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Sample records for sensitive oligonucleotide ligation

  1. Template-Directed Ligation of Peptides to Oligonucleotides

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    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

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

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    Abe, Hiroshi; Kimura, Yasuaki

    2018-01-01

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

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

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    Panpradist, Nuttada; Beck, Ingrid A.; Chung, Michael H.; Kiarie, James N.; Frenkel, Lisa M.; Lutz, Barry R.

    2016-01-01

    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. PMID:26751207

  5. Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.

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    Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood

    2017-09-01

    Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.

  6. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

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    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  7. An accurate method for quantifying and analyzing copy number variation in porcine KIT by an oligonucleotide ligation assay

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    Cho In-Cheol

    2007-11-01

    Full Text Available Abstract Background Aside from single nucleotide polymorphisms, copy number variations (CNVs are the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing, real-time PCR, invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed. Results PCR followed by a quantitative oligonucleotide ligation assay (qOLA was developed for quantifying CNVs. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares of bias and standard deviation of qOLA were 2.09 and 0.45, respectively. These values are less than half of those in the published pyrosequencing assay for analyzing CNV in porcine KIT. Using a combined method of qOLA and another pyrosequencing for quantitative analysis of KIT copies with spliced forms, we confirmed the segregation of KIT alleles in 145 F1 animals with pedigree information and verified the correct assignment of genotypes. In a diagnostic test on 100 randomly sampled commercial pigs, there was perfect agreement between the genotypes obtained by grouping observations on a scatter plot and by clustering using the nearest centroid sorting method implemented in PROC FASTCLUS of the SAS package. In a test on 159 Large White pigs, there were only two discrepancies between genotypes assigned by the two clustering methods (98.7% agreement, confirming that the quantitative ligation assay established here makes genotyping possible through the accurate measurement of high KIT copy numbers (>4 per diploid genome. Moreover, the assay is sensitive enough for use on DNA from hair follicles, indicating that DNA from various sources could be used. Conclusion We have established a high

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

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    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

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

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    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.

  10. Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures

    DEFF Research Database (Denmark)

    Manuguerra, Ilenia; Croce, Stefano; El-Sagheer, Afaf H.

    2018-01-01

    Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA...

  11. 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

    achieved, if small, activated, oligonucleotides are ligated on a template. A template directed ligation can lead to autocatalytic or cross- catalytic replication and thus maintain a certain pool of catalyitc species. Important for these processes is a destabilization of the formed douplex....../multiplex to overcome product inhibition. The latest results of our template directed ligation experiments in the eutectic ice phase are presented. Different activation strategies are compared and an outlook towards applications in molecular evolution and artifical cell systems (« protocells ») will be given. Figure 1....... (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...

  12. Simultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescence.

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    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2010-07-01

    In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis.

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

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    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.

  14. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

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    Tang, Weizhong; Zhou, Huafu; Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

  15. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

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    Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

    2012-01-01

    Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

  16. Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides

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    Ho Antoine

    2011-12-01

    Full Text Available Abstract Background Sequencing-by-ligation (SBL is one of several next-generation sequencing methods that has been developed for massive sequencing of DNA immobilized on arrayed beads (or other clonal amplicons. SBL has the advantage of being easy to implement and accessible to all because it can be performed with off-the-shelf reagents. However, SBL has the limitation of very short read lengths. Results To overcome the read length limitation, research groups have developed complex library preparation processes, which can be time-consuming, difficult, and result in low complexity libraries. Herein we describe a variation on traditional SBL protocols that extends the number of sequential bases that can be sequenced by using Endonuclease V to nick a query primer, thus leaving a ligatable end extended into the unknown sequence for further SBL cycles. To demonstrate the protocol, we constructed a known DNA sequence and utilized our SBL variation, cyclic SBL (cSBL, to resequence this region. Using our method, we were able to read thirteen contiguous bases in the 3' - 5' direction. Conclusions Combining this read length with sequencing in the 5' - 3' direction would allow a read length of over twenty bases on a single tage. Implementing mate-paired tags and this SBL variation could enable > 95% coverage of the genome.

  17. SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

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    Thompson, Christopher M; Bloom, Lee R; Ogiue-Ikeda, Mari; Machida, Kazuya

    2015-06-26

    There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment. We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein

  18. Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

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    Marlowe, Jennifer L; Akopian, Violetta; Karmali, Priya; Kornbrust, Douglas; Lockridge, Jennifer; Semple, Sean

    2017-08-01

    The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

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

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    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

  20. Electronic Structures of LNA Phosphorothioate Oligonucleotides

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    Henrik G. Bohr

    2017-09-01

    Full Text Available 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 are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish 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 descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules. Keywords: LNA phosphorothioate, DNA/LNA oligonucleotide, diastereoisomers, Hartree-Fock calculations, iso-potential surface, anion chromatograms

  1. Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

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    David G Nickens

    Full Text Available BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxyribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2>A(1>A(3>A(4>A(0>A(6>A(8>A(10 and T(2>T(3>T(4>T(6 approximately T(1>T(8>T(10. Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90% joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI did not support ligation when

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

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    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.

  3. Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor.

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    Shishkanova, T V; Volf, R; Krondak, M; Král, V

    2007-05-15

    A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.

  4. Hemodynamic characterization of chronic bile duct-ligated rats: effect of pentobarbital sodium

    International Nuclear Information System (INIS)

    Lee, S.S.; Girod, C.; Braillon, A.; Hadengue, A.; Lebrec, D.

    1986-01-01

    Systemic and splanchnic hemodynamics of the chronic bile duct-ligated rat were characterized by radioactive microspheres. Conscious and pentobarbital sodium-anesthetized, bile duct-ligated and sham-operated rats had cardiac output and regional organ blood flows determined. The conscious bile duct-ligated rat compared with the sham-operated showed a hyperdynamic circulation with an increased cardiac output and portal tributary blood flow. Pentobarbital sodium anesthesia induced marked hemodynamic changes in both sham-operated and bile duct-ligated rats. The latter group was especially sensitive to its effects; thus, comparison of cardiac output and portal tributary blood flow between anesthetized bile duct-ligated and sham-operated rats showed no significant differences. The authors conclude that the rat with cirrhosis due to chronic bile duct ligation is an excellent model for hemodynamic investigations but should be studied in the conscious state, since pentobarbital sodium anesthesia eliminated the hyperdynamic circulation

  5. A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

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    Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

    2016-07-01

    In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip

    International Nuclear Information System (INIS)

    Pal, Sukdeb; Kim, Min Jung; Choo, Jaebum; Kang, Seong Ho; Lee, Kyeong-Hee; Song, Joon Myong

    2008-01-01

    A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes

  7. 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.

  8. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

    Science.gov (United States)

    Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

    2002-01-01

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

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

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte S.; Jensen, Knud J.

    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...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...... 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...

  10. A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

    Science.gov (United States)

    de Vries, Michel; Deijs, Martin; Canuti, Marta; van Schaik, Barbera D. C.; Faria, Nuno R.; van de Garde, Martijn D. B.; Jachimowski, Loes C. M.; Jebbink, Maarten F.; Jakobs, Marja; Luyf, Angela C. M.; Coenjaerts, Frank E. J.; Claas, Eric C. J.; Molenkamp, Richard; Koekkoek, Sylvie M.; Lammens, Christine; Leus, Frank; Goossens, Herman; Ieven, Margareta; Baas, Frank; van der Hoek, Lia

    2011-01-01

    In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material. PMID:21283679

  11. A sensitive assay for virus discovery in respiratory clinical samples.

    Directory of Open Access Journals (Sweden)

    Michel de Vries

    Full Text Available In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454. Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18. The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6. Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.

  12. Torque expression in self-ligating orthodontic brackets and conventionally ligated brackets: A systematic review.

    Science.gov (United States)

    Al-Thomali, Yousef; Mohamed, Roshan-Noor; Basha, Sakeenabi

    2017-01-01

    To evaluate the torque expression of self ligating (SL) orthodontic brackets and conventionally ligated brackets and the torque expression in active and passive SL brackets. Our systematic search included MEDLINE, EMBASE, CINAHL, PsychINFO, Scopus, and key journals and review articles; the date of the last search was April 4th 2016. We graded the methodological quality of the studies by means of the Quality Assessment Tool for Quantitative Studies, developed for the Effective Public Health Practice Project (EPHPP). In total, 87 studies were identified for screening, and 9 studies were eligible. The quality assessment rated one of the study as being of strong quality, 7 (77.78%) of these studies as being of moderate quality. Three out of 7 studies which compared SL and conventionally ligated brackets showed, conventionally ligated brackets with highest torque expression compared to SL brackets. Badawi showed active SL brackets with highest torque expression compared to passive SL brackets. Major and Brauchli showed no significant differences in torque expression of active and passive SL brackets. Conventionally ligated brackets presented with highest torque expression compared to SL brackets. Minor difference was recorded in a torque expression of active and passive SL brackets. Key words: Systematic review, self ligation, torque expression, conventional ligation.

  13. 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

  14. Novel approaches to study low-energy electron-induced damage to DNA oligonucleotides

    International Nuclear Information System (INIS)

    Rackwitz, Jenny; Bald, Ilko; Ranković, Miloš Lj; Milosavljević, Aleksandar R

    2015-01-01

    The novel approach of DNA origami structures as templates for precise quantification of various well- defined oligonucleotides provides the opportunity to determine the sensitivity of complex DNA sequences towards low-energy electrons. (paper)

  15. The Medicinal Chemistry of Therapeutic Oligonucleotides.

    Science.gov (United States)

    Wan, W Brad; Seth, Punit P

    2016-11-10

    Oligonucleotide-based therapeutics have made rapid progress in the clinic for treatment of a variety of disease indications. Unmodified oligonucleotides are polyanionic macromolecules with poor drug-like properties. Over the past two decades, medicinal chemists have identified a number of chemical modification and conjugation strategies which can improve the nuclease stability, RNA-binding affinity, and pharmacokinetic properties of oligonucleotides for therapeutic applications. In this perspective, we present a summary of the most commonly used nucleobase, sugar and backbone modification, and conjugation strategies used in oligonucleotide medicinal chemistry.

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

    Science.gov (United States)

    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.

  17. Identification of clinically relevant viridans streptococci by an oligonucleotide array.

    Science.gov (United States)

    Chen, Chao Chien; Teng, Lee Jene; Kaiung, Seng; Chang, Tsung Chain

    2005-04-01

    Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.

  18. [Self-ligating edgewise brackets. An overview].

    Science.gov (United States)

    Katsaros, C; Dijkman, J F

    2003-01-01

    During the last years both the manufactures and the orthodontists seem to show an increased interest in self-ligating brackets. This paper aims to present the history of self-ligating systems, to describe the three mostly used bracketsystems and to review the relevant literature. It seems from the existing data that self-ligating brackets have certain advantages over conventionally ligated brackets. However, the data are still thin and a high need for well designed clinical trials exist.

  19. Translational inhibition of CTX M extended spectrum β-lactamase in clinical strains of Escherichia coli by synthetic antisense oligonucleotides partially restores sensitivity to cefotaxime.

    Directory of Open Access Journals (Sweden)

    John Benedict Readman

    2016-03-01

    Full Text Available Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25 mer phosphorodiamidate morpholino oligomer (PMO and a 13 mer polyamide (peptide nucleic acid (PNA were designed to target mRNA (positions -4 to +21, and –17 to –5 respectively close to the translational initiation site of the extended spectrum β lactamase resistance genes of CTX M group 1. These antisense oligonucleotides were found to inhibit β lactamase activity by up to 96% in a cell free translation transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0 - 40 nM. The PMO and PNA were covalently bound to the cell penetrating peptide (KFF3K and both significantly (P<0.05 increased sensitivity to cefotaxime in a dose dependent manner (0 - 40 nM in field isolates harbouring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

  20. Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

    2013-07-01

    Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

  1. Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes

    Directory of Open Access Journals (Sweden)

    K. Böhme

    2014-01-01

    Full Text Available Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB hybridization on membranes, coupled to the high specific ligation detection reaction (LDR. First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA. Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

  2. 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...

  3. 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.

  4. Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

    Science.gov (United States)

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

    2013-01-01

    Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

  5. Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

    Science.gov (United States)

    Takita, Eiji; Kohda, Katsunori; Tomatsu, Hajime; Hanano, Shigeru; Moriya, Kanami; Hosouchi, Tsutomu; Sakurai, Nozomu; Suzuki, Hideyuki; Shinmyo, Atsuhiko; Shibata, Daisuke

    2013-01-01

    Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. PMID:23897972

  6. Radio-marking and in vivo imagery of oligonucleotides

    International Nuclear Information System (INIS)

    Kuehnast, Bertrand

    2000-01-01

    This research thesis is part of activities aimed at the development of new molecules like oligonucleotides. Its first objective was the development and validation of a marking method with fluorine-18 of oligonucleotides for their in-vivo pharmacological assessment with positron emission tomography (PET). Further investigations addressed the use of iodine-125 for oligonucleotide marking purpose. This radio-marking, and in vivo and ex vivo imagery techniques are described, and their potential is highlighted for the pharmacological assessment of different oligonucleotides

  7. Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

    Science.gov (United States)

    Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

    2014-07-01

    DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

  8. Spectrum of menstrual problems after tubal ligation

    International Nuclear Information System (INIS)

    Naqvi, S.S.B.; Akthar, S.; Mubeen, A.; Noor, A.

    2014-01-01

    To determine the frequency of menstrual problems after tubal ligation. Study Design: Descriptive study. Place and Duration of Study: This study was carried out in gynae department of PAF Hospital Sargodha, Pakistan from September 2011 to September 2012. Patients and methods: Two hundred patients were included in the study with menstrual problems after tubal ligation. Results: Forty seven percent of patients presented with menorrhagia, 28% presented with metrorrhagia, 13% patients were having complaints of polymenorrhoea and 5% patients presented with dysmenorrhea and 7% had oligomenorrhoea after tubal ligation. Thirty one percent of patients ended up in hysterectomy and 37% of patients had improvement of symptoms with medical treatment. Conclusion: Menstrual problem is a common complaint in our gyn ecological patients. A large number of these patients had history of tubal ligation. Menorrhagia, metrorrhagia, polymenorrhoea and dysmenorrhoea can occur after tubal ligation. Patients giving the history of menstrual problems and wants tubal ligation should be counselled earlier for these symptoms. (author)

  9. 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.

  10. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

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

    2013-01-01

    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...

  11. 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

    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...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

  12. Oligonucleotide-based theranostic nanoparticles in cancer therapy

    Science.gov (United States)

    Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

    2016-01-01

    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. PMID:27102380

  13. Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA.

    Directory of Open Access Journals (Sweden)

    Uffe V Schneider

    Full Text Available The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-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--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules. We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  16. Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides

    Science.gov (United States)

    Bielinska, Anna; Shivdasani, Ramesh A.; Zhang, Liquan; Nabel, Gary J.

    1990-11-01

    Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappaB consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappaB. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappaB-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.

  17. 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

  18. A facile one-step fluorescence method for the quantitation of low-content single base deamination impurity in synthetic oligonucleotides.

    Science.gov (United States)

    Su, Xiaoye; Liang, Ruiting; Stolee, Jessica A

    2018-06-05

    Oligonucleotides are being researched and developed as potential drug candidates for the treatment of a broad spectrum of diseases. The characterization of antisense oligonucleotide (ASO) impurities caused by base mutations (e.g. deamination) which are closely related to the target ASO is a significant analytical challenge. Herein, we describe a novel one-step method, utilizing a strategy that combines fluorescence-ON detection with competitive hybridization, to achieve single base mutation quantitation in extensively modified synthetic ASOs. Given that this method is highly specific and sensitive (LoQ = 4 nM), we envision that it will find utility for screening other impurities as well as sequencing modified oligonucleotides. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Evaluation of Micro-organism in Ligated Metal and Self-ligating Brackets using Scanning Electron Microscopy: An In Vivo Study.

    Science.gov (United States)

    Sunil, P C; Michael, Tony; Raju, Aravind S; Paul, Renji K; Mamatha, J; Ebin, T M

    2015-07-01

    The objective of the study was to determine the sites of plaque accumulation and to compare the plaque accumulated with metal and self-ligating orthodontic brackets in order to know which bracket type had a higher plaque retaining capacity. The study was done on 20 subjects who were scheduled for orthodontic treatment including extraction of four premolars and fixed orthodontic appliances. Mesh-backed edgewise metal brackets ligated with steel ligatures and self-ligating brackets were bonded to the premolars to be extracted using composite (Transbond XT, 3M). The subjects were told to continue their normal oral hygiene regimen. Teeth were extracted at 1, 2, and 3 weeks after bracket bonding. Plaque attached to the buccal surfaces was stained using plaque disclosing agent. The teeth were then immersed in fixative containing 4% formaldehyde and 1% glutaraldehyde in phosphate buffer for 24 h, followed by 0.1 M phosphate buffer for 12 h. The specimens were then mounted on aluminum stubs, and sputter coated with gold prior to Scanning electron microscopy examination. The results showed that increased retention of plaque in metal brackets ligated with steel ligatures and comparatively less in self-ligating brackets at the base of the brackets. This study highlights that higher retention of plaque in 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.

  20. Hemorrhoidectomy: pedicle ligation vs pedicle coagulation

    International Nuclear Information System (INIS)

    Shaikh, B.S.; Balaoch, I.B.; Sohu, K.M.

