSP 47 improves bleomycin-induced pulmonary fibrosis in rats

    Directory of Open Access Journals (Sweden)

    Noguchi Takayuki

    2007-05-01

    Full Text Available Abstract Background The most common pathologic form of pulmonary fibrosis arises from excessive deposition of extracellular matrix proteins such as collagen. The 47 kDa heat shock protein 47 (HSP47 is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen. Objectives To determine whether inhibition of HSP47 could have beneficial effects in mitigating bleomycin-induced pulmonary fibrosis in rats. Methods All experiments were performed with 250–300 g male Wistar rats. Animals were randomly divided into five experimental groups that were administered: 1 saline alone, 2 bleomycin alone, 3 antisense HSP47 oligonucleotides alone, 4 bleomycin + antisense HSP47 oligonucleotides, and 5 bleomycin + sense control oligonucleotides. We investigated lung histopathology and performed immunoblot and immunohistochemistry analyses. Results In rats treated with HSP47 antisense oligonucleotides, pulmonary fibrosis was significantly reduced. In addition, treatment with HSP47 antisense oligonucleotides significantly improved bleomycin-induced morphological changes. Treatment with HSP47 antisense oligonucleotides alone did not produce any significant changes to lung morphology. Immunoblot analyses of lung homogenates confirmed the inhibition of HSP47 protein by antisense oligonucleotides. The bleo + sense group, however, did not exhibit any improvement in lung pathology compared to bleomycin alone groups, and also had no effect on HSP47 expression. Conclusion These findings suggest that HSP47 antisense oligonucleotide inhibition of HSP47 improves bleomycin-induced pulmonary fibrosis pathology in rats.

  14. Ligating Dopamine as Signal Trigger onto the Substrate via Metal-Catalyst-Free Click Chemistry for "Signal-On" Photoelectrochemical Sensing of Ultralow MicroRNA Levels.

    Science.gov (United States)

    Ye, Cui; Wang, Min Qiang; Gao, Zhong Feng; Zhang, Ying; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

    2016-12-06

    The efficiency of photon-to-electron conversion is extremely restricted by the electron-hole recombinant. Here, a new photoelectrochemical (PEC) sensing platform has been established based on the signal amplification of click chemistry (CC) via hybridization chain reaction (HCR) for highly sensitive microRNA (miRNA) assay. In this proposal, a preferred electron donor dopamine (DA) was first assembled with designed ligation probe (probe-N 3 ) via amidation reaction to achieve DA-coordinated signal probe (P DA -N 3 ). The P DA -N 3 served as a flexible trigger to signal amplification through efficiently suppressing the electron-hole recombinant. Specifically, the P DA -N 3 can be successfully ligated into the trapped hairpins (H1 and H2) via the superior ligation method of metal-catalyst-free CC, in which the electron donor DA was introduced into the assay system. Moreover, the enzyme-free HCR, employed as a versatile amplification way, ensures that lots of P DA -N 3 can be attached to the substrate. This PEC sensing for miRNA-141 detection illustrated the outstanding linear response to a concentration variation from 0.1 fM to 0.5 nM and a detection limit down to 27 aM, without additional electron donors. The sensor is further employed to monitor miRNA-141 from prostate carcinoma cell (22Rv1), showing good quantitative detection capability. This strategy exquisitely influences the analytical performance and offers a new PEC route to highly selective and sensitive detection of biological molecules.

  15. Idiopathic chylopericardium treated by percutaneous thoracic duct embolization after failed surgical thoracic duct ligation

    Energy Technology Data Exchange (ETDEWEB)

    Courtney, Malachi; Ayyagari, Raj R. [Yale School of Medicine, Yale New Haven Hospital, New Haven, CT (United States); Division of Interventional Radiology, Department of Radiology, 789 Howard Avenue, P.O. Box 208042, New Haven, CT (United States)

    2015-06-15

    Chylopericardium rarely occurs in pediatric patients, but when it does it is most often a result of lymphatic injury during cardiothoracic surgery. Primary idiopathic chylopericardium is especially rare, with few cases in the pediatric literature. We report a 10-year-old boy who presented with primary idiopathic chylopericardium after unsuccessful initial treatment with surgical lymphatic ligation and creation of a pericardial window. Following readmission to the hospital for a right-side chylothorax resulting from the effluent from the pericardial window, he had successful treatment by interventional radiology with percutaneous thoracic duct embolization. This case illustrates the utility of thoracic duct embolization as a less-invasive alternative to surgical thoracic duct ligation, or as a salvage procedure when surgical ligation fails. (orig.)

  16. Progressive lymphangiectasis and recurrent chylothorax in a dog after thoracic duct ligation.

    Science.gov (United States)

    Kerpsack, S J; Smeak, D D; Birchard, S J

    1995-10-15

    A 2-year-old Bernese Mountain Dog was examined to determine the cause of bilateral pleural effusion. Torsion was diagnosed, and a lobectomy of a lung lobe was performed. Chylothorax developed 12 days after lung lobectomy. Mesenteric lymphangiography revealed lymphangiectasis Lymphangiography immediately after surgical thoracic duct was completely obstructed, but chylothorax persisted after thoracic duct ligation. Lymphangiography was repeated 50 days after ligation of the thoracic duct and revealed multiple patent thoracic duct branches and progressive lymphangiectasis. A second attempt to ligate the thoracic duct caused the effusion to become serosanguineous. A pleuroperitoneal shunt with a manually operated pump chamber was used to remove the pleural effusion. Chylothorax was again detected 50 weeks after placement of shunt. Mesenteric lymphangiography revealed multiple patent thoracic duct branches and a lymphatic plexus that extended across the thoracic cavity.

  17. Synthesis, properties, and NMR studies of a C8-phenylguanine modified oligonucleotide that preferentially adopts the Z DNA conformation.

    Science.gov (United States)

    Gannett, Peter M; Heavner, Sue; Daft, Jonathan R; Shaughnessy, Kevin H; Epperson, Jon D; Greenbaum, Nancy L

    2003-10-01

    Carcinogenic aryl hydrazines produce C8-arylated purine adducts. The effect of these adducts on DNA conformation and their role in hydrazine carcinogenesis are unknown. Here, we describe a new synthetic route to produce these adducts that is also compatible with the synthesis of the corresponding phosphoramidites needed for oligonucleotide synthesis. Two oligonucleotides were prepared, an unmodified oligonucleotide, d((5)(')CGCGCGCGCG(3)(')), and a C8-phenylguanine modified oligonucleotide, d((5)(')CGCGCGCGCG(3)(')) (G = 8-phenylguanine). These oligonucleotides were compared using thermal denaturation, circular dichroism, NMR, and molecular modeling. The phenyl modification destabilizes the B DNA form and stabilizes the Z DNA form such that the B:Z ratio is near one under physiological conditions. In light of recent studies that show a role for Z DNA in gene expression and cell transformation, Z DNA stabilization by C8-arylguanine formation from aryl hydrazines may be relevant to their role in carcinogenesis.

  18. Rabbit model provides new insights in liver regeneration after transection with portal vein ligation.

    Science.gov (United States)

    Liao, Mingheng; Zhang, Tao; Wang, Haichuan; Liu, Ying; Lu, Minxun; Huang, Jiwei; Zeng, Yong

    2017-03-01

    The rabbit model of associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) has not been reported before. New Zealand white rabbits were allocated to two protocols. Protocol 1 involved either liver parenchymal transection (LPT, n = 5) or portal vein ligation (PVL, n = 5). Protocol 2 involved the ligation of different portal vein branches combined with liver partition, including the LPT + 20% PVL group (n = 5; the caudate portal vein was ligated), the LPT + 50% PVL group (n = 5; the left portal vein was ligated), and the LPT + 70% PVL group (n = 10; both veins were ligated). Computed tomography liver volumetry was performed immediately after operation. Blood samples were harvested before surgery and at days 1, 3, 7, or 14 after surgery for liver function evaluation. Most rabbits were humanely euthanized on day 7. The livers were harvested, divided into lobes, and weighed; biopsies of each lobe and immunohistochemical staining were performed. In this article, we present a new rabbit model to simulate ALPPS procedure, with a description of the regional anatomical features, surgical routes, and key techniques. The growth rate of remnant right lobe volume increased with proportionally PVL combined with LPT. Specifically, right lobe volume growth rate of the LPT + 50% PVL group overwhelmed 70% PVL alone. There were putative underlying mechanisms other than portal inflow redistribution in triggering residual liver regeneration after ALPPS procedure. This rabbit model is feasible for further mechanism research of this special clinical phenomenon. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

    DEFF Research Database (Denmark)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj

    2010-01-01

    -high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated...... using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates....

  20. Determination of the antiulcer properties of sodium cromoglycate in pylorus-ligated albino rats

    OpenAIRE

    Srivastava Vivek; Viswanathaswamy A.H.M; Mohan Govind

    2010-01-01

    Objectives : To study the ulcer protective property of sodium cromoglycate in pylorus-ligated rats and the biochemical role in ulcer protection by various biochemical tests. Materials and Methods : The ulcer protective effect of sodium cromoglycate was studied using a Pyloric Ligation Model using Wistar albino rats. The antiulcer effect of sodium cromoglycate 40 mg/kg b.w., i.p., was compared with the reference drug ranitidine 27 mg/kg b.w., i.p. The ulcer index was calculated and other bioch...

  1. Enzymatic Ligation Creates Discrete Multi-Nanoparticle Building Blocks for Self-Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Claridge, Shelley A.; Mastroianni, Alexander J.; Au, Yeung B.; Liang, Huiyang W.; Micheel, Christine M.; Frechet, Jean M.J.; Alivisatos, A. Paul

    2008-05-27

    Enzymatic ligation of discrete nanoparticle?DNA conjugates creates nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than double-stranded DNA as in previous experiments. Ligation is verified by agarose gel and small-angle X-ray scattering. This capability is utilized in two ways: first to create a new class of multiparticle building blocks for nanoscale self-assembly; second to develop a system which can amplify a population of discrete nanoparticle assemblies.

  2. Bis-ligated Ti and Zr complexes of chelating N-heterocyclic carbenes

    KAUST Repository

    El-Batta, Amer

    2011-07-01

    In this communication we report the synthesis of novel titanium and zirconium complexes ligated by bidentate "salicylaldimine-like" N-heterocyclic carbenes (NHC). Double addition of the NHC chelate to either TiCl4(thf)2 or ZrCl4 forms bis-ligated organometallic fragments with a distorted octahedral geometry. These complexes are rare examples of group IV transition-metal NHC adducts. Preliminary catalytic tests demonstrate that in the presence of methylaluminoxane (MAO) these complexes are useful initiators for the polymerization of ethylene and the copolymerization of ethylene with norbornene and 1-octene. © 2011 Elsevier B.V. All rights reserved.

  3. Mechanics of breathing after surgical ligation of patent ductus arteriosus in newborns with respiratory distress syndrome.

    Science.gov (United States)

    Szymankiewicz, Marta; Hodgman, Joan E; Siassi, Bijan; Gadzinowski, Janusz

    2004-01-01

    The aim of the study was to detect changes in pulmonary function following ligation of a patent ductus arteriosus (PDA). Pulmonary function was recorded in 16 newborns (birth weight 1,081 +/- 166 g, gestational age 27.6 +/- 1.7 weeks) before and after ligation. No change in resistance of airways or mean airway pressure was observed. We found an increase in dynamic compliance (Cdyn) of 77% (p ventilation (MV) of 17% (p variation in intubated and spontaneously breathing premature newborns, we recommend the analysis of three main parameters: Cdyn, TV and MV for estimation of pulmonary mechanics in these infants. Copyright 2004 S. Karger AG, Basel

  4. Coronary ligation reduces maximum sustained swimming speed in Chinook salmon, Oncorhynchus tshawytscha

    DEFF Research Database (Denmark)

    Farrell, A P; Steffensen, J F

    1987-01-01

    a statistically significant 35.5% reduction in maximum swimming speed. We conclude that the coronary circulation is important for maximum aerobic swimming and implicit in this conclusion is that maximum cardiac performance is probably necessary for maximum aerobic swimming performance.......The maximum aerobic swimming speed of Chinook salmon (Oncorhynchus tshawytscha) was measured before and after ligation of the coronary artery. Coronary artery ligation prevented blood flow to the compact layer of the ventricular myocardium, which represents 30% of the ventricular mass, and produced...

  5. Pd0-Catalyzed Methyl Transfer on Nucleosides and Oligonucleotides, Envisaged as a PET Tracer

    Directory of Open Access Journals (Sweden)

    Eric Fouquet

    2013-11-01

    Full Text Available The methyl transfer reaction from activated monomethyltin, via a modified Stille coupling reaction, was studied under “ligandless” conditions on fully deprotected 5'-modified nucleosides and one dinucleotide. The reaction was optimized to proceed in a few minutes and quantitative yield, even under dilute conditions, thus affording a rapid and efficient new method for oligonucleotide labelling with carbon-11.

  6. Fatty acid-modified gapmer antisense oligonucleotide and human serum albumin constructs for pharmacokinetic modulation

    DEFF Research Database (Denmark)

    Hvam, Michael Lykke; Cai, Yunpeng; Dagnæs-Hansen, Frederik

    2017-01-01

    oligonucleotides (ASOs)/albumin constructs. We show by an electrophoretic mobility assay that fatty acid-modified gapmer and human serum albumin (HSA) can self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type (either palmitic or myristic acid), number...

  7. Optimizing anti-gene oligonucleotide 'Zorro-LNA' for improved strand invasion into duplex DNA

    DEFF Research Database (Denmark)

    Zaghloul, Eman M; Madsen, Andreas S; Moreno, Pedro M D

    2011-01-01

    Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now...

  8. LNA for Optimization of Fluorescent Oligonucleotide Probes: Improved Spectral Properties and Target Binding

    DEFF Research Database (Denmark)

    Astakhova, Irina V; Ustinov, Alexey V; Korshun, Vladimir A

    2011-01-01

    Mixmer LNA/DNA fluorescent probes containing the 1-(phenylethynyl)pyrene fluorophore attached to 2'-arabino-uridine were synthesized and studied. The conjugates displayed significantly higher hybridization affinity to target DNA, increased fluorescence quantum yields of single-stranded oligonucle......-stranded oligonucleotides and their duplexes, and improved ability to form an interstrand excimer compared to analogous non-LNA probes....

  9. Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Torsvik, V.L.; Torsvik, T.

    1997-01-01

    Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization...

  10. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    Science.gov (United States)

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. Copyright © 2016. Published by Elsevier Ltd.

  11. Efficient targeting of fatty-acid modified oligonucleotides to live cell membranes through stepwise assembly

    OpenAIRE

    Desai, Tejal; Gartner, Zev; Weber, RJ; Liang, Si; Selden, NS; Desai, TA; Gartner, ZJ

    2014-01-01

    © 2014 American Chemical Society.Lipid modifications provide efficient targeting of oligonucleotides to live cell membranes in a range of applications. Targeting efficiency is a function of the rate of lipid DNA insertion into the cell surface and its pers

  12. Properties of amphiphilic oligonucleotide films at the air/water interface and after film transfer

    NARCIS (Netherlands)

    Keller, R.; Kwak, M.; de Vries, J. W.; Sawaryn, C.; Wang, J.; Anaya, M.; Muellen, K.; Butt, H. -J.; Herrmann, A.; Berger, R.; Müllen, K.

    2013-01-01

    The self-assembly of amphiphilic hybrid materials containing an oligonucleotide sequence at the air/water interface was investigated by means of pressure-molecular area (Pi-A) isotherms. In addition, films were transferred onto solid substrates and imaged using scanning force microscopy. We used

  13. Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex

    DEFF Research Database (Denmark)

    Lou, Chenguang; Christensen, Niels Johan; Martos Maldonado, Manuel Cristo

    2017-01-01

    The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided...

  14. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    DEFF Research Database (Denmark)

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...

  15. [Use of a synthetic oligonucleotide (CAC)5 in the genomic "dactyloscopy" method].

    Science.gov (United States)

    Korokhov, N P; Popovskiĭ, A V; Sharonova, D A; Novoselov, V P

    1993-01-01

    Potentialities of a chemically synthetized oligonucleotide of a certain structure in the genomic "dactyloscopy" method were under study. A random sample analysis has demonstrated a high resolving power of this variant of the method. Arguments in favor of introducing this approach in practical forensic medical direct identification of biologic material are presented.

  16. Microfluidic bead trap as a visual bar for quantitative detection of oligonucleotides.

    Science.gov (United States)

    Zhao, Zichen; Bao, Yuanye; Chu, Lok Ting; Ho, John Kin Lim; Chieng, Ching-Chang; Chen, Ting-Hsuan

    2017-09-26

    We demonstrate a microfluidic bead trap capable of forming a dipstick-type bar visible to the naked eye for simple and quantitative detection of oligonucleotides. We use magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are connected and form MMPs-targets-PMPs when target oligonucleotides are present, leaving free PMPs with a number inversely proportional to the amount of targets. Using a capillary flow-driven microfluidic circuitry consisting of a magnetic separator to remove the MMPs-targets-PMPs, the free PMPs can be trapped at the narrowing nozzle downstream, forming a visual bar quantifiable based on the length of PMP accumulation. Such a power-free and instrument-free platform enables a limit of detection at 13 fmol (0.65 nM in 20 μl, S/N = 3) of oligonucleotides and is compatible with single-nucleotide polymorphisms and operation in a complex bio-fluid. Moreover, using DNAzyme as the target oligonucleotide that catalyzes a specific hydrolytic cleavage in the presence of lead ions, we demonstrate a model application that detects lead ions with a limit of detection of 12.2 nM (2.5 μg l-1), providing quantitative and visual detection of lead contamination at resource-limited sites.

  17. nuID: a universal naming scheme of oligonucleotides for Illumina, Affymetrix, and other microarrays

    Directory of Open Access Journals (Sweden)

    Kibbe Warren A

    2007-05-01

    Full Text Available Abstract Background Oligonucleotide probes that are sequence identical may have different identifiers between manufacturers and even between different versions of the same company's microarray; and sometimes the same identifier is reused and represents a completely different oligonucleotide, resulting in ambiguity and potentially mis-identification of the genes hybridizing to that probe. Results We have devised a unique, non-degenerate encoding scheme that can be used as a universal representation to identify an oligonucleotide across manufacturers. We have named the encoded representation 'nuID', for nucleotide universal identifier. Inspired by the fact that the raw sequence of the oligonucleotide is the true definition of identity for a probe, the encoding algorithm uniquely and non-degenerately transforms the sequence itself into a compact identifier (a lossless compression. In addition, we added a redundancy check (checksum to validate the integrity of the identifier. These two steps, encoding plus checksum, result in an nuID, which is a unique, non-degenerate, permanent, robust and efficient representation of the probe sequence. For commercial applications that require the sequence identity to be confidential, we have an encryption schema for nuID. We demonstrate the utility of nuIDs for the annotation of Illumina microarrays, and we believe it has universal applicability as a source-independent naming convention for oligomers. Reviewers This article was reviewed by Itai Yanai, Rong Chen (nominated by Mark Gerstein, and Gregory Schuler (nominated by David Lipman.

  18. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes

    NARCIS (Netherlands)

    Jansen, GJ; Mooibroek, M; Idema, J; Harmsen, HJM; Welling, GW; Degener, JE

    The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial

  19. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

    DEFF Research Database (Denmark)

    Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M

    2014-01-01

    , splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we...

  20. Precise construction of oligonucleotide-Fab fragment conjugate for homogeneous immunoassay using HaloTag technology.

    Science.gov (United States)

    Päkkilä, Henna; Peltomaa, Riikka; Lamminmäki, Urpo; Soukka, Tero

    2015-03-01

    The use of oligonucleotide-protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide-protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

    Science.gov (United States)

    Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

    2014-01-01

    Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

  2. Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

    DEFF Research Database (Denmark)

    Taskova, Maria; Mantsiou, Anna; Astakhova, Kira

    2017-01-01

    The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide–oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described...