    2015-01-01

    Objective: To compare the outcome of pedicle ligation vs pedicle coagulation haemorrhoidectomy. Methodology: This comparative prospective study was carried out at Department of Surgery, Ghulam Muhammad Maher Medcial College Hospital, Sukkur, Pakistan from January 2011 to January 2013 and included 300 patients of hemorrhoids. After routine workup, patients were randomly divided into two equal groups with one group receiving pedicle ligation and other pedicle coagulation for hemorrhoidectomy. Postoperatively they were followed for a period of 8 weeks for complications including pain, urinary retention, bleeding and anal stricture. Pain was recorded up to 10th postoperative day on the basis of visual analogue scale. Results: Mean age was 45 years and male to female ratio was 1.7:1. Mean operative time in pedicle ligation group was 15 min (range 14-20 min) and 17 min (15-25 min) in pedicle coagulation group. In Pedicle ligation group, pain was worst in 35 patients, moderate in 85 and mild in 30 patients; on the other hand in pedicle coagulation group, just 09 patients experienced worst pain. Urinary retention was observed in 44 patients in pedicle ligation group and 19 in pedicle coagulation group. Five patients in pedicle ligation group developed bleeding after their discharge from hospital; 7 patients in pedicle coagulation group reported secondary bleeding. Anal stricture was a rare complication and was found equally common in both the groups. Conclusion: Conventional hemorrhoidectomy with pedicle coagulation is an effective treatment modality for hemorrhoids and is associated with less chance of postoperative anal pain and urinary retention. (author)

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

    African Journals Online (AJOL)

    Pan African Medical Journal ... Introduction: Long-term outcome of patients after band ligation have been poorly defined. ... endoscopic band ligation, liver cirrhosis, complication of band ligation, esophageal varices, secondary prevention ...

  2. Sequence-dependent theory of oligonucleotide hybridization kinetics

    International Nuclear Information System (INIS)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-01-01

    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. 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.

  4. Torque expression in self-ligating orthodontic brackets and conventionally ligated brackets: A systematic review

    OpenAIRE

    Al-Thomali, Yousef; Mohamed, Roshan-Noor; Basha, Sakeenabi

    2017-01-01

    Background To evaluate the torque expression of self ligating (SL) orthodontic brackets and conventionally ligated brackets and the torque expression in active and passive SL brackets. Material and Methods Our systematic search included MEDLINE, EMBASE, CINAHL, PsychINFO, Scopus, and key journals and review articles; the date of the last search was April 4th 2016. We graded the methodological quality of the studies by means of the Quality Assessment Tool for Quantitative Studies, developed fo...

  5. [Friction: self-ligating brackets].

    Science.gov (United States)

    Thermac, Guilhem; Morgon, Laurent; Godeneche, Julien

    2008-12-01

    The manufacturers of self-ligating brackets advertise a reduction of the friction engendered between the wire and the bracket, which is an essential parameter for treatment's speed and comfort. We have compared the friction obtained with four types of self-ligating brackets - In-Ovation R, Damon 3, Smart Clip and Quick - with that of a standard bracket Omniarch associated with an elastomeric ligature. All bracket were tested on a bench of traction with three types of wires: steel .019"x.025", TMA .019"x.025" and NEO sentalloy F300 .020"x.020". The results confirm a clear friction reduction for all tested wire.

  6. Arterial ligation versus embolization in epistaxis management: Counterintuitive national trends.

    Science.gov (United States)

    Sylvester, Michael J; Chung, Sei Y; Guinand, Luis A; Govindan, Aparna; Baredes, Soly; Eloy, Jean Anderson

    2017-05-01

    Arterial ligation and embolization are treatment modalities indicated in severe and refractory epistaxis. The purpose of this study was to examine temporal trends and compare outcomes in treatment of hospitalized epistaxis patients with ligation or embolization. This retrospective cohort analysis utilized the 2008 to 2013 National Inpatient Sample to identify patients admitted with a primary diagnosis of epistaxis, and an associated procedure code for ligation or embolization. A total of 1,813 cases met the inclusion criteria, with 57.1% undergoing ligation. During the study period, treatment with ligation has trended downward, whereas treatment with embolization has remained constant. Overall, ligated patients were older (64.1 vs. 62.4 years; P = 0.027) and had higher rates of congestive heart failure (15.1% vs. 9.8%; P = 0.001). No significant differences in rates of chronic pulmonary disease, coagulopathy, liver disease, or hereditary hemorrhagic telangiectasia were observed between cohorts. No differences were observed in rates of blood transfusion, stroke, blindness, or in-hospital mortality; however, ligated patients had lower rates of intubation/tracheostomy (2.8% vs. 5.3%; P = 0.009). Ligated patients also experienced shorter hospital stays (3.6 vs. 4.0 days; P = 0.014) and incurred lower hospital charges ($33,029 vs. $69,304; P < 0.001). Compared to embolization, ligation is associated with significantly decreased hospital charges and shorter hospital stay, without an increase in complication rates. Counterintuitively, ligation appears to be trending downward nationally in its use relative to embolization. 2C Laryngoscope, 127:1017-1020, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  7. A comparative analysis of measles virus RNA by oligonucleotide fingerprinting

    International Nuclear Information System (INIS)

    Stephenson, J.R.; Meulen, V. ter

    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 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. (Author)

  8. 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.

  9. 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.

  10. Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

    Directory of Open Access Journals (Sweden)

    Christoph Niemietz

    2015-09-01

    Full Text Available The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR, expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP. Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO and small interfering RNA (siRNA designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

  11. A water soluble Cu(I)-NHC for CuAAC ligation of unprotected peptides under open air conditions.

    Science.gov (United States)

    Gaulier, Christelle; Hospital, Audrey; Legeret, Bertrand; Delmas, Agnès F; Aucagne, Vincent; Cisnetti, Federico; Gautier, Arnaud

    2012-04-25

    A reducing agent-free version of CuAAC able to operate under open air conditions is reported. A readily-synthesizable, hydrophilic and highly stable Cu(I)-NHC allows the clean ligations of unprotected peptides comprising sensitive side chains, at millimolar concentrations.

  12. Recovery of testicular blood flow following ligation of testicular vessels

    International Nuclear Information System (INIS)

    Pascual, J.A.; Villanueva-Meyer, J.; Salido, E.; Ehrlich, R.M.; Mena, I.; Rajfer, J.

    1989-01-01

    To determine whether initial ligation of the testicular vessels of the high undescended testis followed by a delayed secondary orchiopexy is a viable alternative to the classical Fowler-Stephens procedure, a series of preliminary experiments were conducted in the rat in which testicular blood flow was measured by the 133-xenon washout technique before, and 1 hour and 30 days after ligation of the vessels. In addition, testicular histology, and testis and sex-accessory tissue weights were measured in 6 control, 6 sham operated and 6 testicular vessel ligated rats 54 days after vessel ligation. The data demonstrate that ligation and division of the testicular blood vessels produce an 80 per cent decrease in testicular blood flow 1 hour after ligation of the vessels. However, 30 days later testis blood flow returns to the control and pre-treatment value. There were no significant changes in testis or sex-accessory tissue weights 54 days after vessel ligation. Histologically, 4 of the surgically operated testes demonstrated necrosis of less than 25 per cent of the seminiferous tubules while 1 testis demonstrated more than 75 per cent necrosis. The rest of the tubules in all 6 testes demonstrated normal spermatogenesis. From this study we conclude that initial testicular vessel ligation produces an immediate decrease in testicular blood flow but with time the collateral vessels are able to compensate and return the testis blood flow to its normal pre-treatment value. These preliminary observations lend support for the concept that initial ligation of the testicular vessels followed by a delayed secondary orchiopexy in patients with a high undescended testis may be a possible alternative to the classical Fowler-Stephens approach

  13. [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.

  14. Differentiation of the seven major lyssavirus species by oligonucleotide microarray.

    Science.gov (United States)

    Xi, Jin; Guo, Huancheng; Feng, Ye; Xu, Yunbin; Shao, Mingfu; Su, Nan; Wan, Jiayu; Li, Jiping; Tu, Changchun

    2012-03-01

    An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

  15. 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....

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

    International Nuclear Information System (INIS)

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

    2002-01-01

    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. Application of a cholesterol stationary phase in the analysis of phosphorothioate oligonucleotides by means of ion pair chromatography coupled with tandem mass spectrometry.

    Science.gov (United States)

    Studzińska, Sylwia; Krzemińska, Katarzyna; Szumski, Michał; Buszewski, Bogusław

    2016-07-01

    The main aim of this study was the investigation of the influence of several ion pair reagents towards both the retention and the mass spectrometry sensitivity of phosphorothioate oligonucleotides. A cholesterol stationary phase was applied for the first time in the analysis of this group of compounds. The mobile phase composition was modified by changing the concentration and the type of amines and acetates or 1,1,1,3,3,3-hexafluoroisopropanol. It has been shown that the increase of amines concentration results in the retention factor increase for each oligonucleotide, on each adsorbent. The only exception was the mobile phase composed of triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol. This is a consequence of interactions taking place between a cholesterol molecule and an alcohol. This effect was convenient when the mass spectrometry detection was applied, since it allowed an increase in the sensitivity. Moreover, optimization of the mobile phase composition and its impact on the efficiency of ionization process and on the sensitivity in mass spectrometry were also presented. The optimization of this new method, based on cholesterol stationary phase coupled with mass spectrometry detection, was finally applied for the determination of phosphorothioate oligonucleotides impurity in a real sample. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. 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.

  19. Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

    Science.gov (United States)

    Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

    2016-01-26

    Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application.

  20. 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

    Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-s...... 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....

  1. Laparoscopic-assisted percutaneous internal ring ligation in children

    African Journals Online (AJOL)

    Annals of Pediatric Surgery ... Patients and methods Laparoscopic percutaneous ligation of internal inguinal ring has been ... The mean operative time in our series was 15 (± 3) and 20 (± 5) min for bilateral cases, without anesthesia time. ... Conclusion Laparoscopic percutaneous ligation of internal inguinal ring repair of ...

  2. 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.

  3. 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.)

  4. Hypotension following patent ductus arteriosus ligation: The role of adrenal hormones

    OpenAIRE

    Clyman, RI; Wickremasinghe, A; Merritt, TA; Solomon, T; McNamara, P; Jain, A; Singh, J; Chu, A; Noori, S; Sekar, K; Lavoie, PM; Attridge, JT; Swanson, JR; Gillam-Krakauer, M; Reese, J

    2014-01-01

    Objective To test the hypothesis that an impaired adrenal response to stress might play a role in the hypotension that follows patent ductus arteriosus (PDA) ligation. Study design We performed a multicenter study of infants born at < 32 weeks' gestation who were about to undergo PDA ligation. Serum adrenal steroids were measured 3 times: before and after a cosyntropin (1.0 μg/kg) stimulation test (performed before the ligation), and at 10-12 hours after the ligation. A standardized approach...

  5. Fully automated parallel oligonucleotide synthesizer

    Czech Academy of Sciences Publication Activity Database

    Lebl, M.; Burger, Ch.; Ellman, B.; Heiner, D.; Ibrahim, G.; Jones, A.; Nibbe, M.; Thompson, J.; Mudra, Petr; Pokorný, Vít; Poncar, Pavel; Ženíšek, Karel

    2001-01-01

    Roč. 66, č. 8 (2001), s. 1299-1314 ISSN 0010-0765 Institutional research plan: CEZ:AV0Z4055905 Keywords : automated oligonucleotide synthesizer Subject RIV: CC - Organic Chemistry Impact factor: 0.778, year: 2001

  6. 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.

  7. Mandibular Dental Arch Changes with Active Self‑ligating Brackets ...

    African Journals Online (AJOL)

    ... with different forms of archwires with a control group in nonextraction cases. ... into three groups: Group I was treated with active self‑ligating brackets (Nexus, ... (SS) wires; Group II was treated with interactive self‑ligating bracket system ...

  8. Effects of Atelocollagen Formulation Containing Oligonucleotide on Endothelial Permeability

    Directory of Open Access Journals (Sweden)

    Koji Hanai

    2012-01-01

    Full Text Available Atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial. To date, atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models. However, the biological effects of this technology are not fully understood. In the present study, we investigated the effects of atelocollagen on endothelial paracellular barrier function. An atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells, and the change was dependent on the molecular size, structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation. An immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins. These changes were induced through p38 MAP kinase signaling. It is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties.

  9. Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

    Science.gov (United States)

    Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

    2011-04-01

    Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

  10. Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

    Science.gov (United States)

    Elzahar, N M; Magdy, N; El-Kosasy, Amira M; Bartlett, Michael G

    2018-05-01

    Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

  11. Chemoselective ligation and antigen vectorization.

    Science.gov (United States)

    Gras-Masse, H

    2001-01-01

    The interest in cocktail-lipopeptide vaccines has now been confirmed by phase I clinical trials: highly diversified B-, T-helper or cytotoxic T-cell epitopes can be combined with a lipophilic vector for the induction of B- and T-cell responses of predetermined specificity. With the goal of producing an improved vaccine that should ideally induce a multispecific response in non-selected populations, increasing the diversity of the immunizing mixture represents one of the most obvious strategies.The selective delivery of antigens to professional antigen-presenting cells represents another promising approach for the improvement of vaccine efficacy. In this context, the mannose-receptor represents an attractive entry point for the targeting to dendritic cells of antigens linked to clustered glycosides or glycomimetics. In all cases, highly complex but fully characterized molecules must be produced. To develop a modular and flexible strategy which could be generally applicable to a large set of peptide antigens, we elected to explore the potentialities of chemoselective ligation methods. The hydrazone bond was found particularly reliable and fully compatible with sulphide ligation. Hydrazone/thioether orthogonal ligation systems could be developed to account for the nature of the antigens and the solubility of the vector systems. Copyright 2001 The International Association for Biologicals.

  12. Custom-Designed MLPA Using Multiple Short Synthetic Probes Application to Methylation Analysis of Five Promoter CpG Islands in Tumor and Urine Specimens from Patients with Bladder Cancer

    DEFF Research Database (Denmark)

    Serizawa, R.R.; Ralfkiaer, U.; Dahl, C.

    2010-01-01

    Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using p...

  13. Time course of collagen peak in bile duct-ligated rats.

    Science.gov (United States)

    Tarcin, Orhan; Basaranoglu, Metin; Tahan, Veysel; Tahan, Gülgün; Sücüllü, Ilker; Yilmaz, Nevin; Sood, Gagan; Snyder, Ned; Hilman, Gilbert; Celikel, Cigdem; Tözün, Nurdan

    2011-04-28

    One of the most useful experimental fibrogenesis models is the "bile duct-ligated rats". Our aim was to investigate the quantitative hepatic collagen content by two different methods during the different stages of hepatic fibrosis in bile duct-ligated rats on a weekly basis. We questioned whether the 1-wk or 4-wk bile duct-ligated model is suitable in animal fibrogenesis trials. Of the 53 male Wistar rats, 8 (Group 0) were used as a healthy control group. Bile duct ligation (BDL) had been performed in the rest. Bile duct-ligated rates were sacrificed 7 days later in group 1 (10 rats), 14 days later in group 2 (9 rats), 21 days later in group 3(9 rats) and 28 days later in group 4 (9 rats). Eight rats underwent sham-operation (Sham). Hepatic collagen measurements as well as serum levels of liver enzymes and function tests were all analysed. The peak level of collagen was observed biochemically and histomorphometricly at the end of third week (P fibrosis in bile duct-ligated rats is transient, i.e. reverses spontaneously after 3 weeks. This contrasts any situation in patients where hepatic fibrosis is progressive and irreversible as countless studies performed by many investigators in the same animal model.

  14. 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.

  15. Ligation versus no ligation of spontaneous portosystemic shunts during liver transplantation: Audit of a prospective series of 66 consecutive patients.

    Science.gov (United States)

    Gomez Gavara, Concepcion; Bhangui, Prashant; Salloum, Chady; Osseis, Michael; Esposito, Francesco; Moussallem, Toufic; Lahat, Eylon; Fuentes, Liliana; Compagnon, Philippe; Ngongang, Norbert; Lim, Chetana; Azoulay, Daniel

    2018-04-01

    The management of large spontaneous portosystemic shunt (SPSS) during liver transplantation (LT) is a matter of debate. The aim of this study is to compare the short-term and longterm outcomes of SPSS ligation versus nonligation during LT, when both options are available. From 2011 to 2017, 66 patients with SPSS underwent LT: 56 without and 10 with portal vein thrombosis (PVT), all of whom underwent successful thrombectomy and could have portoportal reconstruction. The SPSS were either splenorenal (n = 40; 60.6%), left gastric (n = 16; 24.2%), or mesenterico-iliac (n = 10; 15.1%). Following portoportal anastomosis, the SPSS was ligated in 36 (54.4%) patients and left in place in 30 (45.5%) patients, based on the effect of the SPSS clamping/unclamping test on portal vein flow during the anhepatic phase. Intraoperatively, satisfactory portal flow was obtained in both groups. Primary nonfunction (PNF) and primary dysfunction (PDF) rates did not differ significantly between the 2 groups. Nonligation of SPSS was significantly associated with a higher rate of postoperative encephalopathy (P SPSS in LT (ie, PNF and PDF, PVT, and encephalopathy) was present in 16 (44.4%) and 22 (73.3%) patients of the ligated and nonligated shunt group, respectively (P = 0.02). Patient (P = 0.05) and graft (P = 0.02) survival rates were better in the ligated shunt group. In conclusion, the present study supports routine ligation of large SPSS during LT whenever feasible. Liver Transplantation 24 505-515 2018 AASLD. © 2018 by the American Association for the Study of Liver Diseases.

  16. Portal vein ligation is as effective as sequential portal vein and hepatic artery ligation in inducing contralateral liver hypertrophy in a rat model

    NARCIS (Netherlands)

    Veteläinen, Reeta; Dinant, Sander; van Vliet, Arlène; van Gulik, Thomas M.

    2006-01-01

    PURPOSE: Dual embolization of the hepatic artery and portal vein (PV) has been proposed to enhance contralateral liver regeneration before resection. The aim of this study was to evaluate the effect of PV ligation compared with simultaneous or sequential dual ligation on regeneration,

  17. 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

  18. Comparison of frictional resistance of esthetic and semi-esthetic self-ligating brackets

    Directory of Open Access Journals (Sweden)

    M S Kannan

    2015-01-01

    Full Text Available Aim: The frictional resistance encountered during sliding mechanics has been well established in the orthodontic literature, and it consists of complex interactions between the bracket, archwire, and method of ligation the claim of reduced friction with self-ligating brackets is often cited as a primary advantage over conventional brackets. This study was done to compare and evaluate the frictional forces generated between fully esthetic brackets and semi-aesthetic self-ligating brackets, which are of passive form and SEM (scanning electron microscope study of the Brackets after Frictional evaluation. Materials and Methods: Two types of self-ligating esthetic brackets, Damon clear (Ormco made of fully ceramic and Opal (Ultradent Products, USA and, Two types of self-ligating semi-esthetic brackets, Clarity SL (3M Unitek and Damon 3 (Ormco both of which are made of ceramic with metal slot. Arch wires with different dimensions and quality 17 × 25, 19 × 25 Titanium Molybdenum Alloy (TMA and 17 × 25, 19 × 25 stainless steel that came from plain strands of wire were used for frictional comparison test. The brackets used in this study had 0.022 × 0.028 inch slot. Results: The statistical tests showed significantly smaller amount of kinetic frictional forces is generated by Damon 3 (semi-esthetic self-ligating brackets. For each wire used, Damon 3 displayed significantly lower frictional forces (P ≤ 0.05 than any of the self-ligating system, followed by Opal (fully esthetic self-ligating brackets which generated smaller amount of frictional forces but relatively on the higher side when compared with Damon 3. Damon clear (fully esthetic self-ligating brackets generated the maximum amount of kinetic forces with all types of wire dimensions and properties when compared to the other three types of self-ligating system. Clarity SL (semi-esthetic self-ligating brackets generated smaller amount of frictional forces when compared with Damon clear and

  19. Comparison of frictional resistance of esthetic and semi-esthetic self-ligating brackets.

    Science.gov (United States)

    Kannan, M S; Murali, R V; Kishorekumar, S; Gnanashanmugam, K; Jayanth, V

    2015-04-01

    The frictional resistance encountered during sliding mechanics has been well established in the orthodontic literature, and it consists of complex interactions between the bracket, archwire, and method of ligation the claim of reduced friction with self-ligating brackets is often cited as a primary advantage over conventional brackets. This study was done to compare and evaluate the frictional forces generated between fully esthetic brackets and semi-aesthetic self-ligating brackets, which are of passive form and SEM (scanning electron microscope) study of the Brackets after Frictional evaluation. Two types of self-ligating esthetic brackets, Damon clear (Ormco) made of fully ceramic and Opal (Ultradent Products, USA) and, Two types of self-ligating semi-esthetic brackets, Clarity SL (3M Unitek) and Damon 3 (Ormco) both of which are made of ceramic with metal slot. Arch wires with different dimensions and quality 17 × 25, 19 × 25 Titanium Molybdenum Alloy (TMA) and 17 × 25, 19 × 25 stainless steel that came from plain strands of wire were used for frictional comparison test. The brackets used in this study had 0.022 × 0.028 inch slot. The statistical tests showed significantly smaller amount of kinetic frictional forces is generated by Damon 3 (semi-esthetic self-ligating brackets). For each wire used, Damon 3 displayed significantly lower frictional forces (P ≤ 0.05) than any of the self-ligating system, followed by Opal (fully esthetic self-ligating brackets) which generated smaller amount of frictional forces but relatively on the higher side when compared with Damon 3. Damon clear (fully esthetic self-ligating brackets) generated the maximum amount of kinetic forces with all types of wire dimensions and properties when compared to the other three types of self-ligating system. Clarity SL (semi-esthetic self-ligating brackets) generated smaller amount of frictional forces when compared with Damon clear and relatively higher amount of frictional forces

  20. Esophageal variceal ligation for hemostasis of acute variceal bleeding

    African Journals Online (AJOL)

    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 ...

  1. Time course of collagen peak in bile duct-ligated rats

    Directory of Open Access Journals (Sweden)

    Snyder Ned

    2011-04-01

    Full Text Available Abstract Background One of the most useful experimental fibrogenesis models is the "bile duct-ligated rats". Our aim was to investigate the quantitative hepatic collagen content by two different methods during the different stages of hepatic fibrosis in bile duct-ligated rats on a weekly basis. We questioned whether the 1-wk or 4-wk bile duct-ligated model is suitable in animal fibrogenesis trials. Methods Of the 53 male Wistar rats, 8 (Group 0 were used as a healthy control group. Bile duct ligation (BDL had been performed in the rest. Bile duct-ligated rates were sacrificed 7 days later in group 1 (10 rats, 14 days later in group 2 (9 rats, 21 days later in group 3(9 rats and 28 days later in group 4 (9 rats. Eight rats underwent sham-operation (Sham. Hepatic collagen measurements as well as serum levels of liver enzymes and function tests were all analysed. Results The peak level of collagen was observed biochemically and histomorphometricly at the end of third week (P Conclusion We have shown that fibrosis in bile duct-ligated rats is transient, i.e. reverses spontaneously after 3 weeks. This contrasts any situation in patients where hepatic fibrosis is progressive and irreversible as countless studies performed by many investigators in the same animal model.

  2. Graphene Oxide Based Nanocarrier Combined with a pH-Sensitive Tracer: A Vehicle for Concurrent pH Sensing and pH-Responsive Oligonucleotide Delivery.

    Science.gov (United States)

    Hsieh, Chia-Jung; Chen, Yu-Cheng; Hsieh, Pei-Ying; Liu, Shi-Rong; Wu, Shu-Pao; Hsieh, You-Zung; Hsu, Hsin-Yun

    2015-06-03

    We chemically tuned the oxidation status of graphene oxide (GO) and constructed a GO-based nanoplatform combined with a pH-sensitive fluorescence tracer that is designed for both pH sensing and pH-responsive drug delivery. A series of GOs oxidized to distinct degrees were examined to optimize the adsorption of the model drug, poly dT30. We determined that highly oxidized GO was a superior drug-carrier candidate in vitro when compared to GOs oxidized to lesser degrees. In the cell experiment, the synthesized pH-sensitive rhodamine dye was first applied to monitor cellular pH; under acidic conditions, protonated rhodamine fluoresces at 588 nm (λex=561 nm). When the dT30-GO nanocarrier was introduced into cells, a rhodamine-triggered competition reaction occurred, and this led to the release of the oligonucleotides and the quenching of rhodamine fluorescence by GO. Our results indicate high drug loading (FAM-dT30/GO=25/50 μg/mL) and rapid cellular uptake (<0.5 h) of the nanocarrier which can potentially be used for targeted RNAi delivery to the acidic milieu of tumors.

  3. 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.

  4. 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.

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

    International Nuclear Information System (INIS)

    Park, Serk In; Park, Sung-Jun; Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won; Park, Yun Gyu

    2016-01-01

    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.

  6. 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/....

  7. 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.

  8. A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

    Science.gov (United States)

    Lohman, Gregory J S; Bauer, Robert J; Nichols, Nicole M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Evans, Thomas C

    2016-01-29

    DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Thermodynamics of Oligonucleotide Duplex Melting

    Science.gov (United States)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  10. Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

    DEFF Research Database (Denmark)

    Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

    2017-01-01

    Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...

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

    International Nuclear Information System (INIS)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-01-01

    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.

  12. 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.

  13. Time course of collagen peak in bile duct-ligated rats

    OpenAIRE

    Tarcin, Orhan; Basaranoglu, Metin; Tahan, Veysel; Tahan, Gülgün; Sücüllü, Ilker; Yilmaz, Nevin; Sood, Gagan; Snyder, Ned; Hilman, Gilbert; Celikel, Cigdem; Tözün, Nurdan

    2011-01-01

    Abstract Background One of the most useful experimental fibrogenesis models is the "bile duct-ligated rats". Our aim was to investigate the quantitative hepatic collagen content by two different methods during the different stages of hepatic fibrosis in bile duct-ligated rats on a weekly basis. We questioned whether the 1-wk or 4-wk bile duct-ligated model is suitable in animal fibrogenesis trials. Methods Of the 53 male Wistar rats, 8 (Group 0) were used as a healthy control group. Bile duct...

  14. Oligonucleotide-based pharmaceuticals: Non-clinical and clinical safety signals and non-clinical testing strategies.

    Science.gov (United States)

    Mustonen, Enni-Kaisa; Palomäki, Tiina; Pasanen, Markku

    2017-11-01

    Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    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 (Prooster 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.

  16. Milrinone use for hemodynamic instability in patent ductus arteriosus ligation.

    Science.gov (United States)

    Halliday, Matthew; Kavarana, Minoo; Ebeling, Myla; Kiger, James

    2017-03-01

    Determine if prophylactic milrinone improves cardiovascular or long-term clinical outcomes in preterm neonates who receive surgical patent ductus arteriosus (PDA) ligation. Retrospective review of 45 infants over a 4-year period that received a PDA ligation at one institution. Data were collected on morbidity and mortality outcomes for all infants as well as milrinone therapy perioperatively. Of the 45 infants that were studied 15 received milrinone in the perioperative period of PDA ligation and the remaining 30 infants did not receive milrinone. The use of milrinone showed no statistically significant improvement in acute markers of hemodynamic stability. There was also no statistically significant difference in morbidity and mortality outcomes in milrinone group compared to the non-milrinone group. Prophylactic milrinone use for premature infants following PDA ligation does not show a significant cardiovascular or long-term clinical benefit.

  17. Patent Ductus Arteriosus Ligation and Adverse Outcomes: Causality or Bias?

    Science.gov (United States)

    Weisz, Dany E.; McNamara, Patrick J.

    2014-01-01

    Observational studies have associated patent ductus arteriosus (PDA) ligation in preterm infants with increased chronic lung disease (CLD), retinopathy of prematurity, and neurodevelopmental impairment at long-term follow-up. Although the biological rationale for this association is incompletely understood, there is an emerging secular trend toward a permissive approach to the PDA. However, insufficient adjustment for postnatal, pre-ligation confounders, such as intraventricular hemorrhage and the duration and intensity of mechanical ventilation, suggests the presence of residual bias due to confounding by indication, and obliges caution in interpreting the ligation-morbidity relationship. A period of conservative management after failure of medical PDA closure may be considered to reduce the number of infants treated with surgery. Increased mortality and CLD in infants with persistent symptomatic PDA suggests that surgical ligation remains an important treatment modality for preterm infants. PMID:25024972

  18. The mechano-gated channel inhibitor GsMTx4 reduces the exercise pressor reflex in rats with ligated femoral arteries.

    Science.gov (United States)

    Copp, Steven W; Kim, Joyce S; Ruiz-Velasco, Victor; Kaufman, Marc P

    2016-05-01

    Mechanical and metabolic stimuli arising from contracting muscles evoke the exercise pressor reflex. This reflex is greater in a rat model of simulated peripheral arterial disease in which a femoral artery is chronically ligated than it is in rats with freely perfused femoral arteries. The role played by the mechanically sensitive component of the exaggerated exercise pressor reflex in ligated rats is unknown. We tested the hypothesis that the mechano-gated channel inhibitor GsMTx4, a relatively selective inhibitor of mechano-gated Piezo channels, reduces the exercise pressor reflex in decerebrate rats with ligated femoral arteries. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the pressor response to Achilles tendon stretch (a purely mechanical stimulus) but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid (purely metabolic stimuli). Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced both the integrated pressor area (control 535 ± 21, GsMTx4 218 ± 24 mmHg·s; P reflex contributes to the exaggerated exercise pressor reflex during intermittent hindlimb muscle contractions in rats with ligated femoral arteries. Copyright © 2016 the American Physiological Society.

  19. Sphenopalatine Artery Ligation for Epistaxis: Factors Influencing Outcome and Impact of Timing of Surgery.

    Science.gov (United States)

    McDermott, Ailbhe M; O'Cathain, Eadaoin; Carey, Brian William; O'Sullivan, Peter; Sheahan, Patrick

    2016-03-01

    Sphenopalatine artery ligation is a commonly employed surgical intervention for control of posterior epistaxis unresponsive to nasal packing. The objective of the present study was to evaluate the outcome of sphenopalatine artery ligation for control of epistaxis at our institution and the impact of timing and other factors on outcome. Case series with chart review. Academic tertiary referral center. Case notes were reviewed for 45 consecutive patients undergoing sphenopalatine artery ligation for control of epistaxis between October 2008 and October 2014. Forty-one patients had nasal packing prior to sphenopalatine artery ligation, with 33 undergoing ≥2 packings. Postoperatively, 6 patients had rebleeding, which was treated with repacking (n = 4) and return to the operating room (n = 2). The overall success rate of sphenopalatine artery ligation was 87% (39 of 45). Rebleeding rate was not affected by concomitant septoplasty, anterior ethmoidal artery ligation, or postoperative nasal packing. Patients undergoing SPA ligation within the first 24 hours of admission had a significantly shorter hospital length of stay (3 vs 6 days, P = .02) and treatment cost (€5905 vs €10,001, P = .03). Length of stay was not influenced by sphenopalatine artery ligation after ≤1 nasal pack versus ≥2 packs. Timing of sphenopalatine artery ligation did not affect blood transfusion requirement (P = .84). Sphenopalatine artery ligation is an effective management strategy for surgical control of refractory epistaxis. Early timing of sphenopalatine artery ligation may lead to reductions in length of stay. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

  20. Comparative Study of Compensatory Liver Regeneration in a Rat Model: Portal Vein Ligation Only versus Sequential Ligation of the Portal Vein and Hepatic Artery

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Soo Young [Dept. of Pathology, Dongnam Institute of Radiological and Medical Sciences, Busan (Korea, Republic of); Jeon, Gyeong Sik [Dept. of Radiology, CHA Bundang Medical Center, College of Medicine, CHA University, Seongnam (Korea, Republic of); Lee, Byung Mo [Dept. of Surgery, Seoul Paik Hospital, Inje University College of Medicine, Seoul (Korea, Republic of)

    2013-04-15

    To compare the volume change and the regenerative capacity between portal vein ligation (embolization) (PVL) and heterochronous PVL with hepatic artery ligation (HAL) in a rodent model. The animals were separated into three groups: group I, ligation of the left lateral and median portal vein branches; group II, completion of PVL, followed by ligation of the same branches of the hepatic artery after 48 h; control group, laparotomy without ligation was performed. Five rats from each group were sacrificed on 1, 3, 5, and 7 days after the operation. Volume change measurement, liver function tests and immunohistochemical analysis were performed. The volume of the nonligated lobe between groups I and II was not significantly different by day 5 and day 7. Mean alanine aminotransferase and total bilirubin levels were significantly higher in group II, while the albumin level was higher in group I. Both c-kit- and MIB-5-positive cells used in the activity detection of regeneration were more prevalent in group I on day 1, 3, and 5, with statistical significance. There was no operation related mortality. PVL alone is safe and effective in compensatory liver regeneration. Performing both PVL and HAL does not confer any additional benefits.

  1. The detection of platelet derived growth factor using decoupling of quencher-oligonucleotide from aptamer/quantum dot bioconjugates

    International Nuclear Information System (INIS)

    Kim, Gang-Il; Sung, Yun-Mo; Kim, Kyung-Woo; Oh, Min-Kyu

    2009-01-01

    High-sensitivity, high-specificity detection of platelet derived growth factor (PDGF)-BB was realized using the change in fluorescence resonance energy transfer (FRET) occurring between quantum dot (QD) donors and black hole quencher (BHQ) acceptors. CdSe/ZnS QD/mercaptoacetic acid (MAA)/PDGF aptamer bioconjugates were successfully synthesized using ligand exchange. Black hole quencher (BHQ)-bearing oligonucleotide molecules showing partial sequence matching to PDGF aptamer were attached to PDGF aptamers and photoluminescence (PL) quenching was obtained through FRET. By adding target PDGF-BB to the bioconjugates containing BHQs, PL recovery was detected due to detachment of BHQ-bearing oligonucleotide from the PDGF aptamer as a result of the difference in affinity to the PDGF aptamer. The detection limit of the sensor was ∼0.4 nM and the linearity was maintained up to 1.6 nM in the PL intensity versus concentration curve. Measurement of PL recovery was suggested as a strong tool for high-sensitivity detection of PDGF-BB. Epidermal growth factor (EGF), the negative control molecule, did not contribute to PL recovery due to lack of binding affinity to the PDGF aptamers, which demonstrates the selectivity of the biosensor.

  2. Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

    Science.gov (United States)

    Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W

    2016-06-01

    Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.