  3. A new achiral reagent for the incorporation of multiple amino groups into oligonucleotides

    DEFF Research Database (Denmark)

    Behrens, Carsten; Petersen, Kenneth H.; Egholm, Michael

    1995-01-01

    The synthesis of a new functionalized achiral linker reagent (10) for the incorporation of multiple primary amino groups into oligonucleotides is described. The linker reagent is compatible with conventional DNA-synthesis following the phosphoramidite methodology, and the linker can be incorporated...

  4. Species‐specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

    National Research Council Canada - National Science Library

    Park, S.H; Itoh, K

    2005-01-01

    .... Methods and Results:  The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces...

  5. Ultrasensitive detection of Hg{sup 2+} using oligonucleotide-functionalized AlGaN/GaN high electron mobility transistor

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Junjie [Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Division of Nanobiomedicine, Key Laboratory for Nano-Bio Interface Research, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 (China); Li, Jiadong; Miao, Bin; Wu, Dongmin, E-mail: dmwu2008@sinano.ac.cn [i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215125 (China); Key Laboratory of Nanodevices and Applications, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 (China); Wang, Jine; Pei, Renjun, E-mail: rjpei2011@sinano.ac.cn [Division of Nanobiomedicine, Key Laboratory for Nano-Bio Interface Research, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 (China); Wu, Zhengyan, E-mail: zywu@ipp.ac.cn [Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China)

    2014-08-25

    An oligonucleotide-functionalized ion sensitive AlGaN/GaN high electron mobility transistor (HEMT) was fabricated to detect trace amounts of Hg{sup 2+}. The advantages of ion sensitive AlGaN/GaN HEMT and highly specific binding interaction between Hg{sup 2+} and thymines were combined. The current response of this Hg{sup 2+} ultrasensitive transistor was characterized. The current increased due to the accumulation of Hg{sup 2+} ions on the surface by the highly specific thymine-Hg{sup 2+}-thymine recognition. The dynamic linear range for Hg{sup 2+} detection has been determined in the concentrations from 10{sup −14} to 10{sup −8} M and a detection limit below 10{sup −14} M level was estimated, which is the best result of AlGaN/GaN HEMT biosensors for Hg{sup 2+} detection till now.

  6. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L. gene expression oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Paula Fernandez

    Full Text Available Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de. The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons. The resulting Sunflower Unigen Resource (SUR version 1.0 was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01 allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  7. Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays

    Science.gov (United States)

    2013-01-01

    Background Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. Results We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. Conclusions Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different

  8. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  9. Gas-phase Reactivity of meta-Benzyne Analogs Toward Small Oligonucleotides of Differing Lengths

    Science.gov (United States)

    Widjaja, Fanny; Max, Joann P.; Jin, Zhicheng; Nash, John J.; Kenttämaa, Hilkka I.

    2017-07-01

    The gas-phase reactivity of two aromatic carbon-centered σ,σ-biradicals ( meta-benzyne analogs) and a related monoradical towards small oligonucleotides of differing lengths was investigated in a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer coupled with laser-induced acoustic desorption (LIAD). The mono- and biradicals were positively charged to allow for manipulation in the mass spectrometer. The oligonucleotides were evaporated into the gas phase as intact neutral molecules by using LIAD. One of the biradicals was found to be unreactive. The reactive biradical reacts with dinucleoside phosphates and trinucleoside diphosphates mainly by addition to a nucleobase moiety followed by cleavage of the glycosidic bond, leading to a nucleobase radical (e.g., base-H) abstraction. In some instances, after the initial cleavage, the unquenched radical site of the biradical abstracts a hydrogen atom from the neutral fragment, which results in a net nucleobase abstraction. In sharp contrast, the related monoradical mainly undergoes facile hydrogen atom abstraction from the sugar moiety. As the size of the oligonucleotides increases, the rate of hydrogen atom abstraction from the sugar moiety by the monoradical was found to increase due to the presence of more hydrogen atom donor sites, and it is the only reaction observed for tetranucleoside triphosphates. Hence, the monoradical only attacks sugar moieties in these substrates. The biradical also shows significant attack at the sugar moiety for tetranucleoside triphosphates. This drastic change in reactivity indicates that the size of the oligonucleotides plays a key role in the outcome of these reactions. This finding is attributed to more compact conformations in the gas phase for the tetranucleoside triphosphates than for the smaller oligonucleotides, which result from stronger stabilizing interactions between the nucleobases.

  10. Coupling Strategies for the Synthesis of Peptide-Oligonucleotide Conjugates for Patterned Synthetic Biomineralization

    Directory of Open Access Journals (Sweden)

    Joshua D. Carter

    2011-01-01

    Full Text Available This work describes preparation strategies for peptide-oligonucleotide conjugates that combine the self-assembling behavior of DNA oligonucleotides with the molecular recognition capabilities of peptides. The syntheses include a solution-phase fragment coupling reaction and a solid-phase fragment coupling strategy where the oligonucleotide has been immobilized on DEAE Sepharose. The yield of four coupling reagents is evaluated, two reagents in water, EDC (1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride and DMTMM (4-(4,6-dimethoxy[1,3,5]triazin-2-yl-4-methyl-morpholinium chloride, and two in dimethylformamide (DMF, PyBOP ((Benzotriazol-1-yloxy tripyrrolidinophosphonium hexafluorophosphate and HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate, while the oligonucleotide fragment is either in solution or immobilized on DEAE. These coupling strategies rely on an unprotected 5′ amino linker on the oligonucleotide reacting with the peptide C-terminus. The peptide, selected from a combinatorial library for its gold-binding behavior, was 12 amino acids long with an N-terminus acetyl cap. Formation of the conjugates was confirmed by gel electrophoresis and mass spectrometry while molecular recognition functionality of the peptide portion was verified using atomic force microscopy. Solution-phase yields were superior to their solid-phase counterparts. EDC resulted in the highest yield for both solution-phase (95% and solid-phase strategies (24%, while the DMF-based reagents, PyBOP and HBTU, resulted in low yields with reduced recovery. All recoverable conjugates demonstrated gold nanoparticle templating capability.

  11. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

    Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

  12. Gene silencing of CD47 and antibody ligation of thrombospondin-1 enhance ischemic tissue survival in a porcine model: implications for human disease.

    Science.gov (United States)

    Isenberg, Jeff S; Romeo, Martin J; Maxhimer, Justin B; Smedley, Jeremy; Frazier, William A; Roberts, David D

    2008-05-01

    Insufficient tissue perfusion underlies many acute and chronic diseases. Tissue perfusion in turn requires adequate blood flow, determined in large part by the relative state of relaxation or constriction of arterial vessels. Nitric oxide (NO) produced by vascular cells modulates blood flow and tissue perfusion by relaxing and dilating arteries. Recently, we reported that the secreted protein thrombospondin-1 (TSP1), through its cell surface receptor CD47, limits the ability of NO to relax and dilate blood vessels and thus decreases tissue perfusion. In the present study, we tested the hypothesis that blocking TSP1-CD47 signaling increases ischemic tissue survival in random cutaneous porcine flaps. Random cutaneous flaps 2 x 10 cm2 were raised in white hairless Yucatan miniature pigs and were treated with a monoclonal antibody to TSP1, an antisense morpholino oligonucleotide to CD47 or control agents and tissue survival assessed. Primary vascular smooth muscle cell cultured from Yucatan pigs were also treated with the same agents +/- and an NO donor (DEA/NO) and cGMP quantified. Antibody blockade of TSP1 or morpholino suppression of CD47 dramatically enhanced survival of random tissue flaps. These responses correlated with increased blood vessel patency and tissue blood flow on vessel injection studies. NO-stimulated cGMP flux in Yucatan vascular smooth muscle cell was abrogated after antibody or morpholino treatment. Antibody ligation of TSP1 or antisense morpholino knock down of CD47 greatly increased tissue survival to ischemia. Given the similarity between porcine and human soft tissues these results suggest significant therapeutic potential for people.

  13. Binding of Dumbbell Oligonucleotides to MoMuLV Reverse Transcriptase: Inhibitory Properties of RNase H Activity

    Directory of Open Access Journals (Sweden)

    Ajay Kumar

    2010-01-01

    Full Text Available Dumbbell oligonucleotides with loops of various chemistry were synthesized. Incubation of dumbbell oligonucleotides containing phosphorothioate bonds or trimethylene phosphate linkages in loops with S1 nuclease did not result in significant cleavage under conditions which led to the degradation of dumbbell oligonucleotide containing phophodiester bonds in the loops. The binding of reverse transcriptase of Moloney Murine Leukemia Virus (MoMuLV was evaluated with all the five oligonucleotides. The protein binds to all the dumbbell oligonucleotides with similar affinity. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. The inhibitory properties of the retroviral RNase H activity was evaluated using 3H –UTP-labeled RNA:RNA-DNA hybrid. It was found that the best dumbbell oligonucleotide, inhibitor contained phosphorothioate residues in both the loops. Our value studies demonstrated that this particularly designed oligonucleotide displays an IC50 of 18 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

  14. The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

    Directory of Open Access Journals (Sweden)

    Ingrid E C Verhaart

    2014-01-01

    Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

  15. A Mechanism for Oxygen Exchange between Ligated Oxometalloporphinates and Bulk Water

    NARCIS (Netherlands)

    Primus, J.; Teunis, K.; Mandon, D.; Veeger, C.; Rietjens, I.M.C.M.

    2000-01-01

    Oxygen exchange between high-valent metal–oxo complexes and bulk water has been monitored for nonligated model porphyrins (hemin, FeTDCPPS, MnTMPyP) and the axially ligated microperoxidase-8 (MP-8). Exchange extents up to 90 ere measured for MP-8 in spite of the presence of an axial histidine ligand

  16. Catecholamine-resistant hypotension and myocardial performance following patent ductus arteriosus ligation.

    LENUS (Irish Health Repository)

    Noori, S

    2014-08-14

    Objective:We performed a multicenter study of preterm infants, who were about to undergo patent ductus arteriosus ligation, to determine whether echocardiographic indices of impaired myocardial performance were associated with subsequent development of catecholamine-resistant hypotension following ligation.Study Design:A standardized treatment approach for hypotension was followed at each center. Infants were considered to have catecholamine-resistant hypotension if their dopamine infusion was >15 μg kg(-1)min(-1). Echocardiograms and cortisol measurements were obtained between 6 and 14 h after the ligation (prior to the presence of catecholamine-resistant hypotension).Result:Forty-five infants were enrolled, 10 received catecholamines (6 were catecholamine-responsive and 4 developed catecholamine-resistant hypotension). Catecholamine-resistant hypotension was not associated with decreased preload, shortening fraction or ventricular output. Infants with catecholamine-resistant hypotension had significantly lower levels of systemic vascular resistance and postoperative cortisol concentration.Conclusion:We speculate that low cortisol levels and impaired vascular tone may have a more important role than impaired cardiac performance in post-ligation catecholamine-resistant hypotension.Journal of Perinatology advance online publication, 14 August 2014; doi:10.1038\\/jp.2014.151.

  17. PDA Ligation in Adults – A 2-years Experience in Tikur Anbassa ...

    African Journals Online (AJOL)

    Fifty four percent of pts had PDA size 5-8mm.One patient died during reoperation . Conclusion: In developed countries , PDA is exclusively managed at infancy but in developing countries like ours, PDA may present in adults with symptoms and if there is no evidence of significant pulmonary hypertension ,PDA ligation is ...

  18. Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development

    NARCIS (Netherlands)

    Smits, Hermelijn H.; de Jong, Esther C.; Schuitemaker, Joost H. N.; Geijtenbeek, Theo B. H.; van Kooyk, Yvette; Kapsenberg, Martien L.; Wierenga, Eddy A.

    2002-01-01

    Th cell polarization toward Th1 or Th2 cells is strongly driven by exogenous cytokines, in particular IL-12 or IL-4, if present during activation by Ag-presenting dendritic cells (DC). However, additional Th cell polarizing mechanisms are induced by the ligation of cell surface molecules on DC and

  19. Template Directed Oligomer Ligation in Eutectic Phases in Water-Ice

    DEFF Research Database (Denmark)

    Dörr, Mark; Löffler, Philipp M. G.; Wieczorek, Rafal

    2011-01-01

    . (a) Reaction scheme of the condensation reaction of two oligoribonucleotides : The leaving group in this example is imidazole. (b) Illustration of a possible spatial arrangement of a template (15nt) directed ligation. The 7-mer is activated with imidazole at the 5' phosphate (apical moiety...

  20. Management of Fistula‑In‑Ano with Special Reference to Ligation of ...

    African Journals Online (AJOL)

    Context: The surgical management of fistula-in-ano is still debatable and no clear recommendations have been made available until now. The present study analyses the results of ligation of intersphincteric fistula tract (LIFT) technique in treating fistula-in-ano in particular with recurrence, healing time, and continence status.

  1. Management of Fistula‑In‑Ano with Special Reference to Ligation of ...

    African Journals Online (AJOL)

    flap,[6,7] ayurvedic seton,[8] ligation of intersphincteric Fistula tract (LIFT),[9,10] and finally, video‑assisted anal fistula treatment.[11] Several sphincter‑sparing ..... patients were obese. Failure to identify fistula tract occurred more often in an obese patient which suggest that obesity might be a factor for treatment failure.

  2. ANESTHESIA MANAGEMENT OF 775 GRAMS PREMATURE PATIENT DURING PDA LIGATION-A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Gulsen KESKiN

    2016-03-01

    We believe that VLBW preterm infants by having multiple system failure may have PDA ligation in operating room if there is no optimum conditions in ICU by obtaining safe transport, performing ketamine in anesthesia induction and maintenance. [J Contemp Med 2016; 6(1.000: 47-50

  3. Clinical evaluation of endoscopic ligation with nylon snares for adenoma of the major duodenal papilla

    Directory of Open Access Journals (Sweden)

    ZHANG Yingchun

    2015-11-01

    Full Text Available ObjectiveTo evaluate the feasibility, safety, and follow-up results of endoscopic ligation with nylon snares for adenoma of the major duodenal papilla. MethodsTwenty-three patients with adenoma of the major papilla who were treated in our hospital from January 2012 to June 2014 were enrolled as subjects. All patients had biliary and pancreatic duct stents placed by endoscopic cholangiopancreatography, followed by complete ligation of tumors with nylon snares. Endoscopic follow-up evaluation of recurrence was performed regularly. ResultsAll patients had biliary and pancreatic duct stents successfully placed and tumors successfully ligated with nylon snares in their first surgery. Endoscopic reexamination at two weeks after surgery showed that tumors were removed in all patients. Postoperative complications, cholangitis and pancreatitis, were found in one (4.3% and two (8.7% patients, respectively, and there were no bleeding, perforation, or death. A follow-up of more than one year in all patients showed that two patients had local recurrence of adenoma. ConclusionEndoscopic ligation with nylon snares is a safe and effective approach for treating adenoma of the major duodenal papilla.

  4. Cecal ligation and puncture induced sepsis impairs host defense against Enterococcus faecium peritonitis

    NARCIS (Netherlands)

    Leendertse, M.; Willems, R.J.; Giebelen, I.A.; Florquin, S.; van den Pangaart, P.S.; Bonten, M.J.; van der Poll, T.

    2009-01-01

    Multiresistant and vancomycin resistant Enterococcus faecium (VRE) can cause serious infections in hospitalized patients with various co-morbid diseases. We investigated the course of VRE peritonitis after cecal ligation and puncture (CLP)-induced sepsis and compared this to sham operated mice. Mice

  5. Management of anal fistula by ligation of the intersphincteric fistula tract

    DEFF Research Database (Denmark)

    Zirak-Schmidt, Samira; Perdawood, Sharaf

    2014-01-01

    INTRODUCTION: Ligation of the intersphincteric fistula tract (LIFT) is a sphincter-preserving procedure for treatment of anal fistulas described in 2007 by Rojanasakul et al. Several studies have since then assessed the procedure with varied results. This review assesses the relevant literature...

  6. A novel device reduces anal pain after rubber band ligation: a randomized controlled trial

    NARCIS (Netherlands)

    Lam, T.J.; Felt-Bersma, R.J.F.

    2012-01-01

    Background Anal pain is a well-known sequel of rubber band ligation (RBL). A plastic device, the anal cooler which can be frozen in a freezer, has been developed to reduce anal pain. It contains a mixture of glycols and has a minimum temperature of 4 °C. This study was designed to investigate the

  7. Shear bond strength comparison between two orthodontic adhesives and self-ligating and conventional brackets.

    Science.gov (United States)

    Northrup, Rodney G; Berzins, David W; Bradley, Thomas Gerard; Schuckit, William

    2007-07-01

    To evaluate and compare the shear bond strengths of two adhesives using two types of brackets: a conventional and a self-ligating bracket system. Sixty extracted human premolars were collected. The premolars were randomly divided into three groups of 20 teeth. All three groups were direct bonded. Groups 1 and 2 used light-cured adhesive and primer (Transbond XT) with a conventional (Orthos) and a self-ligating bracket (Damon 2), respectively. Group 3 used a light-cured primer (Orthosolo) and a light-cured adhesive (Blūgloo) with a self-ligating bracket (Damon 2). The specimens were stored in distilled water at 37 degrees C for 40 +/- 2 hours, after which they were debonded and inspected for Adhesive Remnant Index (ARI) scoring. The mean shear bond strength was 15.2 MPa for group 1, 23.2 MPa for group 2, and 24.8 MPa for group 3. A one-way analysis of variance and post hoc Tukey test showed significant differences in bond strength (P .05) between groups 2 and 3. A Weibull analysis demonstrated that all three groups provided sufficient bond strength with over 90% survival rate at normal masticatory and orthodontic force levels. A Kruskal-Wallis test showed no significant difference (P > .05) in ARI scores among all three groups. All three groups demonstrated clinically acceptable bond strength. The Damon 2 self-ligating bracket exhibited satisfactory in vitro bond strength with both adhesive systems used.

  8. Opening and closure forces of sliding mechanisms of different self-ligating brackets

    Directory of Open Access Journals (Sweden)

    Paola GANDINI

    2013-06-01

    Full Text Available Self-ligating brackets engage the wire by means of a slide mechanism. Forces that have to be applied to open and close the sliding mechanism of brackets are still unknown. Objective The aim of this study was to measure and compare the opening and closure forces of different self-ligating brackets. Material and Methods Three different stainless steel self-ligating brackets (Carriere LX, Ortho Organizers; F1000, Leone; Damon Q, Ormco were tested. For each different bracket, 20 maxillary right central incisors and 20 mandibular right central incisors were used. Opening and closure forces were measured using an Instron Universal Testing Machine. Statistical analysis was performed and ANOVA and Tukey tests were carried out. Results Opening forces were registered between 1.1 N and 5.6 N, whereas closure forces were recorded between 1.57 N and 4.87 N. Significant differences were detected among the different brackets and between the two prescriptions tested. Conclusion The knowledge of different opening and closure forces of self-ligating brackets can help the orthodontist in the clinical management of these devices.

  9. Diathermy excisional hemorrhoidectomy: a prospective randomized study comparing pedicle ligation and pedicle coagulation.