  3. Multicomponent Reactions in Ligation and Bioconjugation Chemistry.

    Science.gov (United States)

    Reguera, Leslie; Méndez, Yanira; Humpierre, Ana R; Valdés, Oscar; Rivera, Daniel G

    2018-05-25

    Multicomponent reactions (MCRs) encompass an exciting class of chemical transformations that have proven success in almost all fields of synthetic organic chemistry. These convergent procedures incorporate three or more reactants into a final product in one pot, thus combining high levels of complexity and diversity generation with low synthetic cost. Striking applications of these processes are found in heterocycle, peptidomimetic, and natural product syntheses. However, their potential in the preparation of large macro- and biomolecular constructs has been realized just recently. This Account describes the most relevant results of our group in the utilization of MCRs for ligation/conjugation of biomolecules along with significant contributions from other laboratories that validate the utility of this special class of bioconjugation process. Thus, MCRs have proven to be efficient in the ligation of lipids to peptides and oligosaccharides as well as the ligation of steroids, carbohydrates, and fluorescent and affinity tags to peptides and proteins. In the field of glycolipids, we highlight the power of isocyanide-based MCRs with the one-pot double lipidation of glycan fragments functionalized as either the carboxylic acid or amine. In peptide chemistry, the versatility of the multicomponent ligation strategy is demonstrated in both solution-phase lipidation protocols and solid-phase procedures enabling the simultaneous lipidation and biotinylation of peptides. In addition, we show that MCRs are powerful methods for synchronized lipidation/labeling and macrocyclization of peptides, thus accomplishing in one step what usually requires long sequences. In the realm of protein bioconjugation, MCRs have also proven to be effective in labeling, site-selective modification, immobilization, and glycoconjugation processes. For example, we illustrate a successful application of multicomponent polysaccharide-protein conjugation with the preparation of multivalent

  4. 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

    by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex......, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12nm) in solution was revealed to be independent of concentration. The topological folding...

  5. Agmatine attenuates neuropathic pain in sciatic nerve ligated rats: modulation by hippocampal sigma receptors.

    Science.gov (United States)

    Kotagale, Nandkishor Ramdas; Shirbhate, Saurabh Haridas; Shukla, Pradeep; Ugale, Rajesh Ramesh

    2013-08-15

    Present study investigated the influence of the sigma (σ₁ and σ₂) receptors within hippocampus on the agmatine induced antinociception in neuropathic rats. Animals were subjected to sciatic nerve ligation for induction of neuropathic pain and observed the paw withdrawal latency in response to thermal hyperalgesia, cold allodynia and the mechanical hyperalgesia. Intrahippocampal (i.h.) as well as intraperitoneal (i.p.) administration of agmatine attenuated neuropathic pain in sciatic nerve ligated rats. Intrahippocampal administration of σ₁ agonist (+)-pentazocine or σ₂ agonist PB28 sensitized whereas, σ₁ antagonist BD1063 or σ₂ antagonist SM21 potentiated antinociceptive effect of agmatine. The behavioral effects correlated with hippocampal tumor necrosis factor-α (TNF-α) levels observed by western blot analysis. These results suggest that both the σ₁ and σ₂ receptor subunits within hippocampus play an important role in antinociceptive action of agmatine against neuropathic pain. © 2013 Elsevier B.V. All rights reserved.

  6. The clot thickens: an incompletely ligated left  atrial appendage

    Directory of Open Access Journals (Sweden)

    Merrill Thomas

    2018-05-01

    Full Text Available Our patient presented with known mechanical mitral valve endocarditis documented by 2D transesophageal echocardiogram (TOE from a recent hospitalization at an outside facility. On admission to our center, there was no prior knowledge of an incompletely ligated left atrial appendage (LAA according to patient- or family-reported history, review of outside records or the outside facility’s 2D TOE report. A 3D TOE performed at our center to assess her pathology, since a month had passed from her prior hospitalization, revealed a LAA ligation with evidence of communication to the left atrium and with clot present in the appendage. This case report highlights the common finding of incomplete closure of the LAA following surgical ligation, thus making it inadequate for stroke prevention in patients with atrial fibrillation, and that 3D TOE plays a valuable role in assessing the durability of LAA ligation.

  7. Protein-Templated Fragment Ligations-From Molecular Recognition to Drug Discovery.

    Science.gov (United States)

    Jaegle, Mike; Wong, Ee Lin; Tauber, Carolin; Nawrotzky, Eric; Arkona, Christoph; Rademann, Jörg

    2017-06-19

    Protein-templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein-templated fragment ligations are chemical reactions between small molecules ("fragments") utilizing a protein's surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. The approach exploits the molecular recognition of reactive small-molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. In this way, chemical synthesis and bioassay are integrated in one single step. This Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein-templated ligation products. The chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Rubber band ligation and infrared photocoagulation for the outpatient treatment of hemorrhoidal disease.

    Science.gov (United States)

    Ricci, Maurício Pichler; Matos, Délcio; Saad, Sarhan Sydney

    2008-01-01

    To compare the results of rubber band ligation and infrared photocoagulation for the treatment of hemorrhoidal disease through the analysis of the incidence of complications after each treatment and respective success rate. Forty-eight patients with first, second or third degree hemorrhoidal disease were randomized to receive treatment with either rubber band ligation (n=23) or infrared photocoagulation (n=25). Each patient was assessed at 1 week and 4 week intervals after treatment. We compared the incidence of complications and efficiency of each treatment modality and Qui-square, Fisher's Exact Test and Student's t Test were used to statistical analysis. Bleeding occured in eight (34,7%) patients treated with rubber band ligation and in four (16,0%) after infrared photocoagulation (p=0,243). Thirteen (52,0%) patients felt pain during infrared photocoagulation and 9 (39,1%) after rubber band ligation (p=0,546). After rubber band ligation, 14 (60,8%) required medication for pain relief. One patient (4,0%) required medication after infrared photocoagulation (prubber band ligator and 1 (4,0%) treated with infrared photocoagulation had symptomatic mucosal ulcers. Perianal dermatitis occured in two (8,0%) patients treated with infrared photocoagulation and one patient (4,3%) was observed to have prolapsed thrombosed piles after rubber band ligation. One month after treatment, 17 of 23 patients treated with rubber band ligation (73,9%) and 18 of 25 patients treated with infrared photocoagulation were asymptomatic. Rubber band ligation treated bleeding and prolapse in 90,0% and 82,4% respectively. Infrared photocoagulation treats bleeding and prolapse in 93,7% and 87,5% respectively. Those differences are not significant. Rubber band ligation causes significantly more pain than infrared photocoagulation during the first week after the procedures and their success rate are not different after four weeks of treatment.

  9. 21 CFR 876.4400 - Hemorrhoidal ligator.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Surgical Devices § 876.4400 Hemorrhoidal ligator. (a... of a ligature or band placed around the hemorrhoid. (b) Classification. Class II (performance...

  10. 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

    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 of naphtha......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...... 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...

  11. [Comparison of band ligation with sclerotherapy for the treatment of bleeding esophageal varices].

    Science.gov (United States)

    Ríos, Eddy; Sierralta, Armando; Abarzúa, Marigraciela; Bastías, Joaquín; Barra, María Inés

    2012-06-01

    Endoscopic band ligation is the treatment of choice for bleeding esophageal varices. However it is not clear if this procedure is associated with less early and late mortality than sclerotherapy. To assess rates of re-bleeding and mortality in cohorts of patients with bleeding esophageal varices treated with endoscopic injection or band ligation. Analysis of medical records and endoscopy reports of two cohorts of patients with bleeding esophageal varices, treated between 1990 and 2010. Of these, 54 patients were treated with sclerotherapy and 90 patients with band ligation. A third cohort of 116 patients that did not require endoscopic treatment, was included. The mean analyzed follow up period was 2.5 years (range 1-16). Collection of data was retrospective for patients treated with sclerotherapy and prospective for patients treated with band ligation. Rates of re-bleeding and medium term mortality were assessed. During the month ensuing the first endoscopic treatment, re-bleeding was recorded in 39 and 72% of patients treated with band ligation and sclerotherapy, respectively (p < 0.01). The relative risk of bleeding after band ligation was 0.53 (95% confidence limits 0.390.73). Death rates until the end of follow up were 20 and 48% among patients with treated with band ligation and sclerotherapy, respectively (p < 0.01), with a relative risk of dying for patients subjected to band ligation of 0.41 (95% confidence limits 0.25-0.68). Band ligation was associated with lower rates of re-bleeding and mortality in these cohorts of patients.

  12. Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

    Science.gov (United States)

    Mumcuoglu, Didem; Sardan Ekiz, Melis; Gunay, Gokhan; Tekinay, Turgay; Tekinay, Ayse B; Guler, Mustafa O

    2016-05-11

    Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

  13. In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

    Science.gov (United States)

    Machida, Kazuya

    2017-01-01

    Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

  14. Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

    Directory of Open Access Journals (Sweden)

    Kai Hoehlig

    Full Text Available A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

  15. Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

    Science.gov (United States)

    Wang, Fenghua; Zhang, Hefei; Gao, Jingxia; Chen, Fengjiao; Chen, Sijie; Zhang, Cuizhen; Peng, Gang

    2016-01-01

    Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

  16. New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Studzińska, Sylwia; Mounicou, Sandra; Szpunar, Joanna; Łobiński, Ryszard; Buszewski, Bogusław

    2015-01-15

    This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of (31)P may be used to quantify these compounds at the level of 80 μg L(-1), while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L(-1). The limit of detection was in the range of 0.07 and 0.13 mg L(-1). Accuracy varied with concentration, but was in the range of 3%. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes.

    Science.gov (United States)

    Cui, Chunzhi; Park, Dong Hyuk; Kim, Jeongyong; Joo, Jinsoo; Ahn, Dong June

    2013-06-14

    Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation.

  18. Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

    Science.gov (United States)

    Wang, Zheng; Vora, Gary J; Stenger, David A

    2004-07-01

    Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

  19. Dielectric properties of DNA oligonucleotides on the surface of silicon nanostructures

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-10-15

    Planar silicon nanostructures that are formed as a very narrow silicon quantum well confined by δ barriers heavily doped with boron are used to study the dielectric properties of DNA oligonucleotides deposited onto the surface of the nanostructures. The capacitance characteristics of the silicon nanostructures with oligonucleotides deposited onto their surface are determined by recording the local tunneling current–voltage characteristics by means of scanning tunneling microscopy. The results show the possibility of identifying the local dielectric properties of DNA oligonucleotide segments consisting of repeating G–C pairs. These properties apparently give grounds to correlate the segments with polymer molecules exhibiting the properties of multiferroics.

  20. Therapeutic efficacy of self-ligating brackets: A systematic review.

    Science.gov (United States)

    Dehbi, Hasnaa; Azaroual, Mohamed Faouzi; Zaoui, Fatima; Halimi, Abdelali; Benyahia, Hicham

    2017-09-01

    Over the last few years, the use of self-ligating brackets in orthodontics has progressed considerably. These systems have been the subject of numerous studies with good levels of evidence making it possible to evaluate their efficacy and efficiency compared to conventional brackets. The aim of this study was to evaluate the therapeutic efficacy of self-ligating brackets by means of a systematic review of the scientific literature. A systematic study was undertaken in the form of a recent search of the electronic Pubmed database, oriented by the use of several keywords combined by Boolean operators relating to the therapeutic efficacy of self-ligating brackets through the study of tooth alignment, space closure, expansion, treatment duration and degree of discomfort. The search was limited to randomized controlled studies, and two independent readers identified studies corresponding to the selection criteria. The chosen articles comprised 20 randomized controlled trials. The studies analyzed revealed the absence of significant differences between the two types of system on the basis of the clinical criteria adopted, thereby refuting the hypothesis of the superiority of self-ligating brackets over conventional systems. Copyright © 2017 CEO. Published by Elsevier Masson SAS. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. Vitamin K1 attenuates bile duct ligation-induced liver fibrosis in rats.

    Science.gov (United States)

    Jiao, Kun; Sun, Quan; Chen, Baian; Li, Shengli; Lu, Jing

    2014-06-01

    Vitamin K1 is used as a liver protection drug for cholestasis-induced liver fibrosis in China, but the mechanism of vitamin K1's action in liver fibrosis is unclear. In this study, a model of liver fibrosis was achieved via bile duct ligation in rats. The rats were then injected with vitamin K1, and the levels of serum aspartate aminotransferase, alanine transaminase, total bilirubin and the fibrotic grade score, collagen content, the expressions of α-smooth muscle actin (SMA) and cytokeratin 19 (CK19) were measured on day 28 after ligation. The levels of the biochemical parameters, fibrotic score and collagen content were significantly reduced by treatment with vitamin K1 in bile duct-ligated rats. In addition, α-SMA and CK19 expression was significantly reduced by vitamin K1 treatment in bile duct-ligated rats. These results suggested that vitamin K1 may attenuate liver fibrosis by inhibiting hepatic stellate cell activation in bile duct-ligated rats.

  5. Friction behavior of self-ligating and conventional brackets with different ligature systems.

    Science.gov (United States)

    Szczupakowski, Alexandra; Reimann, Susanne; Dirk, Cornelius; Keilig, Ludger; Weber, Anna; Jäger, Andreas; Bourauel, Christoph

    2016-07-01

    Self-ligating brackets are widely believed to offer better clinical efficiency and, in particular, less friction. Thus, the goal of this in vitro investigation was to assess the friction behavior of different bracket/archwire/ligature combinations during simulated canine retraction. An important aspect of this work was to determine whether conventional bracket systems behave differently in passive or active self-ligating brackets used with a Slide™ ligature, an elastic ligature, or a steel ligature. Three conventional (Contour, Class One; Discovery(®), Dentaurum; Mystique MB, GAC) and six self-ligating (Carriere SL, Class One; Clarity™ SL, 3M Unitek; Damon3, Ormco; In-Ovation(®) C, GAC; Speed Appliance, Speed System™; QuicKlear(®), Forestadent(®)) bracket systems were analyzed. All brackets featured a 0.022″ slot (0.56 mm). Each conventional system was tested with a steel ligature (0.25 mm; Remanium(®), Dentaurum), an elastic ligature (1.3 mm in diameter; Dentalastics, Dentaurum), and a modified elastic ligature (Slide™; Leone(®)). Each combination was used with four archwires, including rectangular stainless steel (0.46 × 0.64 mm, 0.018 × 0.025″, Dentaurum), rectangular nickel-titanium with Teflon coating (0.46 × 0.64 mm, 0.018 × 0.025″, Forestadent(®)), round coaxial nickel-titanium (0.46 mm, 0.018″, Speed), and half-round/half-square (D-profile) stainless steel (0.46 mm, 0.018″, Speed). In the orthodontic measurement and simulation system (OMSS), retraction of a canine was simulated on a Frasaco model replicated in resin. Based on the force systems, the respective friction values were determined. For each combination of materials, five brackets of the same type were tested and five single measurements performed. Friction values were found to vary distinctly with the different combinations, modifiers being the ligature systems and the archwire types. Any significant friction differences between the steel-ligated, Slide™-ligated

  6. High-throughput determination of RNA structure by proximity ligation.

    Science.gov (United States)

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-09-01

    We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

  7. 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....

  8. 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.

  9. 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.

  10. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

    Science.gov (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  11. [Comparison of root resorption between self-ligating and conventional brackets using cone-beam CT].

    Science.gov (United States)

    Liu, Yun; Guo, Hong-ming

    2016-04-01

    To analyze the differences of root resorption between passive self-ligating and conventional brackets, and to determine the relationship between passive self-ligating brackets and root resorption. Fifty patients were randomly divided into 2 groups using passive self-ligating brackets or conventional straight wire brackets (0.022 system), respectively. Cone-beam CT was taken before and after treatment. The amount of external apical root resorption of maxillary incisors was measured on CBCT images. Student's t test was performed to analyze the differences of root apical resorption between the 2 groups with SPSS17.0 software package. No significant difference(P> 0.05) in root resorption of maxillary incisors was found between passive self-ligating brackets and conventional brackets. Passive self-ligating brackets and conventional brackets can cause root resorption, but the difference was not significant. Passive self-ligating brackets do not induce more root resorption.

  12. 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.

  13. 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......). Using the EndoGIA stapler 45/2.5 mm, we compared en bloc ligation with individual ligation during LN and nephroureterectomy (LNU) with a special focus on the development of AVF and technique safety. METHODS: This was a retrospective study of all patients undergoing LN or LNU at the Department of Urology......, Roskilde Hospital, Denmark, between January 2010 and April 2014. The follow-up period was minimum six months. RESULTS: A total of 228 patients underwent LN and 56 patients underwent LNU. In the LN group, 77 patients underwent en bloc ligation. The mean surgical time was significantly reduced to 89 minutes...

  14. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insert].

    Science.gov (United States)

    Kupriushkin, M S; Pyshnyĭ, D V

    2012-01-01

    Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

  15. Triplex-forming ability of modified oligonucleotides

    DEFF Research Database (Denmark)

    Højland, Torben; Babu, Bolle Ravindra; Bryld, Torsten

    2007-01-01

    We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA ar...

  16. Experience of rubber band ligation for haemorrhoidal disease

    International Nuclear Information System (INIS)

    Hadi, A.; Aman, Z.; Anwar, F.; Khan, M.; Iqbal, Z.

    2011-01-01

    Objectives: To assess symptomatic relief in haemorrhoidal disease using rubber band ligation. Settings and duration: Surgical unit, Hayatabad Medical Complex, Peshawar from July 2007 to June 2009. Patients and Methods: All patients presenting with rectal bleeding or prolapse underwent proto copy to determine the cause of bleeding. Only patients with first, second and third degree haemorrhoids (bleeding and prolapse) were selected for the study and they underwent rubber band ligation in two sessions, 6 weeks apart. Patients were followed in the OPD at 3 months and one year when their subjective and objective symptoms were noted followed by rectal examination and proctoscopy. Therapeutic response was assessed by symptomatic improvement in bleeding and prolapse of hemorrhoids and any complications. Results: A total of 105 patients underwent rubber band ligation. There were 65(62%) males and 40(38%) females with a male to female ratio of 1.6:1. Age of the patients ranged from 21-65 years (+- 2.4 SD). Twenty three (21.9%) patients had first degree, 50(47.6%) had second degree and 32(30.5%) had third degree haemorrhoids. Forty-eight patients (45.7%) presented with degree, 50(47.6%) had second prolapse, 33(31.4%) had both bleeding and prolapse and 23(21.9%) had bleeding only. At 3 months of follow up rubber band ligation cured 76(72.4%) patients, improved 13(12.4%) and failed in 7(6.7%) patients. At one year follow up, recurrence of bleeding and prolapse occurred in 16(15.2%) patients having third degree haemorrhoids. Overall success was third achieved in 89(84.8%) cases. Complications were seen in 20(19.1%) patients which were of mild to moderate nature and no serious/ life threatening complications were noted. Conclusion: Rubber band legislation is a rapid and safe procedure for testing first, second and and early third degree Conclusions: Rubber band ligation is a rapid and safe non operative procedure for treating first, second and third degree haemorrhoids on out

  17. 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.