    Science.gov (United States)

    Bessa, Samer S

    2011-11-01

    In hemorrhoidectomy, pedicle coagulation has been claimed to be associated with less postoperative pain compared with pedicle ligation. This study was designed to compare the effects of pedicle ligation vs pedicle coagulation on postoperative pain in patients undergoing diathermy excisional hemorrhoidectomy. The study was conducted as a single-blind prospective randomized clinical trial. Patients were treated at a single tertiary-level teaching hospital (Main University Hospital) in Alexandria, Egypt, from February 2009 to October 2010. Patients with symptomatic grade III or IV hemorrhoids were eligible. Patients were randomly allocated to receive either pedicle coagulation or pedicle ligation during 3-quadrant diathermy excision hemorrhoidectomy. Patients reported postoperative pain daily on a visual analog scale (0-10, with 10 corresponding to the most severe pain) during the first 10 postoperative days. On-demand parenteral analgesic requirements were recorded during the first 24 hours after surgery. Operative time, postoperative complications, and wound healing rates at 6 weeks postoperatively were also recorded. No a priori power calculation could be performed, so it was not possible to tell whether nonsignificant differences were real or a result of chance. A total of 136 patients were randomly assigned, and 120 patients completed the study (60 in each group). The overall median pain score for the first 10 postoperative days was significantly lower in the pedicle coagulation group than in the pedicle ligation group (4.65 vs 6.56, P parenteral analgesic requirements during the first 24 hours postoperatively.

  10. Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries.

    Science.gov (United States)

    Matysiak, Stefan; Hellmuth, Klaus; El-Sagheer, Afaf H; Shivalingam, Arun; Ariyurek, Yavuz; de Jong, Marco; Hollestelle, Martine J; Out, Ruud; Brown, Tom

    2017-12-19

    DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.

  11. Esophageal variceal ligation by reloading with inexpensive hemorrhoidal O-ring--is an overtube necessary?

    Science.gov (United States)

    Wong, S Y; Ng, F H; Kng, C

    1999-09-01

    The overtube is the major cause for severe complications during endoscopic variceal ligation with a single-shot ligator. This retrospective study was designed to examine the necessity of the placement of an overtube during elective endoscopic variceal ligation. Thirty-one sessions in 18 patients were analyzed. An overtube was inserted using an over-the-scope technique in 11 sessions (group 1) but was omitted in 20 sessions (group II). The complications, technical difficulties, and operating time were analyzed. Child's grading, the size of the esophageal varices, and the number of rubber bands deployed were comparable in both groups. There was a significantly longer operating time (p < 0.01) and more oropharyngeal injury (p = 0.03) in group I than in group II. Mid esophageal injury, which was associated with resistance in withdrawing the gastroscope from the overtube, occurred in 55% of sessions in group I but in 0% of session in group II. In conclusion, the use of an overtube is associated with more complications, and it can be omitted during elective endoscopic variceal ligation.

  12. Exploring the native chemical ligation concept for highly stereospecific glycosylation reactions.

    Science.gov (United States)

    Hoang, Kim Le Mai; Bai, Yaguang; Ge, Xin; Liu, Xue-Wei

    2013-06-07

    Various O-alkyl glycosides were obtained in a highly stereospecific manner with retention of configuration at the anomeric center. Our method has customized native chemical ligation concept for glycoconjugates synthesis, utilizing a meticulously controlled activating system. To explain the origin of stereoselective preference, an S(N)i mechanism was proposed and corroborated by computational calculations.

  13. Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification (MLPA)

    NARCIS (Netherlands)

    Hochstenbach, R; Meijer, J; van de Brug, J; Vossebeld-Hoff, I; Jansen, R; van der Luijt, R B; Sinke, R J; Page-Christiaens, G C M L; Ploos van Amstel, J-K; de Pater, J M

    2005-01-01

    OBJECTIVE: To test whether multiplex ligation-dependent probe amplification (MLPA) can be used for the detection of aneuploidy of chromosomes 13, 18, 21, X, and Y in uncultured amniocytes. METHODS: We performed a prospective study based on 527 amniotic fluid samples. Chromosome copy numbers were

  14. Ligation of pork skin gelatin with glucose moieties affects the junction zones in gelled networks

    NARCIS (Netherlands)

    Baigts Allende, Diana; Jongh, de H.H.J.

    2015-01-01

    This study aims to investigate the role of the ligation of steric moieties on the formation of junction zones during network formation of gelatin gels. The molecular conformational propensities, heat stability and mechanical properties of gradually chemically modified pork skin gelatin have been

  15. Equilibria and kinetics for pH-dependent axial ligation of alkyl (aquo ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 114; Issue 1. Equilibria and kinetics for H-dependent axial ligation of alkyl(aquo) cobaloximes with aromatic and aliphatic N-donor ligands. V Sridhar D Sudarshan Reddy N Ravikumar Reddy S Satyanarayana. Inorganic and Analytical Volume 114 Issue 1 February ...

  16. Ligation of lymph vessels for the treatment of recurrent inguinal lymphoceles following lymphadenectomy

    DEFF Research Database (Denmark)

    Toyserkani, Navid Mohamadpour; Nielsen, Henrik Toft; Bakholdt, Vivi

    2016-01-01

    BACKGROUND: Recurrent lymphocele following groin dissection is generally a self-limiting condition, but in a few cases, the lymphocele persists and for this, there are not many options. Few reports have proposed the efficacy of lymph vessel ligation with patent blue as a vessel locator. We have u...

  17. Effect of non-specific species competition from total RNA on the static mode hybridization response of nanomechanical assays of oligonucleotides

    Science.gov (United States)

    Mishra, Rohit; Hegner, Martin

    2014-06-01

    We investigate here the nanomechanical response of microcantilever sensors in real-time for detecting a range of ultra-low concentrations of oligonucleotides in a complex background of total cellular RNA extracts from cell lines without labeling or amplification. Cantilever sensor arrays were functionalized with probe single stranded DNA (ssDNA) and reference ssDNA to obtain a differential signal. They were then exposed to complementary target ssDNA strands that were spiked in a fragmented total cellular RNA background in biologically relevant concentrations so as to provide clinically significant analysis. We present a model for prediction of the sensor behavior in competitive backgrounds with parameters that are indicators of the change in nanomechanical response with variation in the target and background concentration. For nanomechanical assays to compete with current technologies it is essential to comprehend such responses with eventual impact on areas like understanding non-coding RNA pharmacokinetics, nucleic acid biomarker assays and miRNA quantification for disease monitoring and diagnosis to mention a few. Additionally, we also achieved a femtomolar sensitivity limit for online oligonucleotide detection in a non-competitive environment with these sensors.

  18. Sciatic nerve ligation causes impairment of mitochondria associated with changes in distribution, respiration, and cardiolipin composition in related spinal cord neurons in rats.

    Science.gov (United States)

    Keilhoff, Gerburg; Becker, Axel; Kropf, Siegfried; Schild, Lorenz

    2016-10-01

    Sciatic nerve irritation is often associated with disturbed Ca(2+) homeostasis in related neurons of the spinal cord. Since mitochondria substantially contribute to Ca(2+) homeostasis and little information is available, we studied the effects of loose sciatic nerve ligation, a chronic constriction injury (CCI), on neuronal mitochondria of the L3-L6 regions. Three groups of rats (untreated, sham operated, and ligated) were explored. For the characterization of mitochondria, specimens of the L3-L6 spinal cord regions were evaluated with respect to intracellular localization using pyruvate dehydrogenase immunohistochemistry and Mitotracker Red, and the ATP producing machinery by LC-MS/MS technique for the analysis of cardiolipin and high-resolution respirometry for the measurement of oxygen consumption. Therefore, the phospholipid cardiolipin supports electron transfer within the respiratory chain as part of mitochondrial respiration and is of high impact on the physical properties of the mitochondrial membrane system. Histological analysis of spinal cord motor neurons revealed clustering of mitochondria in ipsilateral samples from ligated animals 14 days after the insult. This phenomenon was similarly evident in the respective contralateral side. The intensity of MT-Red staining was enhanced exclusively at the ipsilateral side, indicating increased mitochondrial activity. CCI of the sciatic nerve caused massive changes in the composition of cardiolipin reflecting mitochondrial impairment in the early phase followed by regeneration processes as late response. Sciatic nerve CCI caused decrease in the capacity of mitochondrial ATP production that recovered within 14 days after treatment. In conclusion, we provide evidence that clustering of mitochondria, already verified for the spinal cord sensory neurons after CCI, also occurs in the respective motor neurons. Further we have demonstrated transient impairment of the capacity of mitochondrial ATP production in tissue

  19. A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation.

    Science.gov (United States)

    Rashidian, Mohammad; Mahmoodi, Mohammad M; Shah, Rachit; Dozier, Jonathan K; Wagner, Carston R; Distefano, Mark D

    2013-03-20

    Imine-based reactions are useful for a wide range of bioconjugation applications. Although aniline is known to catalyze the oxime ligation reaction under physiological conditions, it suffers from slow reaction kinetics, specifically when a ketone is being used or when hydrazone-oxime exchange is performed. Here, we report on the discovery of a new catalyst that is up to 15 times more efficient than aniline. That catalyst, m-phenylenediamine (mPDA), was initially used to analyze the kinetics of oxime ligation on aldehyde- and ketone-containing small molecules. While mPDA is only modestly more effective than aniline when used in equal concentrations (~2-fold), its much greater aqueous solubility relative to aniline allows it to be used at higher concentrations, resulting in significantly more efficient catalysis. In the context of protein labeling, it was first used to site-specifically label an aldehyde-functionalized protein through oxime ligation, and its kinetics were compared to reaction with aniline. Next, a protein was labeled with an aldehyde-containing substrate in crude cell lysate, captured with hydrazide-functionalized beads and then the kinetics of immobilized protein release via hydrazone-oxime exchange were analyzed. Our results show that mPDA can release and label 15 times more protein than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic factor, a protein with therapeutic potential, was successfully labeled with a fluorophore in only 5 min. Finally, a protein containing the unnatural amino acid, p-acetyl phenylalanine, a ketone-containing residue, was prepared and PEGylated efficiently via oxime ligation using mPDA. This new catalyst should have a significant impact on the field of bioconjugation, where oxime ligation and hydrazone-oxime exchange are commonly employed.

  20. A Highly Efficient Catalyst for Oxime Ligation and Hydrazone-Oxime Exchange Suitable for Bioconjugation

    Science.gov (United States)

    Rashidian, Mohammad; Mahmoodi, Mohammad M.; Shah, Rachit; Dozier, Jonathan K.; Wagner, Carston R.; Distefano, Mark D.

    2014-01-01

    Imine-based reactions are useful for a wide range of bioconjugation applications. Although aniline is known to catalyze the oxime ligation reaction under physiological conditions, it suffers from slow reaction kinetics, specifically when a ketone is being used or when hydrazone-oxime exchange is performed. Here, we report on the discovery of a new catalyst that is up to 15 times more efficient than aniline. That catalyst, m-phenylenediamine (mPDA), was initially used to analyze the kinetics of oxime ligation on aldehyde- and ketone-containing small molecules. While mPDA is only modestly more effective than aniline when used in equal concentrations (~ 2-fold), its much greater aqueous solubility relative to aniline allows it to be used at higher concentrations, resulting in significantly more efficient catalysis. In the context of protein labeling, it was first used to site-specifically label an aldehyde-functionalized protein through oxime ligation, and its kinetics were compared to reaction with aniline. Next, a protein was labeled with an aldehyde-containing substrate in crude cell lysate, captured with hydrazide-functionalized beads and then the kinetics of immobilized protein release via hydrazone-oxime exchange were analyzed. Our results show that mPDA can release and label 15 times more protein than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic factor, a protein with therapeutic potential, was successfully labeled with a fluorophore in only 5 min. Finally, a protein containing the unnatural amino acid, p-acetyl phenylalanine, a ketone-containing residue, was prepared and PEGylated efficiently via oxime ligation using mPDA. This new catalyst should have a significant impact on the field of bioconjugation, where oxime ligation and hydrazone-oxime exchange are commonly employed. PMID:23425124

  1. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

    Directory of Open Access Journals (Sweden)

    Andaine Seguin-Orlando

    Full Text Available Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

  2. Tubal ligation, hysterectomy and epithelial ovarian cancer in the New England Case-Control Study.

    Science.gov (United States)

    Rice, Megan S; Murphy, Megan A; Vitonis, Allison F; Cramer, Daniel W; Titus, Linda J; Tworoger, Shelley S; Terry, Kathryn L

    2013-11-15

    Previous studies have observed that tubal ligation and hysterectomy are associated with a decreased risk of ovarian cancer; however, little is known about whether these associations vary by surgical characteristics, individual characteristics or tumor histology. We used logistic regression to examine tubal ligation, simple hysterectomy and hysterectomy with unilateral oophorectomy in relation to risk of epithelial ovarian cancer in the New England Case-Control Study. Our primary analysis included 2,265 cases and 2,333 controls. Overall, tubal ligation was associated with a lower risk of epithelial ovarian cancer [odds ratio (OR) = 0.82, 95% confidence interval (CI): 0.68-0.97], especially for endometrioid tumors (OR = 0.45, 95% CI: 0.29-0.69). The inverse association between tubal ligation and ovarian cancer risk was stronger for women who had undergone the procedure at the time of last delivery (OR = 0.60, 95% CI: 0.42-0.84) rather than at a later time (OR = 0.93, 95% CI: 0.75-1.15). Overall, simple hysterectomy was not associated with ovarian cancer risk (OR: 1.09, 95% CI: 0.83-1.42), although it was associated with a nonsignificant decreased risk of ovarian cancer among women who underwent the procedure at age 45 or older (RR: 0.64, 95% CI: 0.40-1.02) or within the last 10 years (OR = 0.65, 95% CI: 0.38-1.13). Overall, women who had a hysterectomy with a unilateral oophorectomy had significantly lower risk of ovarian cancer (OR = 0.65, 95% CI: 0.45-0.94). In summary, tubal ligation and hysterectomy with unilateral oophorectomy were inversely associated with ovarian cancer risk in a large population-based case-control study. Additional research is necessary to understand the potential biologic mechanisms by which these procedures may reduce ovarian cancer risk. Copyright © 2013 UICC.

  3. Synthesis, Dynamic Combinatorial Chemistry, and PCR Amplification of 3'-5' and 3'-6' Disulfide-linked Oligonucleotides

    DEFF Research Database (Denmark)

    Hansen, Dennis Jul; Manuguerra, Ilenia; Kjelstrup, Michael Brøndum

    2014-01-01

    Disulfide dithymidines linked 3'-5' or 3'-6' were synthesized and incorporated into oligonucleotides through a combined phosphotriester and phosphoramidite solid-phase oligonucleotide synthesis approach. The disulfide links are cleaved and formed reversibly in the presence of thiols...... and oligonucleotides. This link was shown to be sequence-adaptive in response to given templates in the presence of mercaptoethanol. The artificial 3'-5' and 3'-6' disulfide link was tolerated by polymerases in the polymerase chain reaction (PCR). By using sequencing analysis, we show that single mutations frequently...

  4. Amino acids attached to 2'-amino-LNA: Synthesis of DNA mixmer oligonucleotides with increased duplex stability

    DEFF Research Database (Denmark)

    Johannsen, Marie Willaing; Wengel, Jesper; Wamberg, Michael Chr.

    2010-01-01

    The synthesis of 2'-amino-LNA (locked nucleic acid) opens up exciting possibilities for modification of nucleic acids by conjugation to the 2'-nitrogen. Incorporation of unmodified and N-functionalized 2'-amino-LNA nucleotides improve duplex stability compared to unmodified DNA. 2'-Amino......-LNA nucleosides derivatized with amino acids have been synthesized and incorporated into DNA oligonucleotides. Following oligonucleotide synthesis, peptides have been added using solid phase peptide coupling chem. Modification of oligonucleotides with pos. charged residues greatly improves thermal stability....

  5. Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray.

    Science.gov (United States)

    Utsumi, Yoshinori; Tanaka, Maho; Kurotani, Atsushi; Yoshida, Takuhiro; Mochida, Keiichi; Matsui, Akihiro; Ishitani, Manabu; Sraphet, Supajit; Whankaew, Sukhuman; Asvarak, Thipa; Narangajavana, Jarunya; Triwitayakorn, Kanokporn; Sakurai, Tetsuya; Seki, Motoaki

    2016-07-01

    Cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides f. sp. Manihotis, is a serious disease of cassava (Manihot esculenta) worldwide. In this study, we established a cassava oligonucleotide-DNA microarray representing 59,079 probes corresponding to approximately 30,000 genes based on original expressed sequence tags and RNA-seq information from cassava, and applied it to investigate the molecular mechanisms of resistance to fungal infection using two cassava cultivars, Huay Bong 60 (HB60, resistant to CAD) and Hanatee (HN, sensitive to CAD). Based on quantitative real-time reverse transcription PCR and expression profiling by the microarray, we showed that the expressions of various plant defense-related genes, such as pathogenesis-related (PR) genes, cell wall-related genes, detoxification enzyme, genes related to the response to bacterium, mitogen-activated protein kinase (MAPK), genes related to salicylic acid, jasmonic acid and ethylene pathways were higher in HB60 compared with HN. Our results indicated that the induction of PR genes in HB60 by fungal infection and the higher expressions of defense response-related genes in HB60 compared with HN are likely responsible for the fungal resistance in HB60. We also showed that the use of our cassava oligo microarray could improve our understanding of cassava molecular mechanisms related to environmental responses and development, and advance the molecular breeding of useful cassava plants.

  6. Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy.

    Science.gov (United States)

    Falzarano, Maria Sofia; Passarelli, Chiara; Ferlini, Alessandra

    2014-02-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy.

  7. Long lasting pain hypersensitivity following ligation of the tendon of the masseter muscle in rats: A model of myogenic orofacial pain

    Directory of Open Access Journals (Sweden)

    Dubner Ronald

    2010-07-01

    Full Text Available Abstract Background A major subgroup of patients with temporomandibular joint (TMJ disorders have masticatory muscle hypersensitivity. To study myofacial temporomandibular pain, a number of preclinical models have been developed to induce myogenic pain of the masseter muscle, one of the four muscles involved in mastication. The currently used models, however, generate pain that decreases over time and only lasts from hours to weeks and hence are not suitable for studying chronicity of the myogenic pain in TMJ disorders. Here we report a model of constant myogenic orofacial pain that lasts for months. Results The model involves unilateral ligation of the tendon of the anterior superficial part of the rat masseter muscle (TASM. The ligation of the TASM was achieved with two chromic gut (4.0 ligatures via an intraoral approach. Nocifensive behavior of the rat was assessed by probing the skin site above the TASM with a series of von Frey filaments. The response frequencies were determined and an EF50 value, defined as the von Frey filament force that produces a 50% response frequency, was derived and used as a measure of mechanical sensitivity. Following TASM ligation, the EF50 of the injured side was significantly reduced and maintained throughout the 8-week observation period, suggesting the presence of mechanical hyperalgesia/allodynia. In sham-operated rats, the EF50 of the injured side was transiently reduced for about a week, likely due to injury produced by the surgery. Somatotopically relevant Fos protein expression was indentified in the subnucleus caudalis of the spinal trigeminal sensory complex. In the same region, persistent upregulation of NMDA receptor NR1 phosphorylation and protein expression and increased expression of glial markers glial fibrillary acidic protein (astroglia and CD11b (microglia were found. Morphine (0.4-8 mg/kg, s.c. and duloxetine (0.4-20 mg/kg, i.p., a selective serotonin-norepinephrine reuptake inhibitor

  8. Plaque accumulation and Streptococcus mutans levels around self-ligating bracket clips and elastomeric modules: A randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Dhaval Fadia

    2015-01-01

    Full Text Available Aim: To determine the effect of two different ligating systems that is, elastomeric modules and self-ligating (SL bracket systems (Smartclip - 3M Unitek with respect to harboring bacterial plaque in fixed orthodontic treatment. Objectives: To assess, evaluate, and compare the amount of plaque accumulation and Streptococcus mutans colonization around elastomeric ligation and SL clips in the smart clip appliance. Materials and Methods: A total of 111 orthodontic patients scheduled for fixed orthodontic treatments were selected for this split maxillary arch study. All the patients were bonded with smart-clip (3M Unitek SL brackets, and the wire was placed into the bracket slots, on the randomly selected hemi arch, elastomeric modules were placed for the study to be conducted. Microbial and periodontal plaque accumulation was recorded at 3-time intervals post ligation. Plaque index-by Silness and Loe, modified Quigely Hein index, bleeding on probing were evaluated, and biofilm was collected from the tooth surface after 30 days and placed in petri dishes containing Mitis Salivarius agar for bacterial culturing. Result: It was observed that the side where ligation was done with elastomeric modules accumulated more plaque and increase in S. mutans colony forming units as compared to the side without external ligation (P < 0.05. Conclusion: Reduced bacterial colonization and better plaque control was seen with SL orthodontic bracket appliance system as compared to conventional ligation method.