  18. Cholestasis progression effects on long-term memory in bile duct ligation rats

    Directory of Open Access Journals (Sweden)

    Nasrin Hosseini

    2014-01-01

    Full Text Available Background : There is evidence that cognitive functions are affected by some liver diseases such as cholestasis. Bile duct ligation induces cholestasis as a result of impaired liver function and cognition. This research investigates the effect of cholestasis progression on memory function in bile duct ligation rats. Materials and Methods: Male Wistar rats were randomly divided into five groups, which include: control group for BDL-7, control group for BDL-21, sham group (underwent laparotomy without bile duct ligation, BDL-7 group (7 days after bile duct ligation, and BDL-21 group (21 days after bile duct ligation. Step-through passive avoidance test was employed to examine memory function. In all groups, short-term (7 days after foot shock and long-term memories (21 days after foot shock were assessed. Results: Our results showed that liver function significantly decreased with cholestasis progression (P < 0.01. Also our findings indicated BDL-21 significantly impaired acquisition time (P < 0.05. Memory retrieval impaired 7 (P < 0.05 and 21 days (P < 0.001 after foot shock in BDL-7 and BDL-21 groups, respectively. Conclusion: Based on these findings, liver function altered in cholestasis and memory (short-term and long-term memory impaired with cholestasis progression in bile duct ligation rats. Further studies are needed to better insight the nature of progression of brain damage in cholestatic disease.

  19. Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR.

    Science.gov (United States)

    Knez, Karel; Spasic, Dragana; Delport, Filip; Lammertyn, Jeroen

    2015-05-15

    Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. 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

  1. Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.

    Directory of Open Access Journals (Sweden)

    Michael A Cook

    Full Text Available BACKGROUND: Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM, we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. CONCLUSIONS/SIGNIFICANCE: These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

  2. 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.

  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. THERAPEUTIC ANTISENSE OLIGONUCLEOTIDES AGAINST CANCER: HURDLING TO THE CLINIC

    Directory of Open Access Journals (Sweden)

    Pedro Miguel Duarte Moreno

    2014-10-01

    Full Text Available Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen, oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

  5. Peroxide-mediated desulfurization of phosphorothioate oligonucleotides and its prevention.

    Science.gov (United States)

    Krotz, Achim H; Mehta, Rahul C; Hardee, Gregory E

    2005-02-01

    Desulfurization at the internucleotide phosphorothioate linkage of antisense oligonucleotides (ASOs) in dermatological formulations has been investigated using strong ion exchange chromatography and mass spectroscopy. The formation of phosphate diester linkages appeared to arise from a reaction between the phosphorothioate oligonucleotide and a potent oxidizing agent. Screening of excipients used in the formulation indicated that the cause of desulfurization was related to the presence of polyethylene glycol-derived nonionic surfactants MYRJ 52 or BRIJ 58. Autoxidation of the polyethylene glycol chain is suggested as the probable origin for the observed incompatibility. The ability of various antioxidants to prevent oxidative degradation of ASO-1 in simple test systems and in oil-in-water emulsions is described. It is found that in test systems both lipophilic and hydrophilic antioxidants are effective. However, in cream formulation (oil-in-water emulsions) of ASO-1 the addition of hydrophilic antioxidants L-cysteine or DL-alpha-lipoic acid has been shown to be superior in protecting the oligonucleotide from desulfurization upon storage. Copyright 2004 Wiley-Liss, Inc.

  6. A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

    Directory of Open Access Journals (Sweden)

    Lykke Pedersen

    2014-01-01

    Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

  7. 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.

  8. 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.

  9. 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.

  10. Versatile functionalization of nanoelectrodes by oligonucleotides via pyrrole electrochemistry.

    Science.gov (United States)

    Descamps, Emeline; Nguyen, Khoa; Bouchain-Gautier, Christelle; Filoramo, Arianna; Goux-Capes, Laurence; Goffman, Marcello; Bourgoin, Jean-Philippe; Mailley, Pascal; Livache, Thierry

    2010-11-15

    Surface modification at the nanometer scale is a challenge for the future of molecular electronics. In particular, the precise anchoring and electrical addressing of biological scaffolds such as complex DNA nanonetworks is of importance for generating bio-directed assemblies of nano-objects for nanocircuit purposes. Herein, we consider the individual modification of nanoelectrodes with different oligonucleotide sequences by an electrochemically driven co-polymerization process of pyrrole and modified oligonucleotide sequences bearing pyrrole monomers. We demonstrate that this one-step technique presents the advantages of simplicity, localization of surface modification, mechanical, biological and chemical stability of the coatings, and high lateral resolution.

  11. Cost and efficacy comparison of in vitro fertilization and tubal anastomosis for women after tubal ligation.

    Science.gov (United States)

    Messinger, Lauren B; Alford, Connie E; Csokmay, John M; Henne, Melinda B; Mumford, Sunni L; Segars, James H; Armstrong, Alicia Y

    2015-07-01

    To compare cost and efficacy of tubal anastomosis to in vitro fertilization (IVF) in women who desired fertility after a tubal ligation. Cost-effectiveness analysis. Not applicable. Not applicable. Not applicable. Cost per ongoing pregnancy. Cost per ongoing pregnancy for women after tubal anastomosis ranged from $16,446 to $223,482 (2014 USD), whereas IVF ranged from $32,902 to $111,679 (2014 USD). Across maternal age groups cost effective than IVF for ongoing pregnancy. Sensitivity analyses validated these findings across a wide range of ongoing pregnancy probabilities as well as costs per procedure. Tubal anastomosis was the most cost-effective approach for most women less than 41 years of age, whereas IVF was the most cost-effective approach for women aged ≥41 years who desired fertility after tubal ligation. A model was created that can be modified based on cost and success rates in individual clinics for improved patient counseling. Copyright © 2015 American Society for Reproductive Medicine. All rights reserved.

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

    Science.gov (United States)

    Gupta, P J

    2003-10-01

    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.

  13. Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

    International Nuclear Information System (INIS)

    Maria Lina R; Budiman Bela; Andi Yasmon

    2009-01-01

    Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

  14. Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

    Science.gov (United States)

    Studzińska, Sylwia

    2018-01-01

    Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

    Directory of Open Access Journals (Sweden)

    Mingjie Tang

    Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

  16. Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

    Science.gov (United States)

    Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

    2004-03-01

    A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non

  17. Sieve-based device for MALDI sample preparation. I. Influence of sample deposition conditions in oligonucleotide analysis to achieve significant increases in both sensitivity and resolution.

    Science.gov (United States)

    Molin, Laura; Cristoni, Simone; Crotti, Sara; Bernardi, Luigi Rossi; Seraglia, Roberta; Traldi, Pietro

    2008-11-01

    Spraying of oligonucleotide-matrix solutions through a stainless steel (ss) sieve (38 microm, 450 mesh) leads to the formation, on the matrix-assisted laser desorption/ionization (MALDI) sample holder, of uniformly distributed microcrystals, well separated from each other. When the resulting sample holder surface is irradiated by laser, abundant molecular species form, with a clear increase in both intensity and resolution with respect to values obtained by 'Dried Droplet', 'Double Layer', and 'Sandwich' deposition methods. In addition, unlike the usual situation, the sample is perfectly homogeneous, and identical spectra are obtained by irradiating different areas. On one hand, the data indicate that this method is highly effective for oligonucleotide MALDI analysis, and on the other, that it can be validly employed for fully automated MALDI procedures.

  18. Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

    Science.gov (United States)

    Weinberg, Marc S.; Michod, Richard E.

    2017-01-01

    In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3′ end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life. PMID:28989747

  19. Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

    Science.gov (United States)

    Dhar, Nisha; Weinberg, Marc S.; Michod, Richard E.; Durand, Pierre M.

    2017-09-01

    In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3' end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life.

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

    Science.gov (United States)

    Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

    2015-01-01

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

  1. 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

  2. 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...

  3. Ligation of internal iliac arteries for control of pelvic haemorrhage.

    Directory of Open Access Journals (Sweden)

    Nandanwar Y

    1993-10-01

    Full Text Available This is a retrospective review of the internal iliac ligations (IAL done over a period of 3 years. Both gynaecological and obstetric cases were considered. A total of 46 ligations were performed. The additional treatment required was hysterectomy to control haemorrhage. Intra-and post-operative complications were noted. A comparative review of the effectiveness of IAL in different situations is done and alternative modalities of treatment are considered.

  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

    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. Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

    Science.gov (United States)

    Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

    2017-07-15

    We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Azobenzene Photoswitches for Staudinger-Bertozzi Ligation

    NARCIS (Netherlands)

    Szymanski, Wiktor; Wu, Bian; Poloni, Claudia; Janssen, Dick B.; Feringa, Ben L.

    2013-01-01

    A novel family of azobenzenes containing residues needed for aqueous Staudinger–Bertozzi ligation to azides was designed. The resulting photochromes show stable and reversible switching behavior in water, with a photostationary state (PSS) of up to 95:5 cis/trans. Applications in model systems

  7. Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

    International Nuclear Information System (INIS)

    Sakai, Hiroshi; Tagawa, Yoh-ichi; Tamai, Miho; Motoyama, Hiroaki; Ogawa, Shinichiro; Soeda, Junpei; Nakata, Takenari; Miyagawa, Shinichi

    2010-01-01

    Research highlights: → Hepatic progenitor cells were isolated from the portal branch-ligated liver of mice. → Portal branch ligation-stimulated hepatic progenitor cells (PBLHCs) express Hmga2. → PBLHCs have bidirectional differentiation capability in vitro. -- Abstract: Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

  8. Radiological study on the change of duct-ligated parotid gland

    International Nuclear Information System (INIS)

    Ohnishi, Takashi

    1994-01-01

    The change of the parotid gland with time following ligation of the main duct was investigated. The duct-ligated parotid gland in rabbit was examined by salivary gland scintigraphy with 99m Tc-pertechnetate ( 99m TcO 4 - ), sialography and microscopic observation. The third day after ligation of the main duct, the outward form of the parotid gland on the static scintigram was not well-defined. On the seventh day, 99m TcO 4 - accumulation was decreased slightly. On the 14th day, atrophy of the parotid gland occurred. The degree of atrophy produced by ligation increases as the duration of the ligation increases. On the 42nd day, the presence of the parotid gland was not recorded practically. The main duct was dilated on the third day. On the seventh day, the intraglandular ducts were bent and strictured. Disappearance of the peripheral duct and atrophy of the parotid gland parenchyma was observed. On microscopic observation, the intraglandular tributaries and the lumen were dilated on the third day. And the reticular fiber was observed that was irregularly formed in parts. The acinar cells were pressed by large and small dilated lumen on the seventh day. On the 14th day, the collagenous fiber around the acini and the duct was increased still more. In addition, fibrosis of the lobule interspace was observed. The degree of atrophy of the acini and lobule was increased maximally on the 42nd day. These results of the salivary gland scintigraphy closely connected with sialograms and microscopic findings. The parotid gland tissue decreases and changings of the duct system were indicated by these imaging methods in detail. (author)

  9. 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.

  10. Functional regulation of RNA-induced silencing complex by photoreactive oligonucleotides.

    Science.gov (United States)

    Matsuyama, Yohei; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

    2014-02-01

    We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  11. Effects of cholestasis on learning and locomotor activity in bile duct ligated rats.

    Science.gov (United States)

    Hosseini, Nasrin; Alaei, Hojjatallah; Nasehi, Mohammad; Radahmadi, Maryam; Mohammad Reza, Zarrindast

    2014-01-01

    Cognitive functions are impaired in patients with liver disease. Bile duct ligation causes cholestasis that impairs liver function. This study investigated the impact of cholestasis progression on the acquisition and retention times in the passive avoidance test and on the locomotor activity of rats. Cholestasis was induced in male Wistar rats by ligating the main bile duct. Locomotor activity, learning and memory were assessed by the passive avoidance learning test at day 7, day 14, and day 21 post-bile duct ligation. The serum levels of bilirubin, alanine aminotransferase, and alkaline phosphatase were measured. The results showed that acquisition time and locomotor activity were not affected at day 7 and day 14, but they were significantly (P locomotor activity were impaired at 21 days after bile duct ligation following the progression of cholestasis.

  12. Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

    Directory of Open Access Journals (Sweden)

    Parekh-Olmedo Hetal

    2006-10-01

    Full Text Available Abstract Background Huntington's Disease (HD is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions. Results As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP. Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene. Conclusion Single-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.

  13. 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.

  14. Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

    Science.gov (United States)

    Catron, Brittany; Caruso, Joseph A.; Limbach, Patrick A.

    2012-06-01

    A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as 31P+, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.

  15. i-Genome: A database to summarize oligonucleotide data in genomes

    Directory of Open Access Journals (Sweden)

    Chang Yu-Chung

    2004-10-01

    Full Text Available Abstract Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.

  16. In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

    Science.gov (United States)

    Chow, C W; Clark, M P; Rinaldo, J E; Chalkley, R

    1996-03-01

    In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

  17. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  18. Self-ligating versus conventional metallic brackets on Streptococcus mutans retention: A systematic review.

    Science.gov (United States)

    Longoni, Juliano N; Lopes, Beatriz M; Freires, Irlan A; Dutra, Kamile L; Franco, Ademir; Paranhos, Luiz R

    2017-01-01

    The present study aimed to review the literature systematically and assess comparatively whether self-ligating metallic brackets accumulate less Streptococcus mutans biofilm than conventional metallic brackets. The systematic search was performed following PRISMA guidelines and registration in PROSPERO. Seven electronic databases (Google Scholar, LILACS, Open Grey, PubMed, SciELO, ScienceDirect, and Scopus) were consulted until April 2016, with no restriction of language and time of publication. Only randomized clinical studies verifying S. mutans colonization in metallic brackets (self-ligating and conventional) were included. All steps were performed independently by two operators. The search resulted in 546 records obtained from the electronic databases. Additionally, 216 references obtained from the manual search of eligible articles were assessed. Finally, a total of 5 studies were included in the qualitative synthesis. In 1 study, the total bacterial count was not different among self-ligating and conventional brackets, whereas in 2 studies the amount was lower for self-ligating brackets. Regarding the specific count of S. mutans , 2 studies showed less accumulation in self-ligating than in conventional brackets. Based on the limited evidence, self-ligating metallic brackets accumulate less S. mutans than conventional ones. However, these findings must be interpreted in conjunction with particularities individual for each patient - such as hygiene and dietary habits, which are components of the multifactorial environment that enables S. Mutans to proliferate and keep retained in the oral cavity.

  19. Factors Promoting Tubal Ligation in Females Presenting to Tertiary Care Centers

    Directory of Open Access Journals (Sweden)

    Hammad Ali Qazi

    2009-09-01

    Full Text Available Objective: Despite development of new contraceptive methods, sterilization remained the most widely used method. Our objective was to determine the factors contributing to decision making for tubal ligation in females.Materials and methods: A cross sectional study was conducted in Jinnah Post graduate Medical Centre (JPMC between March - November 2008. About 505 Females using contraceptive measures were consecutively included. Those having any severe debilitating disease and unfamiliar with “urdu” language were excluded. Three trained co researchers conducted structured interviews to determine the frequency and factors associated with tubal ligation.Results: The final multiple logistic regression showed illiteracy [AOR 2.91 95% CI 1.53-5.53], number of children < 3 [AOR 6.15 95% CI 2.61-14.50], age of women < 30 [AOR 0.12 95% CI 0.06-0.22] years and information gained through health worker [AOR 3.04 95% CI 1.60-5.80] to be statistically significant.Conclusion: This highlights tubal ligation was more common in uneducated women of age > 35 years having <3 children. The most common means of promoting tubal ligation was information gained through health workers.

  20. 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.

  1. 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.

  2. Evaluation of frictional forces of polycarbonate self-ligating brackets.

    Science.gov (United States)

    Fernandes, Daniel J; Miguel, José Augusto M; Quintão, Catia C A; Elias, Carlos N

    2010-01-01

    To evaluate the frictional forces generated by ceramic- (Opal, Ultradent) and glass-fiber-reinforced polycarbonate self-ligating brackets (Oyster, Gestenco) and compare the effectiveness of these ligatureless systems with glass-fiber-reinforced polycarbonate conventional brackets (Blonde, Gestenco). The hypothesis is that there is no difference between frictional forces generated by ceramic- and glass-fiber-reinforced polycarbonate self-ligating and glass-fiber-reinforced polycarbonate conventional brackets. Twelve preadjusted 0.022 3 0.028-inch maxillary canine brackets were tested, divided into three groups: Opal, Oyster, and Blonde. Frictional tests were conducted with the Emic DL 10000 testing machine with a 20 N loadcell for 40 seconds at a 0.5 cm/min speed. Each bracket-wire combination was tested five times. The data generated were analyzed by parametric analysis of variance (one-way ANOVA) and Bonferroni tests. Analysis of variance indicated significant differences for the three groups (Pfrictional forces of the Oyster glass-fiber-reinforced polycarbonate self-ligating brackets were significantly lower (37.0 ± 8.9 cN) than those of the Opal ceramic-reinforced polycarbonate self-ligating brackets (49.5 ± 10.1 cN), while the Blonde glass-fiber-reinforced conventional bracket frictional forces were 105.8 ± 6.4 cN. Oyster glass-fiber-reinforced polycarbonate brackets produced less friction than Opal ceramic-reinforced polycarbonate brackets. The polycarbonate ligatureless system showed significantly lower frictional forces compared to Blonde conventional polycarbonate brackets tied with elastomeric ligatures. The study rejected the initial hypothesis because there are significant differences of frictional forces among the tested systems. © 2010 BY QUINTESSENCE PUBLISHING CO, INC.