  9. Analysis of lamb lung development with tracheal ligation in left posterolateral diaphragmatic hernia in cases and controls

    Directory of Open Access Journals (Sweden)

    Mahmoud Ashrafi

    2006-12-01

    Full Text Available BACKGROUND: When we perform surgery in utero, lungs have an appropriate time to decrease magnitude of hypoplasia, with surgery in uterus. METHODS: Six time-dated single-fetus ewes were selected to induce diaphragmatic hernia. Fetal lambs were divided proportionally into 2 groups, namely group 1, diaphragmatic hernia and tracheal ligation (TL group, and group 2, diaphragmatic hernia only (NL group. Morphologic assessments (weight, volume, bronchiolar branching and histological tests (lung maturation phase were performed. RESULTS: Lung weight in relation to lamb weight in the tracheal ligation group differed significantly from the control group (P<0.006 in the right lung and P<0.005 in the left lung. Other parameters were markedly different between the two groups. Lung volumes in relation to lamb weight were 29.88 ± 12.54 in the tracheal ligated group [TL] and 6.44 ± 1.84 in the non-ligated [NL] group in the right lung and 21.52 ± 7.58 in the TL group and 3.86 ± 1.90 in the NL group in the left lung. All parameters were in favor of tracheal ligation. Lung maturation phase arrested in the canalicular phase in the NL group and continued to the saccular phase in the TL group. CONCLUSIONS: Tracheal ligation resulted in more mature lungs in the TL group. KEY WORDS: Congenital diaphragmatic hernia, tracheal ligation, sheep, lamb, fetal surgery.

  10. Ultrahigh molecular recognition specificity of competing DNA oligonucleotide strands in thermal equilibrium

    CERN Document Server

    Schenkelberger, Marc; Mai, Timo; Ott, Albrecht

    2016-01-01

    The specificity of molecular recognition is important to molecular self-organization. A prominent example is the biological cell where, within a highly crowded molecular environment, a myriad of different molecular receptor pairs recognize their binding partner with astonishing accuracy. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide strand can prevail against a disproportionally more concentrated competitor that exhibits reduced affinity due to a mismatch. The magnitude of deviation from the simple picture above may reach several orders of magnitude. In our experiments the effective molecular affinity of a given strand remains...

  11. Thermal Stability of Modified i-Motif Oligonucleotides with Naphthalimide Intercalating Nucleic Acids

    DEFF Research Database (Denmark)

    El-Sayed, Ahmed Ali; Pedersen, Erik B.; Khaireldin, Nahid Y.

    2016-01-01

    of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding......In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion...... and non-crowding). This study indicated a positive effect of the naphthalimide intercalating nucleotides on the stabilities of the i-motif structures compared to the wild-type structure which is in contrast to a previous observation for a pyrene-intercalating nucleotide showing a decrease in Tm values....

  12. Electrochemical study of ellipticine interaction with single and double stranded oligonucleotides.

    Science.gov (United States)

    Tmejova, Katerina; Krejcova, Ludmila; Hynek, David; Adam, Vojtech; Babula, Petr; Trnkova, Libuse; Stiborova, Marie; Eckschlager, Tomas; Kizek, Rene

    2014-02-01

    Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is an alkaloid that has been isolated from plants of an Apocynaceae family. It is one of the simplest naturally occurring alkaloids with a planar structure. Over the past decades, ellipticine became a very promising antitumor agent. Interaction with DNA is one of the most studied ellipticine effects on cell division. This phenomenon is not clearly explained so far. In our experiments we studied interaction of ellipticine with single-stranded and double-stranded oligonucleotides by electrochemical methods on mercury electrode. Differential pulse voltammetry was applied for ellipticine (Elli) and CA peak detection. Square wave voltammetry was applied for G peak detection. The effect of the interaction time and ellipticine concentrations on interactions of ellipticine with single- and double-stranded oligonucleotides was tested too.

  13. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors

    Science.gov (United States)

    Yang, Kyung-Ae; Barbu, Mihaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry M.; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S.; Stojanovic, Milan N.

    2014-11-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and specificity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a nonspecific organometallic receptor. The method is general and enables broad clinical use of aptamers for the detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    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......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...... 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...

  15. Chemical Etiology of Nucleic Acid Structure: The α-Threofuranosyl-(3′→2′) Oligonucleotide System

    National Research Council Canada - National Science Library

    K.-U. Schöning; P. Scholz; S. Guntha; X. Wu; R. Krishnamurthy; A. Eschenmoser

    2000-01-01

    TNAs [(L)-α-threofuranosyl oligonucleotides] containing vicinally connected (3′→2′) phosphodiester bridges undergo informational base pairing in antiparallel strand orientation and are capable of cross-pairing with RNA and DNA...

  16. Crystallization of a member of the recFOR DNA repair pathway, RecO, with and without bound oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Aono, Shelly; Hartsch, Thomas; Schulze-Gahmen, Ursula

    2003-01-22

    RecFOR proteins are important for DNA repair by homologous recombination in bacteria. The RecO protein from Thermus thermophilus was cloned, purified and characterized for its binding to oligonucleotides. The protein was crystallized alone and in complex with a 14-mer oligonucleotide. Both crystal forms grow under different crystallization conditions in the same space group, P3121 or P3221, with almost identical unit cell parameters. Complete data sets were collected to 2.8 Angstrom and 2.5 Angstrom for RecO alone and the RecO-oligonucleotide complex, respectively. Visual comparison of the diffraction patterns between the two crystal forms and calculation of an Rmerge of 33.9 percent on F indicate that one of the crystal forms is indeed a complex of RecO with bound oligonucleotide.

  17. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NARCIS (Netherlands)

    Fantroussi, El S.; Urakawa, H.; Bernhard, A.E.; Kelly, J.J.; Noble, P.A.; Smidt, H.; Yershov, G.M.; Stahl, D.A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of

  18. Site-specific synthesis of oligonucleotides containing malondialdehyde adducts of deoxyguanosine and deoxyadenosine via a postsynthetic modification strategy.

    Science.gov (United States)

    Wang, Hao; Kozekov, Ivan D; Kozekova, Albena; Tamura, Pamela J; Marnett, Lawrence J; Harris, Thomas M; Rizzo, Carmelo J

    2006-11-01

    Malondialdehyde (MDA) and its reactive equivalent, base propenal, are products of oxidative damage to lipids and DNA, respectively; they are mutagenic in bacterial and mammalian systems, and MDA is carcinogenic in rats. MDA adducts of deoxyguanosine (M1dG), deoxyadenosine (OPdA), and deoxycytidine (OPdC) have been characterized. We have developed site-specific syntheses of M1dG and OPdA adducted oligonucleotides that rely on a postsynthetic modification strategy. This work provides an alternative route to the M1dG adducted oligonucleotide and, to date, the only viable strategy for the site-specific synthesis of OPdA-modified oligonucleotides. The stability of the modified oligonucleotides was examined by UV thermal melting studies (Tm). In contrast to the M1dG adduct, OPdA caused very little change in the Tm.

  19. Site-specific synthesis of oligonucleotides containing malondialdehyde adducts of deoxyguanosine and deoxyadenosine via a post-synthetic modification strategy

    Science.gov (United States)

    Wang, Hao; Kozekov, Ivan D.; Kozekova, Albena; Tamura, Pamela; Marnett, Lawrence J.; Harris, Thomas M.; Rizzo, Carmelo J.

    2008-01-01

    Malondialdehyde (MDA) and its reactive equivalent, base propenal, are products of oxidative damage to lipids and DNA, respectively; they are mutagenic in bacterial and mammalian systems and MDA is carcinogenic in rats. MDA adducts of deoxyguanosine (M1dG), deoxyadenosine (OPdA) and deoxycytidine (OPdC) have been characterized. We have developed site-specific syntheses of M1dG and OPdA adducted oligonucleotides that rely on a post-synthetic modification strategy. This work provides an alternative route to the M1dG adducted oligonucleotide and to date, the only viable strategy for the site-specific synthesis of OPdA modified oligonucleotides. The stability of the modified oligonucleotides was examined by UV thermal melting studies (Tm). In contrast to the M1dG adduct, OPdA caused very little change in the Tm. PMID:17112234

  20. Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

    Directory of Open Access Journals (Sweden)

    Mohammed Bakkali

    Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

  1. DNA Damage Response Pathway and Replication Fork Stress During Oligonucleotide Directed Gene Editing

    OpenAIRE

    Bonner, Melissa; Strouse,Bryan; Applegate, Mindy; Livingston, Paula; Kmiec, Eric B.

    2012-01-01

    Single-stranded DNA oligonucleotides (ODNs) can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, desi...

  2. Studies on oligonucleotides and analogues: from drug candidates to components for high-ordered supramolecular structures

    OpenAIRE

    Nici, Fabrizia

    2016-01-01

    Last century advances in molecular biology and biotechnologies enabled us to understand the exceptionally broad repertoire of nucleic acids functions. For a long time DNA was regarded as a rigid molecule with the sole purpose to store and transmit genetic information. However, this idea has been completely overtaken with the discovery of the catalytic activity of specific RNA molecules. In effect, the early 1970s and the embryonic works on antisense oligonucleotides paved the way for our c...

  3. Scalable Amplification of Strand Subsets from Chip-Synthesized Oligonucleotide Libraries (Open Access)

    Science.gov (United States)

    2015-11-16

    oligonucleotides can have practically arbitrary sequences, as fixed sequence domains are cleaved off using nicking endonucleases . Our process is more...with two nicking sites (blue), a restriction site (orange) and one or two orthogonal subpool- specific barcodes of 10 nucleotides (nt) each (black... restriction sites are shared by all strands of the library, barcodes are subpool-specific. The ‘intervening region’ (dark blue) comprises all non-target

  4. Non-absorbable sutures are associated with lower recurrence rates in laparoscopic percutaneous inguinal hernia ligation.

    Science.gov (United States)

    Grimsby, G M; Keays, M A; Villanueva, C; Bush, N C; Snodgrass, W T; Gargollo, P C; Jacobs, M A

    2015-10-01

    Laparoscopic hernia repair with percutaneous ligation of the patent processes vaginalis is a minimally invasive alternative to open inguinal herniorrhaphy in children. With the camera port concealed at the umbilicus, this technique offers an excellent cosmetic result. It is also faster than the traditional laparoscopic repair with no differences in complication rates or hospital stay. The goal of this study was to describe a series of consecutive patients, emphasizing the impact of suture materials (absorbable vs. non-absorbable) on hernia recurrences. A retrospective review was performed of consecutive transperitoneal laparoscopic subcutaneous ligations of a symptomatic hernia and/or communicating hydrocele by 4 surgeons. Patients > Tanner 2 or with prior hernia repair were excluded. The success of the procedure and number of sutures used was compared between cases performed with absorbable vs. non-absorbable suture. Risk factors for surgical failure (age, weight, number of sutures used, suture type) were assessed with logistic regression. 94 patients underwent laparoscopic percutaneous hernia ligation at a mean age of 4.9 years. Outcomes in 85 (90%) patients with 97 hernia repairs at a mean of 8 months after surgery revealed 26% polyglactin vs 4% polyester recurrences (p = 0.004) which occurred at mean of 3.6 months after surgery, Table 1. Repairs performed with non-absorbable suture required only 1 suture more often than those performed with absorbable suture (76% vs 60%, p = 0.163). Logistic regression revealed suture type was an independent predictor for failure (p = 0.017). Weight (p = 0.249), age (p = 0.055), and number of sutures (p = 0.469) were not significantly associated with recurrent hernia. Our review of consecutive hernia repairs using the single port percutaneous ligation revealed a significantly higher recurrent hernia rate with absorbable (26%) versus non-absorbable (4%) suture. This finding remained significant in a logistic regression model

  5. Lower-extremity lymphedema following neck dissection--an uncommon complication after cervical ligation of the thoracic duct.

    Science.gov (United States)

    Raguse, Jan D; Pfitzmann, Robert; Bier, Jürgen; Klein, Martin

    2007-09-01

    Thoracic duct injuries and chylous fistula are well-known complications of neck dissection, occurring in 1-2% of cases. Management of these injuries can be conservative or operative. Conservative treatment consists of fat restricted diet or total parenteral nutrition reducing the volume of chyle production. Operative management includes exploration of the neck or if necessary open thoracotomy to ligate the thoracic duct. Following cervical thoracic duct ligation only few complications like chylothorax or chylous ascites are described in the literature. To the best authors knowledge, this is the first report in the english literature describing lower-extremity lymphedema following cervical thoracic duct ligation.

  6. Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

    OpenAIRE

    Cognet, J A; Gabarro-Arpa, J; Le Bret, M; van der Marel, G A; van Boom, J H; Fazakerley, G V

    1991-01-01

    We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts ...

  7. Proposal for new European pharmaceutical legislation to permit access to custom-made anti-sense oligonucleotide medicinal products.

    Science.gov (United States)

    Johnston, John D; Feldschreiber, Peter

    2014-06-01

    Current European pharmaceutical legislation is not adequate to meet advances in science and technologies that will lead to rapid development of custom-made medicines. Using existing legislation for custom-made medical devices as a template and anti-sense oligonucleotides as model medicinal products, we propose new European pharmaceutical legislation to permit timely access to custom-made anti-sense oligonucleotide medicinal products. The proposals may be more widely applicable to other medicinal products. © 2013 The British Pharmacological Society.

  8. Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2′-Amino-LNA Functionalized with Galactose Units

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar

    2017-05-01

    Full Text Available A convenient method for the preparation of oligonucleotides containing internally-attached galactose and triantennary galactose units has been developed based on click chemistry between 2′-N-alkyne 2′-amino-LNA nucleosides and azido-functionalized galactosyl building blocks. The synthesized oligonucleotides show excellent binding affinity and selectivity towards complementary DNA/RNA strands with an increase in the melting temperature of up to +23.5 °C for triply-modified variants.

  9. Targeting the r(CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides.

    Science.gov (United States)

    Tran, Tuan; Childs-Disney, Jessica L; Liu, Biao; Guan, Lirui; Rzuczek, Suzanne; Disney, Matthew D

    2014-04-18

    We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

  10. Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

    Science.gov (United States)

    Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

    2017-05-01

    Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

  11. Site-Selective Artificial Ribonucleases: Oligonucleotide Conjugates Containing Multiple Imidazole Residues in the Catalytic Domain

    Directory of Open Access Journals (Sweden)

    Natalia G. Beloglazova

    2011-01-01

    Full Text Available Design of site-selective artificial ribonucleases (aRNases is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.

  12. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    Directory of Open Access Journals (Sweden)

    John S. Furey

    2005-04-01

    Full Text Available In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA and Hierarchical Cluster Analysis (HCA were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  13. Probe selection algorithm for oligonucleotide array-based medium-resolution genotyping.

    Science.gov (United States)

    Zhou, Y; Peng, S; Gao, H; Cheng, J

    2004-11-01

    Medium-resolution genotyping has the goal of distinguishing different subgroups instead of each element in a group. An oligonucleotide array provides an inexpensive, high-throughput method to identify differences in DNA sequence among individuals, which is fundamental for genotyping. As the cost and difficulty of designing and fabricating the oligonucleotide array dramatically increase with the number of probes used, it is therefore important to have a design with a minimum number of probes meeting the requirement of medium-resolution genotyping. The first algorithm for designing and selecting probes for oligonucleotide array-based medium-resolution typing is reported. The goal in deriving the algorithm was to select a minimum number of probes from a large probe set on the premise of minimum loss of resolution. The algorithm, which was based on entropy, conditional entropy and mutual information theory, was used to select the minimum number of probes from a large probe set. The algorithm was tested on a human leukocyte antigen (HLA) sequence data set Thirty probes were selected from 390 probes for HLA-A, and 60 probes were selected from 767 probes for HLA-B. Although the number of probes was reduced by almost ten times, the distinguishability was reduced only a little, by 0.45% (from 99.90% to 99.45%) for HLA-A and 0.27% (from 99.84% to 99.57%) for HLA-B, respectively. This is a satisfactory and practical result.

  14. The estimation of quantitative parameters of oligonucleotides immobilization on mica surface

    Science.gov (United States)

    Sharipov, T. I.; Bakhtizin, R. Z.

    2017-05-01

    Immobilization of nucleic acids on the surface of various materials is increasingly being used in research and some practical applications. Currently, the DNA chip technology is rapidly developing. The basis of the immobilization process can be both physical adsorption and chemisorption. A useful way to control the immobilization of nucleic acids on a surface is to use atomic force microscopy. It allows you to investigate the topography of the surface by its direct imaging with high resolution. Usually, to fix the DNA on the surface of mica are used cations which mediate the interaction between the mica surface and the DNA molecules. In our work we have developed a method for estimation of quantitative parameter of immobilization of oligonucleotides is their degree of aggregation depending on the fixation conditions on the surface of mica. The results on study of aggregation of oligonucleotides immobilized on mica surface will be presented. The single oligonucleotides molecules have been imaged clearly, whereas their surface areas have been calculated and calibration curve has been plotted.

  15. Structure/nuclease activity relationships of DNA cleavers based on cationic metalloporphyrin-oligonucleotide conjugates.

    Science.gov (United States)

    Mestre, B; Jakobs, A; Pratviel, G; Meunier, B

    1996-07-16

    The covalent attachment of a managanese-tris(methylpyridiniumyl)porphyrin entity to an antisense oligonucleotide allowed sequence-selective oxidative cleavage of DNA when the metalloporphyrin was activated by potassium monopersulfate (KHSO5). We prepared several structurally modified metallo-porphyrin-oligonucleotide conjugates in order to find out the most efficient compound for in vitro DNA cleavage. The nature and the length of the tether were modulated, the metalloporphyrin entity was modified (metal, ligand), and different ways of activation of the metalloporphyrin were assayed. We noticed that the location of the peptidic bond within the linker could greatly affect the cleavage efficiency of the different conjugates. We showed that the most efficient conjugate for oxidative DNA cleavage was a manganese tetracationic porphyrin-oligonucleotide compound. When the metalloporphyrin moiety was activated by a reducing agent in the presence of molecular oxygen, DNA cleavage was efficient at suitable concentrations of the reducing agent, in order to avoid the reduction of the activated DNA cleaver, a putative high-valent metal-oxo species, by the excess of reducing agent.

  16. Efficient surface patterning of oligonucleotides inside a glass capillary through oxime bond formation.

    Science.gov (United States)

    Dendane, Nabil; Hoang, Antoine; Guillard, Ludovic; Defrancq, Eric; Vinet, Françoise; Dumy, Pascal

    2007-01-01

    The efficient surface patterning of oligonucleotides was accomplished onto the inner wall of fused-silica capillary tubes as well as on the surface of glass slides through oxime bond formation. The robustness of the method was demonstrated by achieving the surface immobilization of up to three different oligonucleotide sequences inside the same capillary tube. The method involves the preparation of surfaces grafted with reactive aminooxy functionalities masked with the photocleavable protecting group, 2-(2-nitrophenyl) propyloxycarbonyl group (NPPOC). Briefly, NPPOC-aminooxy silane 1 was prepared and used to silanize the glass surfaces. The NPPOC group was cleaved under brief irradiation to unmask the reactive aminooxy group on surfaces. These reactive aminooxy groups were allowed to react with aldehyde-containing oligonucleotides to achieve an efficient surface immobilization. The advantage associated with the present approach is that it combines the high-coupling efficiency of oxime bond formation with the convenience associated with the use of photolabile groups. The present strategy thus offers an alternative approach for the immobilization of biomolecules in the microchannels of "labs on a chip" devices.