  3. The effect of ligation method on friction in sliding mechanics.

    Science.gov (United States)

    Hain, Max; Dhopatkar, Ashish; Rock, Peter

    2003-04-01

    During orthodontic tooth movement with the preadjusted edgewise system, friction generated at the bracket/archwire interface tends to impede the desired movement. The method of ligation is an important contributor to this frictional force. This in vitro study investigated the effect of ligation method on friction and evaluated the efficacy of the new slick elastomeric modules from TP Orthodontics (La Porte, Ind), which are claimed to reduce friction at the module/wire interface. Slick modules were compared with regular nonslick modules, stainless steel ligatures, and the SPEED self-ligating bracket system (Strite Industries, Cambridge, Ontario, Canada). The effect of using slick modules with metal-reinforced ceramic (Clarity, 3M Unitek, Monrovia, Calif) and miniature brackets (Minitwin, 3M Unitek) was also examined. Results showed that, when considering tooth movement along a 0.019 x 0.025-in stainless steel archwire, saliva-lubricated slick modules can reduce static friction at the module/archwire interface by up to 60%, regardless of the bracket system. The SPEED brackets produced the lowest friction compared with the 3 other tested bracket systems when regular modules were used. The use of slick modules, however, with all of the ligated bracket types tested significantly reduced friction to below the values recorded in the SPEED groups. Loosely tied stainless steel ligatures were found to generate the least friction.

  4. Probing Nitrosyl Ligation of Surface-Confined Metalloporphyrins by Inelastic Electron Tunneling Spectroscopy

    Science.gov (United States)

    2013-01-01

    Complexes obtained by the ligation of nitric oxide (NO) to metalloporphyrins represent important model systems with biological relevance. Herein we report a molecular-level investigation of surface-confined cobalt tetraphenyl porphyrin (Co-TPP) species and their interaction with NO under ultrahigh vacuum conditions. It is demonstrated that individual NO adducts can be desorbed using the atomically sharp tip of a scanning tunneling microscope, whereby a writing process is implemented for fully saturated regular metalloporphyrin arrays. The low-energy vibrational characteristics of individual Co-TPP-nitrosyl complexes probed by inelastic electron tunneling spectroscopy (IETS) reveal a prominent signature at an energy of ≃31 meV. Using density functional theory-based IETS simulations—the first to be performed on such an extensive interfacial nanosystem—we succeed to reproduce the low-frequency spectrum for the NO-ligated complex and explain the absence of IETS activity for bare Co-TPP. Moreover, we can conclusively assign the IETS peak of NO-Co-TPP to a unique vibration mode involving the NO complexation site, namely, the in-plane Co–N–O rocking mode. In addition, we verify that the propensity rules previously designed on small aromatic systems and molecular fragments hold true for a metal–organic entity. This work notably permits one to envisage IETS spectroscopy as a sensitive tool to chemically characterize hybrid interfaces formed by complex metal–organic units and gaseous adducts. PMID:23718257

  5. Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

    Science.gov (United States)

    Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

    2008-01-01

    Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

  6. Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

    Science.gov (United States)

    Godinho, Bruno M D C; Gilbert, James W; Haraszti, Reka A; Coles, Andrew H; Biscans, Annabelle; Roux, Loic; Nikan, Mehran; Echeverria, Dimas; Hassler, Matthew; Khvorova, Anastasia

    2017-12-01

    Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

  7. Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

    Science.gov (United States)

    Low, Donald W.; Hill, Michael G.; Carrasco, Michael R.; Kent, Stephen B. H.; Botti, Paolo

    2001-01-01

    We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet7]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the α-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet7]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by ≈45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments. PMID:11390992

  8. Transcatheter closure of re-canalized patent ductus arteriosus after surgical ligation

    International Nuclear Information System (INIS)

    Zhang Qingqiao; Jiang Shiliang; Huang Lianjun; Zhao Shihua; Zheng Hong; Ling Jian; Jin Jinglin; Xu Zhongying; Xie Ruolan; Dai Ruping

    2002-01-01

    Objective: To evaluate the effectiveness of transcatheter closure of re-canalized patent ductus arteriosus (PDA) after surgical ligation. Methods: Between June 1995 and November 2000, 14 patients (5 male, 9 female) with re-canalized PDA after surgical ligation underwent transcatheter closure, their median age was 13 years (range 4 to 48 years). The time between surgical ligation and the interventional procedure ranged from one month to twenty-two years. Implantations of Amplatzer duct occluder and Rashkind occluder were performed trans-venously. Cook coil occlusions was performed trans-arterially. Follow-up with X-ray radiograph and echocardiography was made 24 hours, 1, 3, 6 months, and more than 1 year after the procedure. Results: Twelve PDAs were of funnel shape, and the remaining two PDAs were of tubular shape. The median minimum diameter of re-canalized PDA after ligation was 4 mm (range 1 to 8 mm). Aortograms ten minutes after closure showed complete closure and trivial residual shunt in 11 and 3 patients, respectively. The technical success rate was 100%, and there were no complications. Echocardiography showed complete closure in all patients within 24 hours. All patients were discharged in one to two days after the procedure. At a follow-up of one to eighteen months in ten patients, there were no migration of devices and residual PDA. Conclusion: Transcatheter closure using Amplatzer duct occluder, coil (Cook company or Pfm company) and Rashkind occluder was an effective method for patients with re-canalized PDA after surgical ligation. It may be an alternative to second surgery owing to its safety, reliability, min-invasiveness, and short hospitalization

  9. Transcatheter closure of re-canalized patent ductus arteriosus after surgical ligation

    Energy Technology Data Exchange (ETDEWEB)

    Qingqiao, Zhang; Shiliang, Jiang; Lianjun, Huang; Shihua, Zhao; Hong, Zheng; Jian, Ling; Jinglin, Jin; Zhongying, Xu; Ruolan, Xie; Ruping, Dai [Chinese Academy of Medical Science, Beijing Union Medical College, Beijing (China). Cardiovascular Inst. and Fuwai Hospital, Dept. of Radiology

    2002-02-01

    Objective: To evaluate the effectiveness of transcatheter closure of re-canalized patent ductus arteriosus (PDA) after surgical ligation. Methods: Between June 1995 and November 2000, 14 patients (5 male, 9 female) with re-canalized PDA after surgical ligation underwent transcatheter closure, their median age was 13 years (range 4 to 48 years). The time between surgical ligation and the interventional procedure ranged from one month to twenty-two years. Implantations of Amplatzer duct occluder and Rashkind occluder were performed trans-venously. Cook coil occlusions was performed trans-arterially. Follow-up with X-ray radiograph and echocardiography was made 24 hours, 1, 3, 6 months, and more than 1 year after the procedure. Results: Twelve PDAs were of funnel shape, and the remaining two PDAs were of tubular shape. The median minimum diameter of re-canalized PDA after ligation was 4 mm (range 1 to 8 mm). Aortograms ten minutes after closure showed complete closure and trivial residual shunt in 11 and 3 patients, respectively. The technical success rate was 100%, and there were no complications. Echocardiography showed complete closure in all patients within 24 hours. All patients were discharged in one to two days after the procedure. At a follow-up of one to eighteen months in ten patients, there were no migration of devices and residual PDA. Conclusion: Transcatheter closure using Amplatzer duct occluder, coil (Cook company or Pfm company) and Rashkind occluder was an effective method for patients with re-canalized PDA after surgical ligation. It may be an alternative to second surgery owing to its safety, reliability, min-invasiveness, and short hospitalization.

  10. Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating

    International Nuclear Information System (INIS)

    Abel, B.; Aslan, K.

    2012-01-01

    This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization, where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  11. 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.

  12. Debris and friction of self-ligating and conventional orthodontic brackets after clinical use.

    Science.gov (United States)

    Araújo, Raíssa Costa; Bichara, Lívia Monteiro; Araujo, Adriana Monteiro de; Normando, David

    2015-07-01

    To compare the degree of debris and friction of conventional and self-ligating orthodontic brackets before and after clinical use. Two sets of three conventional and self-ligating brackets were bonded from the first molar to the first premolar in eight individuals, for a total of 16 sets per type of brackets. A passive segment of 0.019 × 0.025-inch stainless steel archwire was inserted into each group of brackets. Frictional force and debris level were evaluated as received and after 8 weeks of intraoral exposure. Two-way analysis of variance and Wilcoxon signed-rank test were applied at P brackets (P brackets showed a higher amount of debris compared with the conventional brackets. The frictional force in conventional brackets was significantly higher when compared with self-ligating brackets before clinical use (P brackets, when exposed to the intraoral environment, showed a significant increase in frictional force during the sliding mechanics. Debris accumulation was higher for the self-ligating system.

  13. 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.

  14. Staphylococcus aureus detection in blood samples by silica nanoparticle-oligonucleotides conjugates.

    Science.gov (United States)

    Borsa, Baris A; Tuna, Bilge G; Hernandez, Frank J; Hernandez, Luiza I; Bayramoglu, Gulay; Arica, M Yakup; Ozalp, V Cengiz

    2016-12-15

    A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Ergonomic varicocele ligation: laparoscopic intracorporeal knot-tying

    African Journals Online (AJOL)

    Department of Pediatric Surgery, Sevket Yilmaz Education and Research. Hospital, Bursa, Turkey ... laparoscopic approach to varicocelectomy has been the high associated ... bilateral varicoceles, a 3 mm trocar was placed at the right. McBurney's .... open-high or subinguinal ligation (microsurgical), laparo- scopic methods ...

  16. MR imaging of salivary glands after ductal ligation and stimulation by pilocarpine

    International Nuclear Information System (INIS)

    Patronas, N.J.; Tsuchimoch, M.; Webber, R.; Ruttimann, U.; Fox, P.; Bacher, J.; Schellinger, D.

    1988-01-01

    This paper presents an assessment of the usefulness of MR imaging in pathologic conditions of the salivary gland. The authors performed MR imaging in six dogs after ductal ligation on one side, followed by secretory stimulation with intraperitoneal injection of pilocarpine (5 mg/kg). On the images obtained after ductal ligation and before stimulation, there was no significant change in the signal intensity on either side. After injection of pilocarpine, however, T2-weighted images showed an obvious increase in signal intensity of the ligated gland in every instance. Their results indicate that MR images obtained after pilocarpine stimulation will be useful to study patients with ductal obstruction and that they may provide an objective basis for a noninvasive diagnostic test for unilateral stenosis

  17. Association of Patent Ductus Arteriosus Ligation With Death or Neurodevelopmental Impairment Among Extremely Preterm Infants

    Science.gov (United States)

    Mirea, Lucia; Rosenberg, Erin; Jang, Maximus; Ly, Linh; Church, Paige T.; Kelly, Edmond; Kim, S. Joseph; Jain, Amish; McNamara, Patrick J.; Shah, Prakesh S.

    2017-01-01

    Importance Observational studies have associated patent ductus arteriosus (PDA) ligation among preterm infants with adverse neonatal outcomes and neurodevelopmental impairment in early childhood, with a resultant secular trend away from surgical treatment. However, to our knowledge, studies have inadequately addressed sources of residual bias, including survival bias and major neonatal morbidities arising before exposure to ligation. Objective Evaluate the association between PDA ligation vs medical management and neonatal and neurodevelopmental outcomes. Design, Setting, and Participants This retrospective cohort study of preterm infants younger than 28 weeks gestational age born between January 1, 2006, and December 31, 2012, with clinical and echocardiography diagnoses of hemodynamically significant PDA was conducted at 3 tertiary neonatal intensive care units and affiliated follow-up programs. Exposure Surgical ligation vs medical management. Main Outcomes and Measures The primary outcome was a composite of death or neurodevelopmental impairment (NDI) at 18 to 24 months corrected age. Secondary outcomes included death before discharge, NDI, moderate-severe chronic lung disease, and severe retinopathy of prematurity. Multivariable logistic regression analysis was used to adjust for perinatal and postnatal confounders. Results Of 754 infants with hemodynamically significant PDA (mean [standard deviation] gestational age 25.7 [1.2] weeks and birth weight 813 [183] grams), 184 (24%) underwent ligation. Infants who underwent ligation had a higher frequency of morbidities before PDA closure, including sepsis, necrotizing enterocolitis, and a dependence on mechanical ventilation. After adjusting for perinatal characteristics and preligation morbidities, there was no difference in the odds of death or NDI (adjusted odds ratio (aOR), 0.83; 95% CI, 0.52-1.32), NDI (aOR, 1.27; 95% CI, 0.78-2.06), chronic lung disease (aOR, 1.36; 95% CI, 0.78-2.39) or severe retinopathy of

  18. Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

    Science.gov (United States)

    Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

    2006-01-01

    Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

  19. 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.

  20. The effect of ligation on the load deflection characteristics of nickel titanium orthodontic wire.

    Science.gov (United States)

    Kasuya, Shugo; Nagasaka, Satoshi; Hanyuda, Ai; Ishimura, Sadao; Hirashita, Ayao

    2007-12-01

    This study examined the effect of ligation on the load-deflection characteristics of nickel-titanium (NiTi) orthodontic wire. A modified three-point bending system was used for bending the NiTi round wire, which was inserted and ligated in the slots of three brackets, one of which was bonded to each of the three bender rods. Three different ligation methods, stainless steel ligature (SSL), slot lid (SL), and elastomeric ligature (EL), were employed, as well as a control with neither bracket nor ligation (NBL). The tests were repeated five times under each condition. Comparisons were made of load-deflection curve, load at maximum deflection of 2,000 microm, and load at a deflection of 1,500 microm during unloading. Analysis of Variance (ANOVA) and Dunnett's test were conducted to determine method difference (alpha = 0.05). The interaction between deflection and ligation was tested, using repeated-measures ANOVA (alpha = 0.05). The load values of the ligation groups were two to three times greater than the NBL group at a deflection of 1,500 microm during unloading: 4.37 N for EL, 3.90 N for SSL, 3.02 N for SL, and 1.49 N for NBL (P wire may make NiTi wire exhibit a significantly heavier load than that traditionally expected. NiTi wire exhibited the majority of its true superelasticity with SL, whereas EL may act as a restraint on its superelasticity.

  1. Synthesis of modified oligonucleotides that contain purine and pyrimidine radio-induced base lesions

    International Nuclear Information System (INIS)

    Romieu, Anthony

    1999-01-01

    Different factors as oxidizing or carcinogenesis agents, UV and ionizing radiations,.... can generate a wide, spectrum of DNA base damages. In order to study the biochemical and structural features of these DNA damages, it is important to prepare short DNA fragments (20 to 50 bases long) bearing a single or several modifications at specific sites in their sequences. The chemical synthesis is a powerful tool to prepare such modified DNA fragments. This work focusses on the chemical incorporation of several modified nucleosides formed in DNA by ionizing radiations or by photo-sensitization. The first part of this study describes the preparation of a phosphoramidite synthon of 5-hydroxy-2'-deoxyuridine and its subsequent incorporation into synthetic oligonucleotides ranging from 14 to 33 bases long. In a second part (chapters Ill and IV), the synthesis and incorporation of original radiation-induced tandem lesions: the carbon-bridged cyclo-nucleosides are presented. Both (5'R)- and (5'S) diastereomers of 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine have been individually inserted into various different oligonucleotides (3 to 22 bases long) by using the standard phosphoramidite chemistry. The chemical incorporation of a pyrimidine analogue: (5'S, 6S)-5',6-cyclo-5,6-dihydro-thymidine has been also achieved. The loss of aromaticity of this modified nucleoside and its poor reactivity required the development of a synthetic strategy entirely different from that used for the preparation and subsequent incorporation of the phosphoramidite synthons of 5',8-cyclo-purine-2'-deoxyribo-nucleosides. The third part of this study deals with the synthesis of a phosphoramidite synthon of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine. The two (4R)- and (4S)- diastereomers of this oxidized purine have been separated and individually inserted in several synthetic DNA fragments. No epimerization of C-4 position was observed during the solid-phase synthesis and during the

  2. 18F-labelling of oligonucleotides using succinimido 4-[18F]fluorobenzoat

    International Nuclear Information System (INIS)

    Hedberg, Elisabeth; Laangstroem, Bengt

    1998-01-01

    A general method for the labelling of oligodeoxynucleotide and oligonucleoside phosphorothioates in the 5'-position with the positron-emitting radionuclide 18 F (t 1/2 = 110 min) is described. The label was incorporated by the reaction of succinimido 4 -[ 18 F]fluorobenzoate 4 with oligonucleotides (18- and 20-mers) modified in the 5'-position with a hexylamine linker. Oligodeoxynucleotides 5'-GCT,AAG,CGA,TGC,CTC,CGT-3' (MTCa) and 5'-GAA,CCT,CTG,AGA,GTT,CAT,CT-3' (CROa) were labelled in 20±3 % (MTCa) and 13±3 % (CROa) radiochemical yields (non-isolated, decay-corrected and based on 4). Oligonucleoside phosphorotioates MTCa (S-MTCa) and CROa (S-CROa) were labelled in 9 and 7% isolated radiochemical yield, respectively (decay-corrected and based on 4). Labelled oligonucleotides and phosphorothioate analogues were separated from their unlabelled counterparts using reversed-phase perfusion chromatography. The molecular mass of a labelled oligonucleotide CROa was determined by ESI-MS after a mixed 18 F/ 19 F fluorobenzoate labelling experiment and corresponded with the expected structure. (au)

  3. 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...... precursors. Cross-catalytic product formation followed product inhibited kinetics, but approximately two replication rounds were observed. Analogous but less efficient replication was found for a similar tetrameric system. These results demonstrate that simpler nucleobase replication systems than natural...

  4. Subcutaneous endoscopically assisted ligation using miniport for ...

    African Journals Online (AJOL)

    Background This report describes the first miniport method using subcutaneous endoscopically assisted ligation (SEAL) for the treatment of girls with inguinal hernia. To validate its safety and efficacy, the authors evaluated their early experiences. Methods Between April 2014 and December 2014, 19 SEALs using miniport ...

  5. Recovery of vocal fold immobility following isolated patent ductus arteriosus ligation.

    Science.gov (United States)

    Nichols, Brent G; Jabbour, Jad; Hehir, David A; Ghanayem, Nancy S; Beste, David; Martin, Timothy; Woods, Ronald; Robey, Thomas

    2014-08-01

    Identify laryngoscopic and functional outcomes of infants with vocal fold immobility (VFI) following patent ductus arteriosus (PDA) ligation and identify predictors of recovery. Retrospective review of patients with VFI following PDA ligation from 2001 to 2012 at a single institution. Inclusion criteria were: (1) PDA ligation as only cardiac surgical procedure; (2) left VFI documented by laryngoscopy; (3) minimum follow up 120 days, with at least 2 laryngoscopies performed. Resolution of VFI was determined at follow-up laryngoscopy. Univariate logistic regression models were used to identify variables associated with VFI recovery. 66 subjects were included with median follow up of 3.0 (± 2.1) years. The mean gestational age was 24.5 ± 1.4 weeks, mean birth weight 673 ± 167 g, and mean age at procedure was 18.6 ± 14.3 days. Patients presented with respiratory symptoms (39%), dysphonia (78%) and dysphagia (55%). Resolution of VFI was observed in 2/66 (3%) patients. Recovery was documented at 20 days and 11 months respectively. Respiratory symptoms, dysphagia, and dysphonia persisted at last follow up in 11%, 47%, and 20% of patients. VFI associated with ligation of the ductus arteriosus has a low rate of recovery. Clinical symptoms frequently persist, and as such regular follow-up by otolaryngologists to mitigate morbidity is indicated. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Can interposition of a silicone implant after sapheno-femoral ligation prevent recurrent varicose veins?

    NARCIS (Netherlands)

    de Maeseneer, M. G.; Giuliani, D. R.; van Schil, P. E.; de Hert, S. G.

    2002-01-01

    OBJECTIVES: To investigate whether a silicone implant at the sapheno-femoral ligation site could prevent recurrent varicosities. MATERIALS AND METHODS: Two non-randomised groups of patients were studied prospectively. In group A 173 patients and 212 limbs had sapheno-femoral ligation, while 172

  7. Frictional resistance of self-ligating versus conventional brackets in different bracket-archwire-angle combinations

    Science.gov (United States)

    MONTEIRO, Maria Regina Guerra; da SILVA, Licinio Esmeraldo; ELIAS, Carlos Nelson; VILELLA, Oswaldo de Vasconcellos

    2014-01-01

    Objective To compare the influence of archwire material (NiTi, beta-Ti and stainless steel) and brackets design (self-ligating and conventional) on the frictional force resistance. Material and Methods Two types of brackets (self-ligating brackets - Smartclip, 3M/Unitek - and conventional brackets - Gemini, 3M/Unitek) with three (0, 5, and 10 degrees) slot angulation attached with elastomeric ligatures (TP Orthodontics) were tested. All brackets were tested with archwire 0.019"x0.025" nickel-titanium, beta-titanium, and stainless steel (Unitek/3M). The mechanical testing was performed with a universal testing machine eMIC DL 10000 (eMIC Co, Brazil). The wires were pulled from the bracket slots at a cross-head speed of 3 mm/min until 2 mm displacement. Results Self-ligating brackets produced significantly lower friction values compared with those of conventional brackets. Frictional force resistance values were directly proportional to the increase in the bracket/ wire angulation. With regard to conventional brackets, stainless steel wires had the lowest friction force values, followed by nickel-titanium and beta-titanium ones. With regard to self-ligating brackets, the nickel-titanium wires had the lowest friction values, significantly lower than those of other materials. Conclusion even at different angulations, the self-ligating brackets showed significantly lower friction force values than the conventional brackets. Combined with nickel-titanium wires, the self-ligating brackets exhibit much lower friction, possibly due to the contact between nickel-titanium clips and wires of the same material. PMID:25025564

  8. Frictional resistance of self-ligating versus conventional brackets in different bracket-archwire-angle combinations

    Directory of Open Access Journals (Sweden)

    Maria Regina Guerra MONTEIRO

    2014-06-01

    Full Text Available Objective: To compare the influence of archwire material (NiTi, beta-Ti and stainless steel and brackets design (self-ligating and conventional on the frictional force resistance. Material and Methods: Two types of brackets (self-ligating brackets - Smartclip, 3M/Unitek - and conventional brackets - Gemini, 3M/Unitek with three (0, 5, and 10 degrees slot angulation attached with elastomeric ligatures (TP Orthodontics were tested. All brackets were tested with archwire 0.019"x0.025" nickel-titanium, beta-titanium, and stainless steel (Unitek/3M. The mechanical testing was performed with a universal testing machine eMIC DL 10000 (eMIC Co, Brazil. The wires were pulled from the bracket slots at a cross-head speed of 3 mm/min until 2 mm displacement. Results: Self-ligating brackets produced significantly lower friction values compared with those of conventional brackets. Frictional force resistance values were directly proportional to the increase in the bracket/ wire angulation. With regard to conventional brackets, stainless steel wires had the lowest friction force values, followed by nickel-titanium and beta-titanium ones. With regard to self-ligating brackets, the nickel-titanium wires had the lowest friction values, significantly lower than those of other materials. Conclusion: even at different angulations, the self-ligating brackets showed significantly lower friction force values than the conventional brackets. Combined with nickel-titanium wires, the self-ligating brackets exhibit much lower friction, possibly due to the contact between nickel-titanium clips and wires of the same material.

  9. Comparison of Frictional Forces Generated by a New Ceramic Bracket with the Conventional Brackets using Unconventional and Conventional Ligation System and the Self-ligating Brackets: An In Vitro Study.

    Science.gov (United States)

    Pasha, Azam; Vishwakarma, Swati; Narayan, Anjali; Vinay, K; Shetty, Smitha V; Roy, Partha Pratim

    2015-09-01

    Fixed orthodontic mechanotherapy is associated with friction between the bracket - wire - ligature interfaces during the sliding mechanics. A sound knowledge of the various factors affecting the magnitude of friction is of paramount importance. The present study was done to analyze and compare the frictional forces generated by a new ceramic (Clarity Advanced) bracket with the conventional, (metal and ceramic) brackets using unconventional and conventional ligation system, and the self-ligating (metal and ceramic) brackets in the dry condition. The various bracket wire ligation combinations were tested in dry condition. The brackets used were of 0.022″ × 0.028″ nominal slot dimension of MBT prescription: Stainless steel (SS) self-ligating bracket (SLB) of (SmartClip), SS Conventional bracket (CB) (Victory series), Ceramic SLB (Clarity SL), Conventional Ceramic bracket with metal slot (Clarity Bracket), Clarity Advanced Ceramic Brackets (Clarity(™) ADVANCED, 3M Unitek). These brackets were used with two types of elastomeric ligatures: Conventional Elastomeric Ligatures (CEL) (Clear medium mini modules) and Unconventional Elastomeric Ligatures (UEL) (Clear medium slide ligatures, Leone orthodontic products). The aligning and the retraction wires were used, i.e., 0.014″ nickel titanium (NiTi) wires and 0.019″ × 0.025″ SS wires, respectively. A universal strength testing machine was used to measure the friction produced between the different bracket, archwires, and ligation combination. This was done with the use of a custom-made jig being in position. Mean, standard deviation, and range were computed for the frictional values obtained. Results were subjected to statistical analysis through ANOVA. The frictional resistance observed in the new Clarity Advanced bracket with a conventional elastomeric ligature was almost similar with the Clarity metal slot bracket with a conventional elastomeric ligature. When using the UEL, the Clarity Advanced bracket

  10. Ultrasensitive detection of Hg2+ using oligonucleotide-functionalized AlGaN/GaN high electron mobility transistor

    International Nuclear Information System (INIS)

    Cheng, Junjie; Li, Jiadong; Miao, Bin; Wu, Dongmin; Wang, Jine; Pei, Renjun; Wu, Zhengyan

    2014-01-01

    An oligonucleotide-functionalized ion sensitive AlGaN/GaN high electron mobility transistor (HEMT) was fabricated to detect trace amounts of Hg 2+ . The advantages of ion sensitive AlGaN/GaN HEMT and highly specific binding interaction between Hg 2+ and thymines were combined. The current response of this Hg 2+ ultrasensitive transistor was characterized. The current increased due to the accumulation of Hg 2+ ions on the surface by the highly specific thymine-Hg 2+ -thymine recognition. The dynamic linear range for Hg 2+ detection has been determined in the concentrations from 10 −14 to 10 −8 M and a detection limit below 10 −14 M level was estimated, which is the best result of AlGaN/GaN HEMT biosensors for Hg 2+ detection till now.

  11. 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'...

  12. Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture.

    Science.gov (United States)

    Lin, Da; Hong, Ping; Zhang, Siheng; Xu, Weize; Jamal, Muhammad; Yan, Keji; Lei, Yingying; Li, Liang; Ruan, Yijun; Fu, Zhen F; Li, Guoliang; Cao, Gang

    2018-05-01

    Chromosome conformation capture (3C) technologies can be used to investigate 3D genomic structures. However, high background noise, high costs, and a lack of straightforward noise evaluation in current methods impede the advancement of 3D genomic research. Here we developed a simple digestion-ligation-only Hi-C (DLO Hi-C) technology to explore the 3D landscape of the genome. This method requires only two rounds of digestion and ligation, without the need for biotin labeling and pulldown. Non-ligated DNA was efficiently removed in a cost-effective step by purifying specific linker-ligated DNA fragments. Notably, random ligation could be quickly evaluated in an early quality-control step before sequencing. Moreover, an in situ version of DLO Hi-C using a four-cutter restriction enzyme has been developed. We applied DLO Hi-C to delineate the genomic architecture of THP-1 and K562 cells and uncovered chromosomal translocations. This technology may facilitate investigation of genomic organization, gene regulation, and (meta)genome assembly.

  13. An experimental microangiographic study on injured liver acinus by ligation of common bile duct

    International Nuclear Information System (INIS)

    Park, Jong Yeon; Kim, Yoon Gyu; Moon, Ki Ho; Lee, Suek Hong; Kim, Byung Soo; Han, Gun Taik

    1994-01-01

    The purpose of this study was to evaluate the morphologic changes of the injured hepatic acini following ligation of common bile duct and to investigate the pathophysiologic process of hepatic failure and biliary liver cirrhosis in the extrahepatic cholestasis. The common bile ducts of 18 rabbits were ligated partially. The rabbits were killed and selective microangiography was carried out with infusion of barium suspensio via portal vein 4 to 24 weeks after ligation. Selective microangiography was also carried out in two normal rabbits. The microangiographic findings were evaluated and correlated with histopathologic features. The sinusoids of the liver acinus showed distortion, varying degrees of luminal widening, and irregularities in architecture. Terminal branches of the portal vein (TPV) showed increased number of branches, luminal narrowing, tortuosity, distortion, and beaded appearance. Peribiliary plexi were found as thin curvilinear, barium-filled structures along the wall of the dilated bile duct. The microangiographic findings were well correlated with histopathologic findings. The grades of microangiographic and histopathologic findings were poorly correlated with the duration of the ligation of CBD. Changes in microvasculature of the liver scinus following partial ligation of common bile duct were demonstrated by microangiography. Although the microvascular changes were evoked secondary to the injury, they might have some active roles in the pathophysiologic process in the liver

  14. Air-powder polishing on self-ligating brackets after clinical use: effects on debris levels.

    Science.gov (United States)

    Aragón, Mônica L S Castro; Lima, Leandro Santiago; Normando, David

    2016-01-01

    Debris buildup on brackets and arch surfaces is one of the main factors that can influence the intensity of friction between bracket and orthodontic wire. This study sought to evaluate the effect of air-powder polishing cleaning on debris levels of self-ligating ceramic brackets at the end of orthodontic treatment, compared to the behavior of conventional brackets. Debris levels were evaluated in metal conventional orthodontic brackets (n = 42) and ceramic self-ligating brackets (n = 42) on canines and premolars, arranged in pairs. There were brackets with and without air-powder polishing. At the end of orthodontic treatment, a hemiarch served as control and the contralateral hemiarch underwent prophylaxis with air-powder polishing. Debris buildup in bracket slots was assessed through images, and Wilcoxon test was used to analyze the results. The median debris levels were statistically lower in the conventional metal brackets compared to self-ligating ones (p = 0.02), regarding brackets not submitted to air-powder polishing. Polishing significantly reduced debris buildup to zero in both systems, without differences between groups. Ceramic self-ligating brackets have a higher debris buildup in comparison to conventional metal brackets in vivo, but prophylaxis with sodium bicarbonate jet was effective in reducing debris levels in self-ligating and also in conventional brackets.

  15. OUTCOMES OF HILAR PEDICLE CONTROL USING SUTURE LIGATION DURING LAPAROSCOPIC SPLENECTOMY.

    Science.gov (United States)

    Makgoka, M

    2017-06-01

    Laparoscopic splenectomy is a well described gold standard procedure for various indications. One of the key steps during laparoscopic splenectomy is the hilar pedicle vessels control, which can be challenging in most cases. Most centres around the world recommend the use Ligaclib or endovascular staplers as Methods of choice for hilar pedicle control but the issue is the cost and efficiency of the laparoscopic haemostatic devices. A descriptive retrospective study of patients who had laparoscopic splenectomy from 2013 to present. Hilar splenic vessel control was done with suture ligation. We looked at outcomes of patients offered this technique, complications of this technique, and describing the technique of hilar control in laparoscopic splenectomy. Total of 27 patients had laparoscopic splenectomy with splenic hilar pedicle control with suture ligation. Mean operative time, mean blood volume loss, length of hospital stay, postoperative complications conversion to laparotomy. Laparoscopic hilar pedicle control with suture ligation is safe and effective for the patient in our hospital setting.

  16. Subcutaneous endoscopically assisted ligation using miniport for ...

    African Journals Online (AJOL)

    miniport for the treatment of girls with inguinal hernia. Akinari Hinoki*, Ikeda ... method using subcutaneous endoscopically assisted ligation (SEAL) for the ... the open technique and an additional 2 mm miniport). A ... (unilateral, n = 9) or 42 ± 8 min (bilateral, n = 5). The mean .... Methods of laparoscopic repair have recently ...

  17. Acute kidney injury secondary to iatrogenic bilateral ureteric ligation ...

    African Journals Online (AJOL)

    Acute kidney injury secondary to iatrogenic bilateral ureteric ligation following emergency abdominal hysterectomy. Oluseyi A. Adejumo, Olurotimi S. Ogundiniyi, Ayodeji A. Akinbodewa, Lawrence A. Adesunloro, Oladimeji J. Olafisoye ...

  18. Elevated mitochondrial gene expression during rat liver regeneration after portal vein ligation.

    Science.gov (United States)

    Shimizu, Y; Suzuki, H; Nimura, Y; Onoue, S; Nagino, M; Tanaka, M; Ozawa, T

    1995-10-01

    We explored the molecular basis of mitochondrial energy production during rat liver regeneration after portal vein ligation. Ligation of the left branch of the portal vein induces an increase in the weight of the nonligated lobe, counterbalancing the reduced weight of the ligated lobe. Using this model, we investigated changes in mitochondrial DNA-binding proteins, mitochondrial DNA, and mitochondrial messenger RNA (mRNA) in rat hepatocytes of the nonligated lobes. The amount of mitochondrial DNA-binding protein increased maximally (200% to 300% of the preoperative level) at 12 hours after the operation, before an increase (390%) in mitochondrial DNA content at 24 hours, and parallel to an increase (240%) in mitochondrial mRNA levels at 12 hours. These results suggest that the energy supply for liver regeneration is achieved through enhancement of mitochondrial DNA replication as well as transcription, in which the mitochondrial DNA-binding proteins probably play regulatory roles. We also found that in the nonligated lobes, mRNA levels of hepatocyte growth factor increased to a detectable level only 12 hours after the operation. These molecular biochemical data help explain why preoperative portal vein embolization, which is a modification of portal vein branch ligation, is an effective method to prevent posthepatectomy liver failure.

  19. 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.

  20. Access to bifunctionalized biomolecular platforms using oxime ligation

    Czech Academy of Sciences Publication Activity Database

    Křenek, Karel; Gažák, Radek; Daskhan, G. Ch.; Garcia, J.; Fiore, M.; Dumy, P.; Šulc, Miroslav; Křen, Vladimír; Renaudet, O.