  17. Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

    Science.gov (United States)

    Yokota, Toshifumi; Wood, Matthew J. A.

    2013-01-01

    Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357

  18. Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Yoshitsugu Aoki

    2013-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials.

  19. Detection of Glucose with Atomic Absorption Spectroscopy by Using Oligonucleotide Functionalized Gold Nanoparticle.

    Science.gov (United States)

    Zhang, Hong; Yan, Honglian; Ling, Liansheng

    2016-06-01

    A novel method for the detection of glucose was established with atomic absorption spectroscopy by using the label of gold nanoparticle (AuNP). Silver-coated glass assembled with oligonucleotide 5'-SH-T12-AGA CAA GAG AGG-3' (Oligo 1) was acted as separation probe, oligonucleotide 5'-CAA CAG AGA ACG-T12-SH-3' modified gold nanoparticle (AuNP-Oligo 2) was acted as signal-reporting probe. Oligonucleotide 5'-CGT TCT CTG TTG CCT CTC TTG TCT-3' (Oligo 3) could hybridize with Oligo 1 on the surface of silver-coated glass and AuNP-Oligo 2, and free AuNP-Oligo 2 could be removed by rinsing with buffer. Hence the concentration of Oligo 3 was transformed into the concentration of gold element. In addition, Oligo 3 could be cleaved into DNA fragments by glucose, glucose oxidase and Fe(2+)-EDTA through Fenton reaction. Thereby the concentration of glucose could be transformed to the absorbance of gold element. Under the optimum conditions, the integrated absorbance decreased proportionally to the concentration of glucose over the range from 50.0 μM to 1.0 mM with a detection limit of 40.0 μM. Moreover, satisfactory result was obtained when the assay was used to determinate glucose in human serum.

  20. Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

    Directory of Open Access Journals (Sweden)

    Martina Schleicher

    2012-01-01

    Full Text Available In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2 and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P<0.05. Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization.

  1. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer.

    Science.gov (United States)

    Bates, Paula J; Laber, Damian A; Miller, Donald M; Thomas, Shelia D; Trent, John O

    2009-06-01

    Certain guanine-rich (G-rich) DNA and RNA molecules can associate intermolecularly or intramolecularly to form four stranded or "quadruplex" structures, which have unusual biophysical and biological properties. Several synthetic G-rich quadruplex-forming oligodeoxynucleotides have recently been investigated as therapeutic agents for various human diseases. We refer to these biologically active G-rich oligonucleotides as aptamers because their activities arise from binding to protein targets via shape-specific recognition (analogous to antibody-antigen binding). As therapeutic agents, the G-rich aptamers may have some advantages over monoclonal antibodies and other oligonucleotide-based approaches. For example, quadruplex oligonucleotides are non-immunogenic, heat stable and they have increased resistance to serum nucleases and enhanced cellular uptake compared to unstructured sequences. In this review, we describe the characteristics and activities of G-rich oligonucleotides. We also give a personal perspective on the discovery and development of AS1411, an antiproliferative G-rich phosphodiester oligonucleotide that is currently being tested as an anticancer agent in Phase II clinical trials. This molecule functions as an aptamer to nucleolin, a multifunctional protein that is highly expressed by cancer cells, both intracellularly and on the cell surface. Thus, the serendipitous discovery of the G-rich oligonucleotides also led to the identification of nucleolin as a new molecular target for cancer therapy.

  2. Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay.

    Directory of Open Access Journals (Sweden)

    Andries Blokzijl

    Full Text Available The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.To investigate SOX10 as a potential biomarker for melanoma and vitiligo.In this study we have applied proximity ligation assay (PLA to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

  3. Ultrasensitive monitoring of ribozyme cleavage product using molecular-beacon-ligation system

    Institute of Scientific and Technical Information of China (English)

    MENG XiangXian; TANG ZhiWen; WANG KeMin; TAN WeiHong; YANG XiaoHai; LI Jun; GUO QiuPing

    2007-01-01

    This paper reports a new approach to detect ribozyme cleavage product based on the molecular- beacon-ligation system. The molecular beacon, designed in such a way that one-half of its loop is complementary to ribozyme cleavage product, is used to monitor ligation process of RNA/DNA complex in a homogeneous solution and to convert directly cleavage product information into fluorescence signal. The method need not label ribozyme and ribozyme substrate, which is fast, simple and ultrasensitive for detection of cleavage product. Detection limit of the assay is 0.05 nmol/L. The cleavage product of hammerhead ribozyme against hepatitis C virus RNA (HCV-RNA) was detected perfectly based on this assay. Owing to its ultrasensitivity, excellent specificity, convenience and fidelity, this method might hold out great promise in ribozyme reaction and ribozyme gene therapy.

  4. Oxime ligation in acetic acid: efficient synthesis of aminooxy-peptide conjugates.

    Science.gov (United States)

    Chelushkin, Pavel S; Leko, Maria V; Dorosh, Marina Yu; Burov, Sergey V

    2017-01-01

    Oxime ligation is a powerful tool in various bioconjugation strategies. Nevertheless, high reaction rates and quantitative yields are typically reported for aldehyde-derived compounds. In contrary, keto groups react much slower, with quantitative yields achieved at 5 h for low-molecular weight compounds and more than 15 h for polymers or dendrimers. In this communication, we report that oxime ligation proceeds rapidly with quantitative (>95%) conversion within 1.5-2 h in pure acetic acid. The practical utility of suggested technique is illustrated by the synthesis of peptide-steroid and peptide-polymer conjugates of model aminooxy-peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  5. Tubal ligation and salpingectomy and the risk of epithelial ovarian cancer and borderline ovarian tumors

    DEFF Research Database (Denmark)

    Madsen, C; Baandrup, Louise; Dehlendorff, Christian

    2015-01-01

    sampling. We required that cases and controls have no previous cancer and that controls have no previous bilateral oophorectomy. METHODS: Conditional logistic regression was used to estimate odds ratios and 95% confidence intervals, adjusting for potential confounders. MAIN OUTCOME MEASURES: Epithelial...... ovarian cancer and borderline ovarian tumors stratified according to histology. RESULTS: Tubal ligation reduced overall epithelial ovarian cancer risk (odds ratios 0.87; 95% confidence interval 0.78-0.98). We observed significant risk variation according to histology (p = 0.003) with the strongest risk...... reductions associated with endometrioid cancer (odds ratios 0.66; 95% confidence interval 0.47-0.93) and epithelial ovarian cancer of "other" histology (odds ratios 0.60; 95% confidence interval 0.43-0.83). Tubal ligation was not associated with risk of borderline ovarian tumors. Finally, bilateral...

  6. Building photoswitchable 3,4'-AMPB peptides: Probing chemical ligation methods with reducible azobenzene thioesters

    Directory of Open Access Journals (Sweden)

    Gehad Zeyat

    2012-06-01

    Full Text Available Photoswitchable peptides were synthesized by using cysteine- and auxiliary-based native chemical ligation reactions. For this purpose, the two regioisomeric azobenzene building blocks 3,4'-AMPB thioester 1b and 4,4'-AMPB thioester 2b were employed in the ligation reactions. While 4,4'-AMPB requires the 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary to minimize reduction of the diazene unit, 3,4'-AMPB can be used in combination with the 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary as well as the Nα-2-mercaptoethyl auxiliary. Thus, 3,4'-AMPB derivatives/peptides proved to be significantly less prone to reduction by aliphatic and aromatic thiols than were the 4,4'-AMPB compounds.

  7. Extrahepatic bile duct ligation in broiler chickens: ultrastructural study of Ito cell

    Directory of Open Access Journals (Sweden)

    Ekowati Handharyani

    2004-12-01

    Full Text Available The Ito cell (fat-storing cell is a cell lying in perisinusoidal space of liver. The function of Ito cell is expanding from a site of fat-storing site to a center of extracellular matrix metabolism and mediator production in the liver. This study was performed in order to evaluate the Ito cells in cholestatic condition. The artificial cholestatic was conducted by ligation of extrahepatic bile ducts (bile duct ligation = BDL in broilers. The results showed that BDL induced bile congestion, fibrosis, proliferation of Ito cells and intrahepatic bile ductules. Immunohistochemistry demonstrated that Ito cells were scattered throughout the fibrotic areas, and larger in size with more extensive immunoreactivity than those in normal livers. Ultrastructural study demonstrated that Ito cells were closely associated with the production of extracellular collagen fibers. Ito cells actively react against hepatocytic injuries, especially in fibrogenesis of cholestatic livers.

  8. Contraception methods, beyond oral contraceptives and tubal ligation, and risk of ovarian cancer.

    Science.gov (United States)

    Ness, Roberta B; Dodge, Rhiannon C; Edwards, Robert P; Baker, Julie A; Moysich, Kirsten B

    2011-03-01

    Few studies have examined methods of contraception, beyond oral contraceptives (OCs) and tubal ligation, in relation to ovarian cancer risk. Nine hundred two cases with incident ovarian/peritoneal/tubal cancer were compared with 1800 population-based control subjects. Women self-reported all methods of contraception by using life calendars. Each of the contraceptive methods examined reduced the risk of ovarian cancer as compared with use of no artificial contraception. Comparing ever versus never use, after adjustment for potentially confounding factors and all other methods of contraception, the methods of contraception that emerged as protective were OCs (adjusted odds ratio [adj OR] 0.75, 95% confidence interval [CI] 0.61-0.93); tubal ligation (adj OR 0.63, 95% CI 0.51-0.77); intrauterine devices (IUDs) (adj OR 0.75, 95% CI 0.59-0.95); and vasectomy (adj OR 0.77, 95% CI 0.61-0.99). Although for OCs and tubal ligation we found that the longer the duration of use, the greater the effect, for IUDs the pattern was reversed: significant protection occurred with short duration and progressively greater risk (albeit nonsignificant) was seen with longer duration of use. In the largest case-control study to date, a range of effective methods of contraception reduced the risk for ovarian cancer. OCs and tubal ligation reduced ovarian cancer risk with lower odds ratios with longer duration of use, whereas IUDs reduced risk overall, having the greatest impact with short duration of use. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. An enhanced method for sequence walking and paralog mining: TOPO® Vector-Ligation PCR

    Directory of Open Access Journals (Sweden)

    Davis Thomas M

    2010-03-01

    Full Text Available Abstract Background Although technological advances allow for the economical acquisition of whole genome sequences, many organisms' genomes remain unsequenced, and fully sequenced genomes may contain gaps. Researchers reliant upon partial genomic or heterologous sequence information require methods for obtaining unknown sequences from loci of interest. Various PCR based techniques are available for sequence walking - i.e., the acquisition of unknown DNA sequence adjacent to known sequence. Many such methods require rigid, elaborate protocols and/or impose narrowly confined options in the choice of restriction enzymes for necessary genomic digests. We describe a new method, TOPO® Vector-Ligation PCR (or TVL-PCR that innovatively integrates available tools and familiar concepts to offer advantages as a means of both targeted sequence walking and paralog mining. Findings TVL-PCR exploits the ligation efficiency of the pCR®4-TOPO® (Invitrogen, Carlsbad, California vector system to capture fragments of unknown sequence by creating chimeric molecules containing defined priming sites at both ends. Initially, restriction enzyme-digested genomic DNA is end-repaired to create 3' adenosine overhangs and is then ligated to pCR4-TOPO vectors. The ligation product pool is used directly as a template for nested PCR, using specific primers to target orthologous sequences, or degenerate primers to enable capture of paralogous gene family members. We demonstrated the efficacy of this method by capturing entire coding and partial promoter sequences of several strawberry Superman-like genes. Conclusions TVL-PCR is a convenient and efficient method for DNA sequence walking and paralog mining that is applicable to any organism for which relevant DNA sequence is available as a basis for primer design.

  10. Effects of self-ligating and conventional brackets on halitosis and periodontal conditions.

    Science.gov (United States)

    Kaygisiz, Emine; Uzuner, Fatma Deniz; Yuksel, Sema; Taner, Levent; Çulhaoğlu, Rana; Sezgin, Yasemin; Ateş, Can

    2015-05-01

    To evaluate the effects of fixed orthodontic treatment with steel-ligated conventional brackets and self-ligating brackets on halitosis and periodontal health. Sixty patients, at the permanent dentition stage aged 12 to 18 years, who had Angle Class I malocclusion with mild-to-moderate crowding were randomly selected. Inclusion criteria were nonsmokers, without systematic disease, and no use of antibiotics and oral mouth rinses during the 2-month period before the study. The patients were subdivided into three groups randomly: the group treated with conventional brackets (group 1, n  =  20) ligated with steel ligature wires, the group treated with self-ligating brackets (group 2, n  =  20), and the control group (group 3, n  =  20). The periodontal records were obtained 1 week before bonding (T1), immediately before bonding (T2), 1 week after bonding (T3), 4 weeks after bonding (T4), and 8 weeks after bonding (T5). Measurements of the control group were repeated within the same periods. The volatile sulfur components determining halitosis were measured with the Halimeter at T2, T3, T4, and T5. A two-way repeated measures of analysis of variance (ANOVA) was used to compare the groups statistically. No statistically significant group × time interactions were found for plaque index, gingival index, pocket depth, bleeding on probing, and halitosis, which means three independent groups change like each other by time. The risk of tongue coating index (TCI) being 2 was 10.2 times higher at T1 than at T5 (P halitosis.

  11. Comparison between Ultroid and Rubber Band Ligation in Treatment of Internal Hemorrhoids

    Directory of Open Access Journals (Sweden)

    Rasoul Azizi

    2010-11-01

    Full Text Available "nHemorrhoid is one of the most common surgical diseases and different methods are available for its treatment. This study is a comparison between two methods of treatment of internal hemorrhoid, Monopolar low voltage instrument (Ultroid and Rubber Band Ligation. This method has been carried out prospectively in which 50 patients who were treated with rubber band ligation and 50 patients with Ultroid were compared according to the incidence of complications, post-operative pain and treatment response. According to this study complete success rate with Ultroid was 82% and partial success rate was 10% and no response to treatment was seen in 8%. In Rubber Band method the complete response rate was 94% (P=0.2. With Ultroid, 74% of patient reported no postoperative pain, 24% reported mild and moderate pain and 2% of patients complained of severe pain. With Rubber band ligation, 72% of patients reported no post-operative pain, 26% reported mild and moderate pain and 1% complained of severe pain (P=0.00. Rubber Band ligation and Ultroid are both considered as outpatient procedures for treatment of hemorrhoids. Both methods are mostly used for grade 1, 2 and sometime grade 3 hemorrhoids. In Ultroid method the operator is required to hold the probe for a period of time, and in most cases, the surgeon should spend between 20-25 minutes for the coagulation of three piles. Some surgeons do not have patience for this modality of internal hemorrhoid treatment. In this study we achieved acceptable results comparable with those of other techniques.

  12. Comparison between Ultroid and Rubber Band Ligation in Treatment of Internal Hemorrhoids

    Directory of Open Access Journals (Sweden)

    Rasoul Azizi

    2010-12-01

    Full Text Available Hemorrhoid is one of the most common surgical diseases and different methods are available for its treatment. This study is a comparison between two methods of treatment of internal hemorrhoid, Monopolar low voltage instrument (Ultroid and Rubber Band Ligation. This method has been carried out prospectively in which 50 patients who were treated with rubber band ligation and 50 patients with Ultroid were compared according to the incidence of complications, post-operative pain and treatment response. According to this study complete success rate with Ultroid was 82% and partial success rate was 10% and no response to treatment was seen in 8%. In Rubber Band method the complete response rate was 94% (P=0.2. With Ultroid, 74% of patient reported no postoperative pain, 24% reported mild and moderate pain and 2% of patients complained of severe pain. With Rubber band ligation, 72% of patients reported no post-operative pain, 26% reported mild and moderate pain and 1% complained of severe pain (P=0.00. Rubber Band ligation and Ultroid are both considered as outpatient procedures for treatment of hemorrhoids. Both methods are mostly used for grade 1, 2 and sometime grade 3 hemorrhoids. In Ultroid method the operator is required to hold the probe for a period of time, and in most cases, the surgeon should spend between 20-25 minutes for the coagulation of three piles. Some surgeons do not have patience for this modality of internal hemorrhoid treatment. In this study we achieved acceptable results comparable with those of other techniques.

  13. TUBAL LIGATION, REPRODUCTIVE EXPERIENCES AND RISK OF OVARIAN CANCER- A PROSPECTIVE STUDY

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    Bessy Binu Sam

    2017-03-01

    Full Text Available BACKGROUND Ovarian cancer is the most lethal malignancy of the female reproductive system. Risk of ovarian cancer increases with age, but the rate of increase slows after the menopause. Tubal ligation confers long-term protection against ovarian cancer. This observational study examines the factors affecting the ovarian cancer risk and also studies the correlation between ovarian cancer risks with reproductive experiences. MATERIALS AND METHODS This study was conducted at Department of Obstetrics and Gynaecology, Government Medical College, Kottayam, Kerala, for a period of one year. Information was collected from 112 women diagnosed with ovarian cancer as treatment group and 336 women without ovarian cancer as control group. A binary logit regression analysis was conducted to study the factors that are affecting the ovarian cancer. The Chi-square test was done to find the association of ovarian cancer risk with different reproductive experiences. RESULTS We found that months of lactation, tubal ligation and oral contraceptive pills had a negative impact on ovarian cancer risk. Our study also proved that age of first pregnancy, age of menarche and age of menopause had a significant association with ovarian cancer risk. CONCLUSION Our findings signify the importance of providing awareness to the public regarding the prevalence, symptomatology, screening/diagnostic techniques, treatment modalities and prognosis of ovarian cancer. The dual benefits of tubal ligation need to be made aware among the public and tubal sterilisation rates have to be enhanced. We recommend the promotion of tubal ligation as a permanent method of contraception in those who have completed their families.

  14. Banding ligation versus beta-blockers for primary prevention in oesophageal varices in adults

    DEFF Research Database (Denmark)

    Gluud, Lise Lotte; Krag, Aleksander

    2012-01-01

    Non-selective beta-blockers are used as a first-line treatment for primary prevention in patients with medium- to high-risk oesophageal varices. The effect of non-selective beta-blockers on mortality is debated and many patients experience adverse events. Trials on banding ligation versus non......-selective beta-blockers for patients with oesophageal varices and no history of bleeding have reached equivocal results....

  15. STUDY OF ANTIULCER ACTIVITY OF ROOTS OF ALANGIUM SALVIFOLIUM LINN. IN PYLORUS LIGATED RATS

    OpenAIRE

    Mohanty P.K.; Panda S.K.; Mishra S.K.; Panda P.K.; Jaliwala Y.A.; Parle Milind

    2011-01-01

    Different extracts of Alangium salvifolium Linn. roots were tested for its antiulcer activity in aspirin induced and pylorus ligated rats model. The effect was assessed by parameters like Total acidity(TA),Free acidity(FA),Peptic activity(PA), Ulcer Index(UI) .The antiucler activity of all extracts were compared to the standard drug ranitidine. The study showed that all extracts were showing reductions TA, PA, FA and UI among which the extract of petroleum ether showed significant reduction ...

  16. Cardioprotective effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats.

    Science.gov (United States)

    Gao, Yan; Gao, Jianping; Chen, Changxun; Wang, Huilin; Guo, Juan; Wu, Rong

    2015-05-01

    The purpose of this study was to explore the effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats and to elucidate the underlying mechanisms. A rat model of ventricular remodeling after myocardial infarction was established by left coronary artery ligation. Rats with coronary artery ligation were randomly divided into five groups: control, plus 40 mg/kg captopril, plus 25 mg/kg polydatin, plus 50 mg/kg polydatin, and plus 100 mg/kg polydatin. The sham-operated group was used as a negative control. Rats were administered intragastrically with the corresponding drugs or drinking water for seven weeks. At the end of the treatment, the left ventricular weight index and heart weight index were assessed. The cross-sectional size of cardiomyocytes was measured by staining myocardium tissue with hematoxylin and eosin. Collagen content was counted by Sirius red in aqueous saturated picric acid. The concentrations of angiotensin I, angiotensin II, aldosterone, and endothelin 1 in myocardium or serum were determined by radioimmunoassay. Hydroxyproline and nitric oxide concentrations and glutathione peroxidase and catalase activities in serum were measured by ultraviolet spectrophotometry. Our results showed that seven weeks of polydatin treatment resulted in a significantly reduced left ventricular weight index, heart weight index, serum concentrations of hydroxyproline and aldosterone, an increased concentration of nitric oxide as well as enhanced activities of glutathione peroxidase and catalase. Myocardial angiotensin I, angiotensin II, and endothelin 1 levels were also reduced. The cardiomyocyte cross-sectional area and collagen deposition diminished. This study suggests that polydatin may attenuate ventricular remodeling after myocardial infarction in coronary artery ligation rats through restricting the excessive activation of the renin-angiotensin-aldosterone system and inhibiting peroxidation. Georg Thieme

  17. Outcomes of Foam Sclerotherapy plus Ligation versus Foam Sclerotherapy Alone for Venous Ulcers in Lower Extremities.

    Science.gov (United States)

    Li, Xin; Fan, Lin; Ren, Shiyan; Li, Xianlun

    2017-11-01

    Foam sclerotherapy (FS) is a safe and effective approach for managing patients with varicose veins and venous ulcers in lower extremities. But, recanalization of the ablated varicose veins and phlebitis are common postoperative complications that jeopardize its clinical effects. We hypothesize that ligation of the ablated varicose veins after FS will improve the outcomes of patients with varicose veins and venous ulcer. This study was aimed to evaluate the clinical efficacy of ligation after FS in comparison with FS alone for the management of patients with varicose veins and venous ulcers in lower extremities. Eighteen patients underwent FS plus ligation (FSL) and 15 patients received FS alone. Aberdeen varicose veins questionnaire (AVVQ) and the revised venous clinical severity score (rVCSS), venous disability scores (VDSs), duplex sonography, ulcer healing rate, and ulcer healing time were documented to compare the outcomes in both groups. The ulcer healing time in patients treated with FSL was shorter than that in patients who received FS (P = 0.022; log-rank test). The average healing time was significantly shorter in FSL group than in FS group (35.67 ± 24.62 days vs. 62.86 ± 47.43, P = 0.042). The mean rVCSS, VDS, and AVVQ at 3 months after treatment in both groups decreased significantly in comparison with baseline, respectively. There were no severe complications or side effects in both groups. Ligation of the treated varicose veins after FS can improve the outcomes of patients with venous ulcers in comparison with FS alone. FSL is a safe, effective, and technically feasible procedure and can be used as a day surgery. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Impact of aberrant left hepatic artery ligation on the outcome of liver transplantation.

    Science.gov (United States)

    Montalti, Roberto; Benedetti Cacciaguerra, Andrea; Nicolini, Daniele; Alì Ahmed, Emad; Coletta, Martina; De Pietri, Lesley; Risaliti, Andrea; Troisi, Roberto Ivan; Mocchegiani, Federico; Vivarelli, Marco

    2017-12-06

    The preservation of a graft's aberrant left hepatic artery (LHA) during liver transplantation (LT) ensures optimal vascularization of the left liver but can also be considered a risk factor for hepatic artery thrombosis. In contrast, ligation of an aberrant LHA may lead to hepatic ischaemia with the potential risk of graft dysfunction and biliary complications. The aim of this study was to prospectively analyse the impact on the surgical strategy for LT of 5 tests performed to establish whether an aberrant LHA was an accessory or a replaced artery, thus leading to the design of a decisional algorithm. From 8/2005 to 12/2016, 395 whole LTs were performed in 376 patients. Five parameters were evaluated to determine whether an aberrant LHA was an accessory or a replaced artery. Based on our decision algorithm, an aberrant LHA was ligated during surgery when assessed as accessory and preserved when assessed as replaced. A total of 138 anatomical variants of hepatic arterial vascularization occurred in 120/395 (30.4%) grafts. Overall, the incidence of an aberrant LHA was 63/395 (15.9%). The LHA was ligated in 33 patients (52.4%) and preserved in 30 patients (47.6%). After a mean follow-up period of 46.2±35.9 months, the incidence of hepatic artery thrombosis, primary non-function, early allograft dysfunction, biliary stricture or leaks and overall survival was similar in the two groups. Once shown to be an accessory, an LHA can be safely ligated without clinical consequences on the outcome of LT. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

  19. A comparison of vas occlusion techniques: cautery more effective than ligation and excision with fascial interposition

    OpenAIRE

    Labrecque Michel; Chen-Mok Mario; Irsula Belinda; Sokal David; Barone Mark A

    2004-01-01

    Abstract Background Vasectomy techniques have been the subject of relatively few rigorous studies. The objective of this analysis was to compare the effectiveness of two techniques for vas occlusion: intraluminal cautery versus ligation and excision with fascial interposition. More specifically, we aimed to compare early failure rates, sperm concentrations, and time to success between the two techniques. Methods We compared semen analysis data from men following vasectomy using two occlusion ...

  20. Anesthetic management of an orangutan (Pongo abelii/pygmaeus) undergoing laparoscopic tubal ligation.

    Science.gov (United States)

    Hendrix, Paula K

    2006-12-01

    An adult hybrid orangutan (Pongo abelii/pygmaeus) was presented to a veterinary teaching hospital for laparoscopic tubal ligation. The orangutan was immobilized with the use of injectable anesthetic agents, then orotracheally intubated. Anesthesia was maintained with the use of isoflurane in oxygen, and positive-pressure ventilation was used to ensure adequate gas exchange. Parameters monitored included arterial blood pressure, ECG, capnometry, and arterial blood gases. Anesthesia was uneventful, and recovery was smooth.

  1. Are self-ligating brackets related to less formation of Streptococcus mutans colonies? A systematic review

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    Leonard Euler Andrade Gomes do Nascimento

    2014-01-01

    Full Text Available OBJECTIVE: To verify, by means of a systematic review, whether the design of brackets (conventional or self-ligating influences adhesion and formation of Streptococcus mutans colonies. METHODS: Search strategy: four databases (Cochrane Central Register of Controlled Trials, Ovid ALL EMB Reviews, PubMed and BIREME were selected to search relevant articles covering the period from January 1965 to December 2012. Selection Criteria: in first consensus by reading the title and abstract. The full text was obtained from publications that met the inclusion criteria. Data collection and analysis: Two reviewers independently extracted data using the keywords: conventional, self-ligating, biofilm, Streptococcus mutans, and systematic review; and independently evaluated the quality of the studies. In case of divergence, the technique of consensus was adopted. RESULTS: The search strategy resulted in 1,401 articles. The classification of scientific relevance revealed the high quality of the 6 eligible articles of which outcomes were not unanimous in reporting not only the influence of the design of the brackets (conventional or self-ligating over adhesion and formation of colonies of Streptococcus mutans, but also that other factors such as the quality of the bracket type, the level of individual oral hygiene, bonding and age may have greater influence. Statistical analysis was not feasible because of the heterogeneous methodological design. CONCLUSIONS: Within the limitations of this study, it was concluded that there is no evidence for a possible influence of the design of the brackets (conventional or self-ligating over colony formation and adhesion of Streptococcus mutans.

  2. Histamine receptors expressed in circulating progenitor cells have reciprocal actions in ligation-induced arteriosclerosis.

    Science.gov (United States)

    Yamada, Sohsuke; Wang, Ke-Yong; Tanimoto, Akihide; Guo, Xin; Nabeshima, Atsunori; Watanabe, Takeshi; Sasaguri, Yasuyuki

    2013-09-01

    Histamine is synthesized as a low-molecular-weight amine from L-histidine by histidine decarboxylase (HDC). Recently, we demonstrated that carotid artery-ligated HDC gene-deficient mice (HDC(-/-)) showed less neointimal formation than wild-type (WT) mice, indicating that histamine participates in the process of arteriosclerosis. However, little is known about the roles of histamine-specific receptors (HHRs) in arteriosclerosis. To define the roles of HHRs in arteriosclerosis, we investigated intimal remodeling in ligated carotid arteries of HHR-deficient mice (H1R(-/-) or H2R(-/-)). Quantitative analysis showed that H1R(-/-) mice had significantly less arteriosclerogenesis, whereas H2R(-/-) mice had more, as compared with WT mice. Bone marrow transplantation from H1R(-/-) or H2R(-/-) to WT mice confirmed the above observation. Furthermore, the increased expression of monocyte chemoattractant protein (MCP-1), platelet-derived growth factor (PDGF), adhesion molecules and liver X receptor (LXR)-related inflammatory signaling factors, including Toll-like receptor (TLR3), interleukin-1 receptor (IL-1R) and tumor necrosis factor receptor (TNF-R), was consistent with the arteriosclerotic phenotype of H2R(-/-) mice. Peripheral progenitor cells in H2R(-/-) mice accelerate ligation-induced arteriosclerosis through their regulation of MCP-1, PDGF, adhesion molecules and LXR-related inflammatory signaling factors. In contrast, peripheral progenitor cells act to suppress arteriosclerosis in H1R(-/-) mice, indicating that HHRs reciprocally regulate inflammation in the ligation-induced arteriosclerosis. © 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

  3. The Association between Endometriosis, Tubal Ligation, Hysterectomy and Epithelial Ovarian Cancer: Meta-Analyses

    Directory of Open Access Journals (Sweden)

    Chunpeng Wang

    2016-11-01

    Full Text Available To investigate the association between endometriosis, tubal ligation, hysterectomy and epithelial ovarian cancer. Relevant published literatures were searched in PubMed, ProQuest, Web of Science and Medline databases during 1995–2016. Heterogeneity was evaluated by I2 statistic. Publication bias was tested by funnel plot and Egger’s test. Odds ratio and 95% CI were used to assess the association strength. The statistical analyses in this study were accomplished by STATA software package. A total of 40,609 cases of epithelial ovarian cancer and 368,452 controls in 38 publications were included. The result suggested that endometriosis was associated with an increased risk of epithelial ovarian cancer (OR = 1.42, 95% CI = 1.28–1.57, tubal ligation was associated with a decreased risk of epithelial ovarian cancer (OR = 0.70, 95% CI = 0.60–0.81, while hysterectomy show no relationship with epithelial ovarian cancer (OR = 0.97, 95% CI = 0.81–1.14. A stratified analysis showed there were associations between endometriosis and the increased risk of epithelial ovarian cancer for studies conducted in USA and Europe. Meanwhile, there were associations between tubal ligation and the decreased risk of epithelial ovarian cancer for studies conducted in USA, Asia, Europe and Australia. The result indicated that endometriosis was a risk factor of epithelial ovarian cancer whereas tubal ligation was a protective risk factor of epithelial ovarian cancer, hysterectomy may have no relationship with epithelial ovarian cancer.

  4. Cannabidiol ameliorates cognitive and motor impairments in mice with bile duct ligation.

    Science.gov (United States)

    Magen, Iddo; Avraham, Yosefa; Ackerman, Zvi; Vorobiev, Lia; Mechoulam, Raphael; Berry, Elliot M

    2009-09-01

    The endocannabinoid system in mice plays a role in models of human cirrhosis and hepatic encephalopathy (HE), induced by a hepatotoxin. We report now the therapeutic effects of cannabidiol (CBD), a non-psychoactive constituent of Cannabis sativa, on HE caused by bile duct ligation (BDL), a model of chronic liver disease. CBD (5mg/kg; i.p.) was administered over 4weeks to mice that had undergone BDL. Cognitive function in the eight arm maze and the T-maze tests, as well as locomotor function in the open field test were impaired by the ligation and were improved by CBD. BDL raised hippocampal expression of the TNF-alpha-receptor 1 gene, which was reduced by CBD. However, BDL reduced expression of the brain-derived neurotrophic factor (BDNF) gene, which was increased by CBD. The effects of CBD on cognition, locomotion and on TNF-alpha receptor 1 expression were blocked by ZM241385, an A(2)A adenosine receptor antagonist. BDL lowers the expression of this receptor. The effects of BDL apparently result in part from down-regulation of A(2)A adenosine receptor. CBD reverses these effects through activation of this receptor, leading to compensation of the ligation effect.

  5. Psychosocial implications of tubal ligation in a rural health district: A phenomenological study

    Directory of Open Access Journals (Sweden)

    Lutala Prosper M

    2011-12-01

    Full Text Available Abstract Background Tubal ligation is the most popular family planning method worldwide. While its benefits, such as effectiveness in protecting against pregnancies, minimal need for long-term follow-up and low side-effects profile are well documented, it has many reported complications. However, to date, these complications have not been described by residents in Congo. Therefore, the study aimed at exploring the experience of women who had undergone tubal ligation, focusing on perceptions of physical, psychological and contextual experiences of participants. Methods This qualitative study used a semi-structured questionnaire in a phenomenological paradigm to collect data. Fifteen participants were purposefully selected among sterilized women who had a ligation procedure performed, were aged between 30 and 40 years, and were living within the catchment area of the district hospital. Data were collected by two registered nurses, tape-recorded, and transcribed verbatim. Reading and re-reading cut and paste techniques, and integration were used to establish codes, categories, themes, and description. Results Diverse and sometimes opposite changes in somatic symptoms, psychological symptoms, productivity, ecological relationships, doctor-client relationships, ethical issues, and change of life style were the major problem domains. Conclusions Clients reported conflicting experiences in several areas of their lives after tubal sterilization. Management, including awareness of the particular features of the client, is needed to decrease the likelihood of psychosocial morbidity and/or to select clients in need of sterilization.

  6. Psychosocial implications of tubal ligation in a rural health district: A phenomenological study

    Science.gov (United States)

    2011-01-01

    Background Tubal ligation is the most popular family planning method worldwide. While its benefits, such as effectiveness in protecting against pregnancies, minimal need for long-term follow-up and low side-effects profile are well documented, it has many reported complications. However, to date, these complications have not been described by residents in Congo. Therefore, the study aimed at exploring the experience of women who had undergone tubal ligation, focusing on perceptions of physical, psychological and contextual experiences of participants. Methods This qualitative study used a semi-structured questionnaire in a phenomenological paradigm to collect data. Fifteen participants were purposefully selected among sterilized women who had a ligation procedure performed, were aged between 30 and 40 years, and were living within the catchment area of the district hospital. Data were collected by two registered nurses, tape-recorded, and transcribed verbatim. Reading and re-reading cut and paste techniques, and integration were used to establish codes, categories, themes, and description. Results Diverse and sometimes opposite changes in somatic symptoms, psychological symptoms, productivity, ecological relationships, doctor-client relationships, ethical issues, and change of life style were the major problem domains. Conclusions Clients reported conflicting experiences in several areas of their lives after tubal sterilization. Management, including awareness of the particular features of the client, is needed to decrease the likelihood of psychosocial morbidity and/or to select clients in need of sterilization. PMID:22176816

  7. Impairment of the organization of locomotor and exploratory behaviors in bile duct-ligated rats

    DEFF Research Database (Denmark)

    Leke, Renata; de Oliveira, Diogo L; Mussulini, Ben Hur M.

    2012-01-01

    Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different ...... from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior....... rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult...... female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure...

  8. Development of a vivo rabbit ligated intestinal Loop Model for HCMV infection.

    Science.gov (United States)

    Tang, Jin; Wu, Qiaoxing; Tang, Xinming; Shi, Ruihan; Suo, Jingxia; Huang, Guangping; An, Junqing; Wang, Jingyuan; Yang, Jinling; Hao, Wenzhuo; She, Ruiping; Suo, Xun

    2016-01-01

    Human Cytomegalovirus (HCMV) infections can be found throughout the body, especially in epithelial tissue. Animal model was established by inoculation of HCMV (strain AD-169) or coinoculation with Hepatitis E virus (HEV) into the ligated sacculus rotundus and vermiform appendix in living rabbits. The specimens were collected from animals sacrificed 1 and a half hours after infection. The virus was found to be capable of reproducing in these specimens through RT-PCR and Western-blot. Severe inflammation damage was found in HCMV-infected tissue. The viral protein could be detected in high amounts in the mucosal epithelium and lamina propria by immunohistochemistry and immunofluorescense. Moreover, there are strong positive signals in lymphocytes, macrophages, and lymphoid follicles. Quantitative statistics indicate that lymphocytes among epithlium cells increased significantly in viral infection groups. The results showed that HCMV or HEV + HCMV can efficiently infect in rabbits by vivo ligated intestine loop inoculation. The present study successfully developed an infective model in vivo rabbit ligated intestinal Loop for HCMV pathogenesis study. This rabbit model can be helpful for understanding modulation of the gut immune system with HCMV infection.

  9. Controlling Peptide Self-Assembly through a Native Chemical Ligation/Desulfurization Strategy.

    Science.gov (United States)

    Rasale, Dnyaneshwar B; Konda, Maruthi; Biswas, Sagar; Das, Apurba K

    2016-03-18

    Self-assembled peptides were synthesized by using a native chemical ligation (NCL)/desulfurization strategy that maintained the chemical diversity of the self-assembled peptides. Herein, we employed oxo-ester-mediated NCL reactions to incorporate cysteine, a cysteine-based dipeptide, and a sterically hindered unnatural amino acid (penicillamine) into peptides. Self-assembly of the peptides resulted in the formation of self-supporting gels. Microscopy analysis indicated the formation of helical nanofibers, which were responsible for the formation of gel matrices. The self-assembly of the ligated peptides was governed by covalent and non-covalent interactions, as confirmed by FTIR, CD, fluorescence spectroscopy, and MS (ESI) analyses. Peptide disassembly was induced by desulfurization reactions with tris(2-carboxyethyl)phosphine (TCEP) and glutathione at 80 °C. Desulfurization reactions of the ligated peptides converted the Cys and penicillamine functionalities into Ala and Val moieties, respectively. The self-supporting gels showed significant shear-thinning and thixotropic properties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

    Directory of Open Access Journals (Sweden)

    Tingshuang Xu

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD, we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk, a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

  11. Arterial Ligation for Infected Femoral Psuedo-Aneurysm in Drug Injecting Abusers

    Directory of Open Access Journals (Sweden)

    Mohammadzade Mohammad Ali

    2009-10-01

    Full Text Available Pseudo-aneurysm of the femoral artery is the most common arterial complication in drug injecting abusers. Scholars in vascular surgery have published debating statements regarding techniques of successful surgical management during last two decades. We present the results of simple arterial ligation in a series of 32 patients presenting with infected femoral pseudo-aneurysm. Most of the patients were males (89%. Young persons in the age group of 15-44 years were mostly affected. Site of lesion included common femoral artery in 65% , superficial femoral artery 28% and at bifurcation 6.2%. celulitis in 14 (53%, abscess & "ncelulitis in 6 (19%, necrosing fasciitis in 2 (6.2% and vascular abscess in 7 (22% cases were the forms of associated local infection. There was no hemorrhage, vascular thrombosis, amputation, or mortality. Claudicating were the only complications identified in 2 patients with Tripe ligation. Ligation is the optimal management for infected pseudo-aneurysms because it is easy, cost-effective, and safe. Early reconstruction is not recommended, since there is an extended infection in the location of the pseudo-aneurysm.

  12. Recovery of the Zika virus through an in vitro ligation approach.

    Science.gov (United States)

    Deng, Cheng-Lin; Zhang, Qiu-Yan; Chen, Dong-Dong; Liu, Si-Qing; Qin, Cheng-Feng; Zhang, Bo; Ye, Han-Qing

    2017-07-01

    In this study, an in vitro ligation method was developed to assemble a full-length infectious cDNA clone of the Zika virus (ZIKV). Four contiguous cDNA subclones covering the complete ZIKV genome were constructed with unique BglI restriction sites at the ends of each fragment. The BglI restriction sites only allow in vitro ligation to happen between interconnecting restriction sites from adjacent cDNA fragments, resulting in an intact full-length cDNA of ZIKV. RNA transcripts derived from the full-length cDNA were infectious. The recombinant virus replicated as efficiently as the wild-type virus with similar growth kinetics and plaque morphologies in Vero and C6/36 cells. Both viruses were inhibited by NITD008 treatment. This in vitro ligation method will facilitate manipulation of the viral genome through genetic modifications of four separated subclones of ZIKV for the rapid and rational development of candidate vaccines and viral replication study.

  13. Evaluation of force released by deflection of orthodontic wires in conventional and self-ligating brackets

    Directory of Open Access Journals (Sweden)

    Rodrigo Hitoshi Higa

    Full Text Available ABSTRACT Introduction: The aim of the study was to evaluate deflection forces of rectangular orthodontic wires in conventional (MorelliTM, active (In-Ovation RTM and passive (Damon 3MXTM self-ligating brackets. Material and Methods: Two brands of stainless steel and nickel-titanium (NiTi wires (MorelliTM and GACTM, in addition to OrmcoTM copper-nickel-titanium wires were used. Specimens were assembled in a clinical simulation device especially designed for this study and tested in an Instron universal testing machine. For the testing procedures, an acrylic structure representative of the maxillary right central incisor was lingually moved in activations of 0 to 1 mm, with readings of the force released by deflection in unloading of 0.5, 0.8 and 1 mm at a constant speed of 2 mm/min. Inter-bracket forces with stainless steel, NiTi and CuNiTi were individually compared by two-way ANOVA, followed by Tukey’s tests. Results: Results showed that there were lower forces in conventional brackets, followed by active and passive self-ligating brackets. Within the brands, only for NiTi wires, the MorelliTM brand presented higher forces than GACTM wires. Conclusions: Bracket systems provide different degrees of deflection force, with self-ligating brackets showing the highest forces.

  14. TWIK-Related Spinal Cord K+ Channel Expression Is Increased in the Spinal Dorsal Horn after Spinal Nerve Ligation

    National Research Council Canada - National Science Library

    Hwang, Hee Youn; Zhang, Enji; Park, Sangil; Chung, Woosuk; Lee, Sunyeul; Kim, Dong Woon; Ko, Youngkwon; Lee, Wonhyung

    2015-01-01

    .... Because there have been no reports on the TRESK expression or its function in the dorsal horn of the spinal cord in neuropathic pain, we analyzed TRESK expression in the spinal dorsal horn in a spinal nerve ligation (SNL) model...

  15. Modified distal revascularization with interval ligation procedure for steal syndrome after arteriovenous fistula creation for hemodialysis access

    NARCIS (Netherlands)

    van der Meer, Saskia; Zeebregts, Clark; Tielliu, Ignacc; Verhoeven, Eric; van den Dungen, Jan

    2007-01-01

    Patients diagnosed with steal syndrome after hemodialysis access surgery have a few options for symptom relief while maintaining vascular access. These include fistula lengthening, banding, distal revascularization with interval ligation (DRIL), revision using distal inflow (RUDI) or proximalization

  16. QSAR and Molecular Docking Studies of Oxadiazole-Ligated Pyrrole Derivatives as Enoyl-ACP (CoA) Reductase Inhibitors

    National Research Council Canada - National Science Library

    Asgaonkar, Kalyani D; Mote, Ganesh D; Chitre, Trupti S

    2014-01-01

    A quantitative structure-activity relationship model was developed on a series of compounds containing oxadiazole-ligated pyrrole pharmacophore to identify key structural fragments required for anti-tubercular activity. Two-dimensional (2D...

  17. Reversible Exchange of L-Type and Bound-Ion-Pair X-Type Ligation on Cadmium Selenide Quantum Belts.

    Science.gov (United States)

    Zhou, Yang; Buhro, William E

    2017-09-20

    CdSe quantum belts of composition {CdSe[n-octylamine]0.53} and protic acids HX (X = Cl, Br, NO3, acetate (OAc), and benzoate (OBz)) react to exchange the L-type amine ligation to bound-ion-pair X-type ligation. The latter ligation has X- anions bound to the nanocrystal surfaces and closely associated LH+ counter-cations (protonated n-octylamine or tri-n-octylphosphine (TOP) to balance the surface charges. The compositions of the exchanged QBs are {CdSe[Br]0.44[n-octylammonium]0.41}, {CdSe[NO3]0.10[TOPH]0.12}, {CdSe[OBz]0.08[n-octylammonium]0.02[TOPH]0.06}, and {CdSe[OAc]0.16[n-octylammonium]0.02[TOPH]0.14}. (The HCl-exchanged QBs are insufficiently stable for elemental analysis.) The bound-ion-pair X-type ligation is fully reversed to L-type n-octylamine ligation in the cases of X = NO3, acetate, and benzoate. The ligand exchanges are monitored by absorption spectroscopy, and the exchanged, bound-ion-pair X-type ligated nanocrystals are characterized by a range of methods.

  18. The leveling effectiveness of self-ligating and conventional brackets for complex tooth malalignments.

    Science.gov (United States)

    Fansa, Magali; Keilig, Ludger; Reimann, Susanne; Jäger, Andreas; Bourauel, Christoph

    2009-07-01

    The transfer of forces and moments between the bracket and archwire is decisive in the multi-band/bracket technique. New developments in bracket design and ligation method aim to optimize the transfer of forces and moments and improve leveling effectiveness. We thus aimed in this study to investigate whether leveling behavior is influenced by different bracket systems, or by the ligation method. The baseline situation for this examination was a complex tooth malalignment. Using the orthodontic measurement and simulation system (OMSS), we tested the leveling effectiveness of nine self-ligating bracket systems made by various manufacturers (Forestadent-Quick, in active and passive variants, Dentsply GAC In-Ovation, adenta TIME, Ormco Damon 2 and Damon 3MX, UP-Dental Opal-M and Opal-2, Strite SPEED) in the 0.022 inch slot system. A conventional bracket system (Dentaurum discovery) was used for reference purposes. We also used a multistranded steel archwire (Ormco Tripleflex, 0.44 mm round) and four nickel-titanium archwires of various diameters (Forestadent BioStarter 0.30 mm round, BioStarter 0.40 mm round, Titanol Low Force 0.40 x 0.40 mm(2) and Titanol Low Force 0.40 x 0.56 mm(2)). The leveling task consisted of correcting a complex malalignment (infraocclusion and vestibular displacement, each of 2 mm) of tooth 21. We analyzed the forces and torque movements that arose during the leveling phase. The test of the ten bracket systems revealed no significant difference in terms of their leveling effectiveness. Both selfligating brackets and conventional brackets behaved similarly, and we observed roughly 80% of the infraocclusions to have been corrected. Vestibular displacement was corrected with all the bracket systems by as much as 100% or even more due to a developing torque movement. The influence of wire material and wire diameter became apparent in relation to existing forces durconvening the leveling stage; those factors' influence was clearly greater than that

  19. Bilateral uterine vessel ligation as a model of intrauterine growth restriction in mice

    Science.gov (United States)

    2014-01-01

    Background Intrauterine Growth Restriction (IUGR) occurs in up to 10% of pregnancies and is considered as a major risk to develop various diseases in adulthood, such as cardiovascular diseases, insulin resistance, hypertension or end stage kidney disease. Several IUGR models have been developed in order to understand the biological processes linked to fetal growth retardation, most of them being rat or mouse models and nutritional models. In order to reproduce altered placental flow, surgical models have also been developed, and among them bilateral uterine ligation has been frequently used. Nevertheless, this model has never been developed in the mouse, although murine tools display multiple advantages for biological research. The aim of this work was therefore to develop a mouse model of bilateral uterine ligation as a surgical model of IUGR. Results In this report, we describe the set up and experimental data obtained from three different protocols (P1, P2, P3) of bilateral uterine vessel ligation in the mouse. Ligation was either performed at the cervical end of each uterine horn (P1) or at the central part of each uterine horn (P2 and P3). Time of surgery was E16 (P1), E17 (P2) or E16.5 (P3). Mortality, maternal weight and abortion parameters were recorded, as well as placentas weights, fetal resorption, viability, fetal weight and size. Results showed that P1 in test animals led to IUGR but was also accompanied with high mortality rate of mothers (50%), low viability of fetuses (8%) and high resorption rate (25%). P2 and P3 improved most of these parameters (decreased mortality and improved pregnancy outcomes; improved fetal viability to 90% and 27%, respectively) nevertheless P2 was not associated to IUGR contrary to P3. Thus P3 experimental conditions enable IUGR with better pregnancy and fetuses outcomes parameters that allow its use in experimental studies. Conclusions Our results show that bilateral uterine artery ligation according to the protocol we

  20. Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

    Science.gov (United States)

    Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

    2016-08-01

    This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  1. [Commemorative lecture of receiving Imamura Memorial Prize. II. Mode of action of oligonucleotide fraction extracted from Mycobacterium bovis BCG].

    Science.gov (United States)

    Yamamoto, S

    1994-09-01

    A fraction extracted from Mycobacterium bovis BCG was found to exhibit strong antitumor activity. This fraction, which was designated MY-1, caused some animal tumors to regress and/or prevent metastasis very effectively. MY-1 after digestion with DNase had almost completely reduced activity, while MY-1 digested with RNase did not. MY-1 also augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factors which showed anti-viral activity and rendered macrophages cytotoxic towards tumor cells. The function of the factor to activate macrophages was destroyed by treatment with anti-interferon (IFN)-gamma antibody, while the anti-viral activity was destroyed by treatment with anti-INF alpha/beta antibody. The oligonucleotides contained in MY-1 distributed in a broad range of molecular size, and peaked at 45 nucleotides. We synthesized 13 kinds of 45-mer nucleotides with sequence present in the known cDNA encoding various BCG proteins. Six out of these oligonucleotides, which contained one or more hexameric palindromic structures, showed strong antitumor activity, while the other without palindrome did not. These active oligonucleotides possessed the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotide to augment NK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects

    National Research Council Canada - National Science Library

    Radelof, U; Hennig, S; Seranski, P; Steinfath, M; Ramser, J; Reinhardt, R; Poustka, A; Francis, F; Lehrach, H

    1998-01-01

    .... To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select...

  3. Sequence-dependent structural changes in a self-assembling DNA oligonucleotide.

    Science.gov (United States)

    Saoji, Maithili; Paukstelis, Paul J

    2015-12-01

    DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.

  4. Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs

    Directory of Open Access Journals (Sweden)

    Lanfranchi Gerolamo

    2002-10-01

    Full Text Available Abstract Background We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences. This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene, with each probe evaluated for hybridization kinetics (Tm and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. Results Hybridization of human muscle cRNAs to this MuscleChip (33 samples showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41% were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39% could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. Conclusion Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.

  5. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

    Science.gov (United States)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D.; Otero, Carolina

    2016-02-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  6. Analysis of oligonucleotide array experiments with repeated measures using mixed models

    Directory of Open Access Journals (Sweden)

    Getchell Thomas V

    2004-12-01

    Full Text Available Abstract Background Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease or absence (Control of the disease, and brain regions including olfactory bulb (OB or cerebellum (CER. In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. Results In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH procedure of controlling false discovery rate (FDR at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the α-level (αnew = 0.0033 determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD procedure at the level of αnew to control the family-wise error rate (FWER for each gene examined. Conclusions A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

  7. Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

    Directory of Open Access Journals (Sweden)

    Reich Marlis

    2009-01-01

    Full Text Available Abstract Background In forest ecosystems, communities of ectomycorrhizal fungi (ECM are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot

  8. Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

    Directory of Open Access Journals (Sweden)

    Harish Bokkasam

    Full Text Available Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

  9. Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis.

    Science.gov (United States)

    Sudor, J; Foret, F; Bocek, P

    1991-12-01

    The separation of oligonucleotides by capillary zone electrophoresis (CZE) was studied in fused silica separation capillaries filled by linear (noncrosslinked) polyacrylamide (PAA) solutions, introduced into the capillary from the stock by pressure after each analysis. The time-consuming in-capillary polymerization step could thus be avoided, and fast and reproducible repetition of the analyses was assured. The PAA concentrations varied within the range of 3-10% and both the reproducibility of the analyses and the stability of the solution in the capillary, with and without a chemically treated inner wall, were tested. Ferguson plots were used to assess the size selectivity of the separation.

  10. Chemosensitization of Human Renal Cell Cancer Using Antisense Oligonucleotides Targeting the Antiapoptotic Gene Clusterin

    Directory of Open Access Journals (Sweden)

    Tobias Zellweger

    2001-01-01

    Full Text Available BACKGROUND: Renal cell cancer (RCC is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO, has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo. METHODS: Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks. RESULTS: Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98% and an overexpression, compared to normal tissue, in a majority of RCC (69%. Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dosedependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo

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

    DEFF Research Database (Denmark)

    1997-01-01

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

  12. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Solov'yov, Ilia A.; Balboni, Imelda M.

    2016-01-01

    molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best...... oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest...

  13. Factors influencing the electrokinetic injection of oligonucleotides in capillary gel electrophoresis when using laser-induced fluorescence detection.

    Science.gov (United States)

    Chen, Buyun; Chen, Guanhua; Bartlett, Michael G

    2014-03-01

    Capillary gel electrophoresis (CGE) is a powerful tool for the analysis of oligonucleotides owing to its extraordinary resolving power. However, the only feasible injection mode for CGE, electrokinetic injection, can cause bias of the injected amount and thus reproducibility issues for CGE methods. Although the source of the bias in electrokinetic injection for analysis of small molecules by capillary zone electrophoresis has long been identified, there are very few studies on electrokinetic injection issues for biological molecules analyzed by CGE. In this study, we report three issues related to electrokinetic injection for oligonucleotides. First, the relationship between the injection amount and the sample solution resistance is not always linear for oligonucleotides, as has been observed for small molecules. Second, the injecting water prior to an oligonucleotide sample dramatically improves the reproducibility of both the injected amount and resolution through a 'stacking-like' mechanism. Third, optimizing the gel concentration dramatically increases the amount of oligonucleotide that is injected into the column. Copyright © 2013 John Wiley & Sons, Ltd.

  14. Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

    Directory of Open Access Journals (Sweden)

    Eric B Alsop

    Full Text Available Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

  15. Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics.

    Science.gov (United States)

    Kor, Kamalodin; Turner, Anthony P F; Zarei, Kobra; Atabati, Morteza; Beni, Valerio; Mak, Wing Cheung

    2016-02-01

    A single-probe strip test for the rapid and sensitive detection of miRNA-21 mimics is reported herein. Highly specific structurally responsive bi-functional, thiol and biotin, DNA/LNA oligonucleotide probes (molecular beacons-MB) were designed and conjugated with gold nanoparticles (AuNPs) (i.e. biotin-MB-AuNPs). The proposed design had the ability to modulate the accessibility of the biotin group as a function of the presence of a miRNA target allowing the interaction of the boilable with the streptavidin test zone only in the presence of the miRNA-21 mimics. For quantitative evaluation, images of the strip tests were recorded using a flatbed scanner (Epson Perfection V370 Photo). The colour intensities of the test zones of the strip tests were analysed with the ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity. The response of the strip test was linear over the range 0.5 to 20 nM miRNA-21 (limit of detection of 115 pM) and showed good reproducibility (intra and inter CVs below 8%); furthermore, the assay was shown to be highly selective, discriminating other interference miRNAs mimics (e.g. miRNA-221 and miRNA-205). Finally, the proposed strip test was used for detection of miRNA-21 mimics in spiked serum samples, demonstrating its potential for point-of-care clinical applications. Main advantages of the single-probe strip test design are its versatility, simplicity and robustness, which can be easily extended to other miRNA targets by tuning the sequence of the single probe. Furthermore, the use of the structurally responsive single probe is particularly relevant in the case of short-length targets, such as miRNA, whereas a conventional sandwich approach might require a careful control of assay conditions such as hybridization temperature and salt concentration.

  16. Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

    Directory of Open Access Journals (Sweden)

    Xing Xian Yu

    Full Text Available Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4 in peripheral tissues. Treatment of diet-induce obese (DIO mice with FGFR4 antisense oligonucleotides (ASO specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

  17. Molecular diagnosis of bloodstream infections with a new dual-priming oligonucleotide-based multiplex PCR assay.

    Science.gov (United States)

    Carrara, Lucrecia; Navarro, Ferran; Turbau, Miquel; Seres, Montse; Morán, Indalecio; Quintana, Isabel; Martino, Rodrigo; González, Yesica; Brell, Albert; Cordon, Oscar; Diestra, Karol; Mata, Caterina; Mirelis, Beatriz; Coll, Pere

    2013-11-01

    Mortality from bloodstream infections (BSIs) correlates with diagnostic delay and the use of inappropriate empirical treatment. Early PCR-based diagnosis could decrease inappropriate treatment, improving patient outcome. The aim of the present study was to assess the clinical utility of this molecular technology to diagnose BSIs. We assessed a new dual-priming oligonucleotide-based multiplex PCR assay, the Magicplex Sepsis Test (MST) (Seegene), along with blood culture (BC). A total of 267 patients from the intensive care unit and haematology and emergency departments were enrolled. Clinical data were also used by physicians to determine the likelihood of infection. Ninety-eight (37 %) specimens were positive: 29 (11 %) by both the MST and BC, 29 (11 %) by the MST only, and 40 (15 %) by BC only. The proportion of agreement between the two methods was 73 % (Cohen's κ: 0.45; 0.28-0.6; indicating fair to moderate agreement). According to clinical assessment, 63 (64 %) positive specimens were considered BSIs: 23 (36 %) were positive by both the MST and BC, 22 (35 %) were positive only by BC, and 18 (29 %) were positive only by the MST. Thirty-eight (14 %) positive specimens by the MST and/or BC were considered as contaminants. Of 101 specimens collected from patients receiving antibiotics, 20 (20 %) were positive by the MST and 32 (32 %) by BC. Sensitivity and specificity were 65 % and 92 %, respectively, for the MST and 71 % and 88 %, respectively for BC. We concluded that the MST shows a high specificity but changes in design are needed to increase bacteraemia detection. For viability in clinical laboratories, technical improvements are also required to further automate the process.

  18. Traceless chemical ligation from S-, O-, and N-acyl isopeptides.

    Science.gov (United States)

    Panda, Siva S; Hall, C Dennis; Oliferenko, Alexander A; Katritzky, Alan R

    2014-04-15

    Peptides are ubiquitous in nature where they play crucial roles as catalysts (enzymes), cell membrane ion transporters, and structural elements (proteins) within biological systems. In addition, both linear and cyclic peptides have found use as pharmaceuticals and components of various conjugate molecular systems. Small wonder then that chemists throughout the ages have sought to mimic nature by synthesis of the amide polymers known as peptides and proteins. The fundamental reaction in the formation of a peptide bond is condensation of an amine of one amino acid with the activated carbonyl group of another. This "fragment condensation" has been achieved in many ways both in solution and by solid-phase peptide synthesis (SPSS) on resin. The most successful method for in-solution coupling is known as native chemical ligation (NCL), and the technique dates back to the pioneering work of Wieland (1953) and subsequently Kent (1994) among many others. This Account builds on the established principles of NCL as applied specifically to S-, O-, and N-isopeptides, molecules that are generally more soluble and less prone to aggregation than native peptides. This Account also covers NCL of isopeptides containing terminal and nonterminal S-acylated cysteine units, reactions that enable the synthesis of native peptides from S-acyl peptides without the use of auxiliaries. With C-terminal S-acyl isopeptides, NCL was carried out under microwave irradiation in phosphate buffer (pH 7.3) at 50 °C. Intramolecular acyl migration was observed through 5-19-membered transition states with relative rates, as assessed by product analysis, in the order, 5 > 10 > 11 > 14, 16, or 17 > 12 > 13, 15, or 19 > 18 ≫ 9 > 8. The rate/pH profile for the 15-membered TS showed a maximum for ligated product versus transacylation at pH 7.0-7.3 presumably associated with the pKa of the N-nucleophile in the hydrogen-bonded TS. Cysteine occurs at low abundance (1.7%) in natural peptides and is rarely

  19. Desmistificando os braquetes autoligáveis Demystifying self-ligating brackets

    Directory of Open Access Journals (Sweden)

    Renata Sathler

    2011-04-01

    Full Text Available Atualmente, os braquetes autoligáveis têm sido associados a tratamentos mais rápidos e eficazes, o que desperta a curiosidade em compará-los ao sistema convencional. Ao contrário dos braquetes tradicionais, os autoligáveis não necessitam de ligaduras, sejam elásticas ou metálicas. A literatura é farta em concluir que essa característica diminui, ostensivamente, a resistência do atrito durante as mecânicas de deslize. Além disso, existem alegações sobre a dimimuição da necessidade de extrações e de expansão maxilar com o uso desses acessórios. Portanto, o objetivo dessa revisão de literatura foi buscar os mais novos estudos a respeito dos aparelhos autoligáveis atualmente utilizados nos tratamentos ortodônticos, confirmando ou retificando as especulações vigentes.Currently self-ligating brackets have been associated to faster and more efficient treatments, which arouse the curiosity to compare them to the conventional system. Unlike traditional appliances, self-ligating brackets do not require elastomeric or metal ligatures. The literature is abundant in concluding that this feature decreases, ostensibly, the friction resistance during the sliding mechanics. Moreover, there are reports on minimizing the need of extractions and maxillary expansion using these accessories. Therefore, the purpose of this literature review was to seek the newest studies about the self-ligating brackets currently used in orthodontic treatments, confirming or correcting current speculations.

  20. Association between incomplete surgical ligation of left atrial appendage and stroke and systemic embolization.

    Science.gov (United States)

    Aryana, Arash; Singh, Steve K; Singh, Sheldon M; O'Neill, P Gearoid; Bowers, Mark R; Allen, Shelley L; Lewandowski, Sammi L; Vierra, Eleanor C; d'Avila, André

    2015-07-01

    Surgical exclusion of the left atrial appendage (LAA) can frequently yield incomplete closure. We evaluated the ischemic stroke/systemic embolization (SSE) risk in patients with atrial fibrillation (AF) and complete LAA closure (cLAA) vs incompletely surgically ligated LAA (ISLL) and LAA stump after surgical suture ligation. Seventy-two patients (CHA2DS2-VASc score 4.1 ± 1.9) underwent surgical LAA ligation in conjunction with mitral valve/AF surgery and postoperative LAA evaluation using computerized tomographic angiography. Overall, cLAA was detected in 46 of 72 patients (64%), ISLL in 17 patients (24%), and LAA stump in 9 patients (12%). The incidences of oral anticoagulation (OAC) and recurrent AF were similar among the 3 groups during 44 ± 19 months of follow-up. SSE occurred in 2% of patients with cLAA vs 24% with ISLL and 0% with LAA stump (P = .006). None of the patients with SSE were receiving OAC, and all had recurrent AF during follow-up. Additionally, patients with SSE exhibited a significantly smaller ISLL neck diameter (2.8 ± 1.0 vs 7.1 ± 2.1 mm; P = .002). The annualized SSE risk was 1.9% (entire cohort), 6.5% (ISLL patients), 14.4% (ISLL patients not receiving OAC), and 19.0% (ISLL neck diameter ≤5.0 mm) per 100 patient-years of follow-up. The latter risk was nearly 5 times greater than predicted by conventional risk-stratification schemes. Moreover, ISLL emerged as an independent predictor of SSE in univariate analyses and as the sole predictor of SSE in a multivariate analysis. In patients with AF, ISLL is a predictor of SSE, independent of conventional risk stratification schemes. Consequently, OAC should be strongly considered in this high-risk cohort. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  1. A comparison of vas occlusion techniques: cautery more effective than ligation and excision with fascial interposition

    Directory of Open Access Journals (Sweden)

    Labrecque Michel

    2004-10-01

    Full Text Available Abstract Background Vasectomy techniques have been the subject of relatively few rigorous studies. The objective of this analysis was to compare the effectiveness of two techniques for vas occlusion: intraluminal cautery versus ligation and excision with fascial interposition. More specifically, we aimed to compare early failure rates, sperm concentrations, and time to success between the two techniques. Methods We compared semen analysis data from men following vasectomy using two occlusion techniques. Data on intraluminal cautery came from a prospective observational study conducted at four sites. Data on ligation and excision with fascial interposition came from a multicenter randomized controlled trial that evaluated the efficacy of ligation and excision with versus without fascial interposition. The surgical techniques used in the fascial interposition study were standardized. The surgeons in the cautery study used their customary techniques, which varied among sites in terms of type of cautery, use of fascial interposition, excision of a short segment of the vas, and use of an open-ended technique. Men in both studies had semen analyses two weeks after vasectomy and then approximately every four weeks. The two outcome measures for the analyses presented here are (a time to success, defined as severe oligozoospermia, or 10 million sperm/mL at week 12 or later. Results Vasectomy with cautery was associated with a significantly more rapid progression to severe oligozoospermia and with significantly fewer early failures (1% versus 5%. Conclusion The use of cautery improves vasectomy outcomes. Limitations of this comparison include (a the variety of surgical techniques in the cautery study and differences in methods of fascial interposition between the two studies, (b the uncertain correlation between sperm concentrations after vasectomy and the risk of pregnancy, and (c the use of historical controls and different study sites.

  2. Application of a pig ligated intestinal loop model for early Lawsonia intracellularis infection

    Directory of Open Access Journals (Sweden)

    Agerholm Jørgen S

    2010-02-01

    Full Text Available Abstract Background Porcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI. However, knowledge of the initial in vivo interaction between porcine intestinal epithelium and the bacterium is limited. The aims of the present study were to evaluate the usefulness of a ligated small intestinal loop model to study L. intracellularis infections and to obtain information on the very early L. intracellularis-enterocyte interactions. Methods A ligated small intestinal loop model using three different L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1 wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2 crude vaccine bacteria (Enterisol® Ileitis Vet, and 3 vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively. Results Although at a low level, close contact between bacteria and the enterocyte brush border including intracellular uptake of bacteria in mature enterocytes was seen at 3 and 6 h PI for the vaccine and the propagated vaccine inocula. Interaction between the wild-type bacteria and villus enterocytes was scarce and only seen at 6 h PI, where a few bacteria were found in close contact with the brush border. Conclusions The ligated intestinal loop model was useful with respect to maintaining an intact intestinal morphology for up to 6 h. Furthermore, the study demonstrated that L. intracellularis interacts with villus enterocytes within 3 to 6 h after inoculation into intestinal loops and that the bacterium, as shown for the vaccine bacteria, propagated as well as non-propagated, was able to invade mature enterocytes. Thus, the study demonstrates

  3. Mean and variance of the Gibbs free energy of oligonucleotides in the nearest neighbor model under varying conditions.

    Science.gov (United States)

    Rahmann, Sven; Gräfe, Christine

    2004-11-22

    In order to assess the stability of DNA-DNA hybridizations-for example during PCR primer design or oligonucleotide selection for microarrays-one needs to predict the change in Gibbs free energy DeltaG during hybridization. The nearest neighbor model provides a good compromise between accuracy and computational simplicity for this task. To determine optimal combinations of reaction parameters (temperature, salt concentration, oligonucleotide length and GC-content), one would like to understand how DeltaG depends on all of these parameters simultaneously. We derive analytic results about the distribution of nearest neighbor DeltaG values for a Bernoulli random sequence model (specified by oligonucleotide length and average GC-content) under given experimental conditions. We find that the distribution of DeltaG values is approximately Gaussian and provide exact formulas for expectation and variance.

  4. FLUORESCENT OLIGONUCLEOTIDES CONTAINING A NOVEL PERYLENE 2 '-AMINO-alpha-L-LNA MONOMER: SYNTHESIS AND ANALYTICAL POTENTIAL

    DEFF Research Database (Denmark)

    Astakhova, I. V.; Kumar, T. S.; Wengel, J.

    2011-01-01

    efficiency of the resulting perylene-2'-amino-alpha-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards...... incorporations of monomers T* was quenched (quantum yield Phi(F) = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Phi(F) = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues...... sequences containing monomers T* at 5'- and 3'-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose...

  5. Successful treatment of familial congenital chylothorax by ligation of the thoracic duct: A case report

    Directory of Open Access Journals (Sweden)

    Leopoldini Dori

    2017-01-01

    Full Text Available A full term boy was admitted with respiratory distress in the fourth week of his life due to spontaneous chylothorax in his right hemithorax. Spontaneous chylothorax occurred previously in a first cousin of the neonate establishing that way the final diagnosis of familial idiopathic congenital pneumothorax. Failure of the conservative treatment consisting of chest tube drainage, discontinuation of oral diet and administration of total parenteral nutrition in combination with octreotide for one month was followed by the successful ligation of the thoracic duct through a right thoracotomy. The boy still remains free of symptoms and without recurrence of the chylothorax two years later.

  6. Successful management of uterine arteriovenous malformation by ligation of feeding artery after unsuccessful uterine artery embolization.

    Science.gov (United States)

    Yokomine, Daisaku; Yoshinaga, Mitsuhiro; Baba, Yasutaka; Matsuo, Takashi; Iguro, Yoshifumi; Nakajo, Masayuki; Douchi, Tsutomu

    2009-02-01

    Uterine arteriovenous malformation (AVM) is a rare and potentially life-threatening disease. The present report describes a postmenopausal patient with uterine AVM manifesting recurrent, massive genital bleeding. Uterine artery embolization (UAE) was scheduled before hysterectomy, but UAE was unsuccessful due to the dilated, tortuous internal iliac arteries, and extremely rapid arterial blood flow. Hysterectomy appeared to carry a potential risk of massive blood loss due to multiple dilated vessels around the uterine corpus and cervix. Therefore, six arteries feeding the uterus were surgically ligated. At 10 months after the operation there have been no episodes of atypical genital bleeding.

  7. Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

    Science.gov (United States)

    Blasius, Melanie; Buob, Rebecca; Shevelev, Igor V; Hubscher, Ulrich

    2007-01-01

    Background Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. Results In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. Conclusion Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the

  8. A new resorbable device for ligation of blood vessels - A pilot study

    Directory of Open Access Journals (Sweden)

    Borg Niklas

    2011-07-01

    Full Text Available Abstract Background During surgery, controlled haemostasis to prevent blood loss is vital for a successful outcome. It can be difficult to ligate vessels located deep in the abdomen. A device that is easy to use and enables secure ligatures could be beneficial. Cable ties made of nylon have been used for ligation but the non-resorbable material caused tissue reactions. The objective of this study was to use a resorbable material to construct a device with a self-locking mechanism and to test its mechanical strength and ligation efficiency. Methods The device was manufactured by injection moulding of polydioxanone, a resorbable polymer used for suture materials. Polydioxanone with inherent viscosities of 1.9 dL/g and 1.3 dL/g were tested. The device consisted of a perforated flexible band which could be pulled through a case with a locking mechanism. After a first version of the device had been tested, some improvements were made. The locking case was downsized, corners were rounded off, the band was made thicker and the mould was redesigned to produce longer devices. Tensile tests were performed with the second version. The first version of the device was used to ligate the ovarian pedicle in a euthanized dog and to test echogenicity of the device with ultrasound. Compression of vessels of the ovarian pedicle was examined by histology. Both versions of the device were tested for haemostasis of and tissue grip on renal arteries in six anaesthetised pigs. Results The tensile strength of the flexible band of the devices with inherent viscosity of 1.9 dL/g was 50.1 ± 5.5 N (range 35.2-62.9 N, n = 11 and the devices with inherent viscosity of 1.3 dL/g had a tensile strength of 39.8 ± 8.1 N (range 18.6-54.2 N, n = 11. Injection moulding of the polymer with lower inherent viscosity resulted in a longer flow distance. Both versions of the device had an effective tissue grip and complete haemostasis of renal arteries was verified. The device attached

  9. Essure hysteroscopic sterilization versus interval laparoscopic bilateral tubal ligation: a comparative effectiveness review.

    Science.gov (United States)

    Ouzounelli, Myrsini; Reaven, Nancy L

    2015-01-01

    A comparative effectiveness analysis was performed to examine the risks and benefits of laparoscopic bilateral tubal ligation compared with hysteroscopic sterilization using the Essure Permanent Birth Control System (Bayer HealthCare AG, Whippany, NJ). Existing evidence shows that both LBTL and Essure are safe and effective methods of female sterilization. Both have high rates of efficacy and low rates of complications although when complications do occur, those related to the Essure procedure are more likely to be minor in nature. The analysis was limited by the restricted number of studies involving head-to-head comparisons of the 2 approaches. Copyright © 2015 AAGL. Published by Elsevier Inc. All rights reserved.

  10. Gastroprotective Effect of Rubia cordifolia Linn. on Aspirin Plus Pylorus-Ligated Ulcer

    Directory of Open Access Journals (Sweden)

    R. S. Deoda

    2011-01-01

    Full Text Available The present study was designed to investigate the effect of Rubia cordifolia (Rubiaceae against experimentally induced gastric ulcer and compare activity with its fractions by employing aspirin plus pylorus-ligated ulcer screening model in Wistar rats. Total acidity, volume of gastric acid secretion, total acid output, and pepsin activity show significant reduction, when compared with the control group. The present study confirmed that chloroform fraction showed the significant activity at lower doses compared to parent extract. The mechanism can be attributed to decrease in gastric acid secretary activity along with strengthening of mucosal defensive mechanism by prostaglandin synthesis and antioxidant potential.

  11. Blunt traumatic celiac artery avulsion managed with celiac artery ligation and open aorto-celiac bypass

    Directory of Open Access Journals (Sweden)

    Matthew D. Kronick

    2017-10-01

    Full Text Available Traumatic celiac artery injuries are rare and highly lethal with reported mortality rates of 38–62%. The vast majority are caused by penetrating trauma with only 11 reported cases due to blunt trauma (Graham et al., 1978; Asensio et al., 2000, 2002. Only 3 of these cases were complete celiac artery avulsions. Management options described depend upon the type of injury and have included medical therapy with anti-platelet agents or anti-coagulants, endovascular stenting, and open ligation. We report a case of a survivor of complete celiac artery avulsion from blunt trauma managed by open bypass.

  12. Prevention of type II endoleak by laparoscopic inferior mesenteric artery ligation.

    Science.gov (United States)

    Brenes, Robert A; Panait, Lucian; Abbas, Hussain M A; Tapias, Leonidas; Tripodi, Giuseppe; Ajemian, Michael S; Macaron, Shady H

    2013-08-01

    Abdominal aortic aneurysm repair by endovascular techniques have gained wide acceptance as a treatment option. A potential well-known complication of endovascular repair includes endoleak. Specifically, type II endoleak, which is described as retrograde flow into the aneurysm sac through collateral vessels, can occur in up to 30% of patients. Certain preoperative factors can predict which patients may develop type II endoleak. This article describes laparoscopic inferior mesenteric artery ligation prior to endovascular abdominal aortic aneurysm repair as a viable treatment option in the prevention of type II endoleak.

  13. Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

    Directory of Open Access Journals (Sweden)

    Shevelev Igor V

    2007-08-01

    Full Text Available Abstract Background Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. Results In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. Conclusion Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a

  14. A Novel Label-Free Optical Biosensor Using Synthetic Oligonucleotides from E. coli O157:H7: Elementary Sensitivity Tests

    National Research Council Canada - National Science Library

    Bahşi, Zehra Banu; Büyükaksoy, Aligül; Olmezcan, Sinan Mert; Simşek, Fethi; Aslan, Muhammed Hasan; Oral, Ahmet Yavuz

    2009-01-01

    .... Refractive index values of the films were measured using a Metricon 2010 prism coupler. On the surface of each film, 12 different spots were taken for measurement and calculation of the mean refractive index values with their standard deviations...

  15. A Novel Label-Free Optical Biosensor Using Synthetic Oligonucleotides from E. coli O157:H7: Elementary Sensitivity Tests

    Directory of Open Access Journals (Sweden)

    Ahmet Yavuz Oral

    2009-06-01

    Full Text Available SiO2-TiO2 thin films for use as fiber optic guiding layers of optical DNA biosensors were fabricated by the sol-gel dip coating technique. The chemical structure and the surface morphology of the films were characterized before immobilization. Single probe DNA strands were immobilized on the surface and the porosity of the films before the hybridization process was measured. Refractive index values of the films were measured using a Metricon 2010 prism coupler. On the surface of each film, 12 different spots were taken for measurement and calculation of the mean refractive index values with their standard deviations. The increased refractive index values after the immobilization of single DNA strands indicated that immobilization was successfully achieved. A further refractive index increase after the hybridization with target single DNA strands showed the possibility of detection of the E. coli O157:H7 EDL933 species using strands of 20-mers (5’-TAATATCGGTTGCGGAGGTG -3’ sequence.

  16. A Novel Label-Free Optical Biosensor Using Synthetic Oligonucleotides from E. coli O157:H7: Elementary Sensitivity Tests.

    Science.gov (United States)

    Bahşi, Zehra Banu; Büyükaksoy, Aligül; Olmezcan, Sinan Mert; Simşek, Fethi; Aslan, Muhammed Hasan; Oral, Ahmet Yavuz

    2009-01-01

    SiO(2)-TiO(2) thin films for use as fiber optic guiding layers of optical DNA biosensors were fabricated by the sol-gel dip coating technique. The chemical structure and the surface morphology of the films were characterized before immobilization. Single probe DNA strands were immobilized on the surface and the porosity of the films before the hybridization process was measured. Refractive index values of the films were measured using a Metricon 2010 prism coupler. On the surface of each film, 12 different spots were taken for measurement and calculation of the mean refractive index values with their standard deviations. The increased refractive index values after the immobilization of single DNA strands indicated that immobilization was successfully achieved. A further refractive index increase after the hybridization with target single DNA strands showed the possibility of detection of the E. coli O157:H7 EDL933 species using strands of 20-mers (5'-TAATATCGGTTGCGGAGGTG -3') sequence.

  17. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  18. Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection

    Directory of Open Access Journals (Sweden)

    Lewis A. Fraser

    2015-12-01

    Full Text Available The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR and Fluorescence Activated Cell Sorting (FACS to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.

  19. Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

    Directory of Open Access Journals (Sweden)

    Shu-ichi Nakano

    2014-01-01

    Full Text Available Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol, small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds.

  20. Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection.

    Science.gov (United States)

    Fraser, Lewis A; Kinghorn, Andrew B; Tang, Marco S L; Cheung, Yee-Wai; Lim, Bryce; Liang, Shaolin; Dirkzwager, Roderick M; Tanner, Julian A

    2015-12-01

    The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR) and Fluorescence Activated Cell Sorting (FACS) to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS) whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.