    2014-01-01

    Roč. 393, JUL 2014 (2014), s. 9-14 ISSN 0008-6215 R&D Projects: GA MŠk(CZ) LD13042 Institutional support: RVO:61388971 Keywords : Chemoselective ligation * Glycocluster * Cyclopeptide Subject RIV: CC - Organic Chemistry Impact factor: 1.929, year: 2014

  1. Evaluation of common vaginal tunic ligation during field castration in draught colts.

    Science.gov (United States)

    Carmalt, J L; Shoemaker, R W; Wilson, D G

    2008-09-01

    The objective of this study was to determine if ligation of the common vaginal tunic could prevent or reduce the incidence of omental herniation and eventration in draught colts undergoing routine field castration. It was found that common vaginal tunic ligation, while not completely preventing omental herniation and evisceration, significantly reduced the incidence of these complications and should be considered in those males deemed at increased risk of significant post castration complications.

  2. The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

    Science.gov (United States)

    2013-01-01

    Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

  3. DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY – HIGH RESOLUTION MASS SPECTROMETRY

    Science.gov (United States)

    Nikcevic, Irena; Wyrzykiewicz, Tadeusz K.; Limbach, Patrick A.

    2010-01-01

    Summary An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described. Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts

  4. Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations.

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

    2004-11-09

    We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

  5. Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

    NARCIS (Netherlands)

    Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.

    2015-01-01

    Background Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that

  6. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

    Science.gov (United States)

    Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

    2010-01-01

    The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

  7. 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.

  8. Friction between various self-ligating brackets and archwire couples during sliding mechanics.

    Science.gov (United States)

    Stefanos, Sennay; Secchi, Antonino G; Coby, Guy; Tanna, Nipul; Mante, Francis K

    2010-10-01

    The aim of this study was to evaluate the frictional resistance between active and passive self-ligating brackets and 0.019 × 0.025-in stainless steel archwire during sliding mechanics by using an orthodontic sliding simulation device. Maxillary right first premolar active self-ligating brackets In-Ovation R, In-Ovation C (both, GAC International, Bohemia, NY), and SPEED (Strite Industries, Cambridge, Ontario, Canada), and passive self-ligating brackets SmartClip (3M Unitek, Monrovia, Calif), Synergy R (Rocky Mountain Orthodontics, Denver, Colo), and Damon 3mx (Ormco, Orange, Calif) with 0.022-in slots were used. Frictional force was measured by using an orthodontic sliding simulation device attached to a universal testing machine. Each bracket-archwire combination was tested 30 times at 0° angulation relative to the sliding direction. Statistical comparisons were performed with 1-way analysis of variance (ANOVA) followed by Dunn multiple comparisons. The level of statistical significance was set at P <0.05. The Damon 3mx brackets had significantly the lowest mean static frictional force (8.6 g). The highest mean static frictional force was shown by the SPEED brackets (83.1 g). The other brackets were ranked as follows, from highest to lowest, In-Ovation R, In-Ovation C, SmartClip, and Synergy R. The mean static frictional forces were all statistically different. The ranking of the kinetic frictional forces of bracket-archwire combinations was the same as that for static frictional forces. All bracket-archwire combinations showed significantly different kinetic frictional forces except SmartClip and In-Ovation C, which were not significantly different from each other. Passive self-ligating brackets have lower static and kinetic frictional resistance than do active self-ligating brackets with 0.019 × 0.025-in stainless steel wire. Copyright © 2010 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  9. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hongguang Sun

    2014-01-01

    Full Text Available Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

  10. 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.

  11. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  12. Increased secretion of insulin and proliferation of islet β-cells in rats with mesenteric lymph duct ligation

    International Nuclear Information System (INIS)

    Nagino, Ko; Yokozawa, Junji; Sasaki, Yu; Matsuda, Akiko; Takeda, Hiroaki; Kawata, Sumio

    2012-01-01

    Highlights: ► Insulin secretion was increased during the OGTT or IVGTT in mesenteric lymph duct-ligated rats. ► Proliferation of islet β-cells was upregulated in lymph duct-ligated rats. ► Mesenteric lymph duct flow has a role in glucose metabolism. -- Abstract: Background and aims: It has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet β-cells in rats. Methods: Male Sprague–Dawley rats (10 weeks old) were divided into two groups: one underwent ligation of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of β-cells was assessed immunohistochemically using antibodies against insulin and Ki-67. Results: During the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p < 0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p < 0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p < 0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p < 0.05) and 120 min (2.5-fold; p < 0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2 min (more than 1.4-fold; p < 0.05). Immunohistochemistry showed that the ratios of

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  14. 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

    2016-04-27

    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.

  15. Sphenopalatine artery ligation: technical note.

    Science.gov (United States)

    Pothier, David D; Mackeith, Samuel; Youngs, Robin

    2005-10-01

    Epistaxis is a common problem. Most patients presenting to hospital will stop bleeding with simple first-aid measures or with nasal packing. Those who do not stop will usually require surgical management. For persistent posterior epistaxis, the sphenopalatine artery may be ligated as the artery leaves the sphenopalatine foramen to enter the nasal mucosa of the lateral wall of the nose. This may be performed endoscopically. We describe the anatomy of the area and the surgical technique. We also present a brief review of the literature on this technique.

  16. Reliability and applications of statistical methods based on oligonucleotide frequencies in bacterial and archaeal genomes

    DEFF Research Database (Denmark)

    Bohlin, J; Skjerve, E; Ussery, David

    2008-01-01

    with here are mainly used to examine similarities between archaeal and bacterial DNA from different genomes. These methods compare observed genomic frequencies of fixed-sized oligonucleotides with expected values, which can be determined by genomic nucleotide content, smaller oligonucleotide frequencies......, or be based on specific statistical distributions. Advantages with these statistical methods include measurements of phylogenetic relationship with relatively small pieces of DNA sampled from almost anywhere within genomes, detection of foreign/conserved DNA, and homology searches. Our aim was to explore...... the reliability and best suited applications for some popular methods, which include relative oligonucleotide frequencies (ROF), di- to hexanucleotide zero'th order Markov methods (ZOM) and 2.order Markov chain Method (MCM). Tests were performed on distant homology searches with large DNA sequences, detection...

  17. A "typodont" study of rate of orthodontic space closure: self-ligating systems vs. conventional systems.

    Science.gov (United States)

    Saporito, I; Butti, A C; Salvato, A; Biagi, R

    2011-01-01

    The aim of this study was to compare the effectiveness of space closure of two bracket systems, self-ligating and conventional ones, considering two systems with the same design and prescription. The experimental model of this study aims to identify in general if one type of system is more suitable for planning the extractive therapy, without considering the clinical aspect necessary for an adequate orthodontic approach. A resin maxilla without the first premolars was used to test the self-ligating and conventional brackets system. Space closure was achieved on 0.016x0.022" in stainless steel wires with nickel-titanium coil springs 150 grams in strength and 10 mm in length. Two experiment settings have been prepared. The first setting was made bonding conventional bracket on the right side and self-ligating on the left; the last one was made bonding self-ligating bracket on the right side and conventional on the left. All measurements (runs) have been repeated seven times for both settings. During the closing phase of extraction spaces both systems are equable since there are no significant statistical differences (P=0.70). Our typodont model showed no significant difference in the efficiency of space closure between the self-ligating bracket and conventional bracket tied with stainless steel ligatures.

  18. Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes

    DEFF Research Database (Denmark)

    Andersson, A M; Gaardsvoll, H; Giladi, E

    1990-01-01

    A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA...... oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized...... the five NCAM mRNAs in rat brain....

  19. 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.

  20. 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

    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...... 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....

  1. 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.

  2. Demonstration of metabolic and cellular effects of portal vein ligation using multi-modal PET/MRI measurements in healthy rat liver.

    Directory of Open Access Journals (Sweden)

    András Fülöp

    Full Text Available OBJECTIVES: In the early recognition of portal vein ligation (PVL induced tumor progression, positron emission tomography and magnetic resonance imaging (PET/MRI could improve diagnostic accuracy of conventionally used methods. It is unknown how PVL affects metabolic patterns of tumor free hepatic tissues. The aim of this preliminary study is to evaluate the effect of PVL on glucose metabolism, using PET/MRI imaging in healthy rat liver. MATERIALS AND METHODS: Male Wistar rats (n=30 underwent PVL. 2-deoxy-2-(18Ffluoro-D-glucose (FDG PET/MRI imaging (nanoScan PET/MRI and morphological/histological examination were performed before (Day 0 and 1, 2, 3, and 7 days after PVL. Dynamic PET data were collected and the standardized uptake values (SUV for ligated and non-ligated liver lobes were calculated in relation to cardiac left ventricle (SUVVOI/SUVCLV and mean liver SUV (SUVVOI/SUVLiver. RESULTS: PVL induced atrophy of ligated lobes, while non-ligated liver tissue showed compensatory hypertrophy. Dynamic PET scan revealed altered FDG kinetics in both ligated and non-ligated liver lobes. SUVVOI/SUVCLV significantly increased in both groups of lobes, with a maximal value at the 2nd postoperative day and returned near to the baseline 7 days after the ligation. After PVL, ligated liver lobes showed significantly higher tracer uptake compared to the non-ligated lobes (significantly higher SUVVOI/SUVLiver values were observed at postoperative day 1, 2 and 3. The homogenous tracer biodistribution observed before PVL reappeared by 7th postoperative day. CONCLUSION: The observed alterations in FDG uptake dynamics should be taken into account during the assessment of PET data until the PVL induced atrophic and regenerative processes are completed.

  3. 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

  4. 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.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    KRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription fa...

  6. Extrahepatic portal venous obstruction: The effects of early ligation of splenic artery during splenectomy.

    Science.gov (United States)

    Gazula, Suhasini; Pawar, D K; Seth, T; Bal, C S; Bhatnagar, V

    2009-10-01

    To objectively demonstrate the gain in blood volume and blood components following early ligation of splenic artery during splenectomy and splenorenal shunts in children with extra hepatic portal venous obstruction (EHPVO). Twenty-eight children (20 males and 8 females, mean age: 9.9 (+/-3.2) years) with EHPVO and hypersplenism were recruited. We followed a protocol of systematically locating and ligating the splenic artery first, followed by a 30-minute waiting period to allow the massive spleen to decongest via the splenic vein and venous collaterals and then completing the splenectomy by standard procedure. No intravenous fluid was administered during this 30-minute period. Blood samples were drawn just prior to splenic artery ligation and soon after splenectomy for the estimation of hematological and biochemical parameters. We noticed a highly significant increase in the hemoglobin, hematocrit, leukocyte, platelet, and RBC counts by early ligation of the splenic artery (p platelet count was equivalent to a platelet transfusion of atleast 4 units of platelet concentrates in an adult. There is a positive correlation between the splenic weight and the platelet gain (p= 0.0568) and the splenic volume on preoperative imaging and the platelet gain (p= 0.0251). Early ligation of the splenic artery during splenectomy results in passive splenic decongestion and thereby a significant gain in blood components. This protocol appears to be a feasible blood conservation method to avoid blood transfusions in this group of hypersplenic EHPVO patients.

  7. Enzymatic Ligation of Large Biomolecules to DNA

    DEFF Research Database (Denmark)

    Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David Henning

    2013-01-01

    The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale s....... As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures....

  8. Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides

    Science.gov (United States)

    Basiri, Babak; Murph, Mandi M.; Bartlett, Michael G.

    2017-08-01

    Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences.

  9. Orchidopexy san ligation technique of orchidopexy | Jain | African ...

    African Journals Online (AJOL)

    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 ...

  10. Evaluation of mesenteric lymphangiography and thoracic duct ligation in cats with chylothorax: 19 cases (1987-1992)

    International Nuclear Information System (INIS)

    Kerpsack, S.J.; McLoughlin, M.A.; Birchard, S.J.; Smeak, D.D.; Biller, D.S.

    1994-01-01

    Mesenteric lymphangiography and thoracic duct ligation were performedon 19 cats with chylothorax between 1987 to 1992. Chylothorax was diagnosed on the basis of detection of chylomicrons in the pleural effusion or determination of a cholesterol concentration:triglyceride concentration ratio of 12 months after surgery. Four cats died between 2 and 13 days after thoracic duct ligation, but pleural effusion had resolved in 3 of these 4 cats at the time of death. Five cats were euthanatized 8 to 36 days after surgery because of persistent chylous effusion after thoracic duct ligation

  11. Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

    Science.gov (United States)

    Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

    2015-02-07

    The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

  12. Respirable antisense oligonucleotides: a new drug class for respiratory disease

    Directory of Open Access Journals (Sweden)

    Tanaka Makoto

    2000-12-01

    Full Text Available Abstract Respirable antisense oligonucleotides (RASONs, which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.

  13. 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.

  14. Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation.

    Science.gov (United States)

    Pentelute, Brad L; Kent, Stephen B H

    2007-02-15

    Increased versatility for the synthesis of proteins and peptides by native chemical ligation requires the ability to ligate at positions other than Cys. Here, we report that Raney nickel can be used under standard conditions for the selective desulfurization of Cys in the presence of Cys(Acm). This simple and practical tactic enables the more common Xaa-Ala junctions to be used as ligation sites for the chemical synthesis of Cys-containing peptides and proteins. [reaction: see text].

  15. Rubber band ligation and infrared photocoagulation for the outpatient treatment of hemorrhoidal disease

    OpenAIRE

    Ricci,Maurício Pichler; Matos,Délcio; Saad,Sarhan Sydney

    2008-01-01

    PURPOSE: To compare the results of rubber band ligation and infrared photocoagulation for the treatment of hemorrhoidal disease through the analysis of the incidence of complications after each treatment and respective success rate. METHODS: Forty-eight patients with first, second or third degree hemorrhoidal disease were randomized to recieve treatment with either rubber band ligation (n=23) or infrared photocoagulation (n=25). Each patient was assessed at 1 week and 4 week intervals after t...

  16. 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.)

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

    International Nuclear Information System (INIS)

    Courtney, Malachi; Ayyagari, Raj R.

    2015-01-01

    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.)

  18. Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

    Science.gov (United States)

    Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

    2018-04-01

    Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Management of IVC Injury: Repair or Ligation? A Propensity Score Matching Analysis Using the National Trauma Data Bank.

    Science.gov (United States)

    Matsumoto, Shokei; Jung, Kyoungwon; Smith, Alan; Coimbra, Raul

    2018-03-01

    Ligation can be used as part of damage-control operations under critical conditions after IVC injury. Inferior vena cava ligation could potentially yield greater survival benefit compared with repair after injury. We hypothesized that ligation significantly improves outcomes compared with repair. The National Trauma Data Bank dataset for 2007-2014 was reviewed. Eligible patients included those sustaining IVC injury who underwent surgical ligation or repair. Data on demographics, outcomes, and complications were collected. Comparative analysis of demographic characteristics, complications and outcomes were performed. There were 4,865 patients identified in the National Trauma Data Bank with IVC injury. A total of 1,316 patients met inclusion criteria. Four hundred and forty-seven patients (34.0%) underwent ligation and 869 (66.0%) underwent repair. Before matching, the ligation group was sicker than the repair group and the in-hospital mortality was significantly higher in the ligation group (43.8% vs 36.2%; odds ratio [OR] 1.37; 95% CI 1.09 to 1.73). One to one propensity score matching generated 310 pairs. After propensity score matching, in-hospital mortality was similar (41.3% vs 39.0%; OR 1.10; 95% CI 0.80 to 1.52). However, IVC ligation was associated with significantly higher complication rates of extremity compartment syndrome (OR 5.23; 95% CI 1.50 to 18.24), pneumonia (OR 1.76; 95% CI 1.08 to 2.86), deep venous thrombosis (OR 2.83 95% CI 1.70 to 4.73), pulmonary embolism (OR 3.63; 95% CI 1.18 to 11.17), and longer hospital length of stay (17.0 days [interquartile range 1.0 to 35.0 days] vs 9.0 days [interquartile range 1.0 to 22.0 days]; p = 0.002). Inferior vena cava ligation is not superior to repair in terms of decreasing mortality in patients with IVC injury, but it is associated with higher complication rates and hospital LOS. Copyright © 2018 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

  20. Increased secretion of insulin and proliferation of islet {beta}-cells in rats with mesenteric lymph duct ligation

    Energy Technology Data Exchange (ETDEWEB)

    Nagino, Ko; Yokozawa, Junji; Sasaki, Yu; Matsuda, Akiko; Takeda, Hiroaki [Department of Gastroenterology, Faculty of Medicine, Yamagata University, Yamagata 990-9585 (Japan); Kawata, Sumio, E-mail: Sumio_Kawata@pref.hyogo.lg.jp [Department of Gastroenterology, Faculty of Medicine, Yamagata University, Yamagata 990-9585 (Japan); Hyogo Prefectural Nishinomiya Hospital, 13-9 Rokutanji-cho, Nishinomiya 662-0918 (Japan)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Insulin secretion was increased during the OGTT or IVGTT in mesenteric lymph duct-ligated rats. Black-Right-Pointing-Pointer Proliferation of islet {beta}-cells was upregulated in lymph duct-ligated rats. Black-Right-Pointing-Pointer Mesenteric lymph duct flow has a role in glucose metabolism. -- Abstract: Background and aims: It has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet {beta}-cells in rats. Methods: Male Sprague-Dawley rats (10 weeks old) were divided into two groups: one underwent ligation of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of {beta}-cells was assessed immunohistochemically using antibodies against insulin and Ki-67. Results: During the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p < 0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p < 0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p < 0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p < 0.05) and 120 min (2.5-fold; p < 0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2

  1. Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H

    DEFF Research Database (Denmark)

    Labuda, T; Gerwien, J; Ødum, Niels

    1999-01-01

    and the TCR-CD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis....... In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src...... family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests...

  2. Root resorption during orthodontic treatment with self-ligating or conventional brackets: a systematic review and meta-analysis.

    Science.gov (United States)

    Yi, Jianru; Li, Meile; Li, Yu; Li, Xiaobing; Zhao, Zhihe

    2016-11-21

    The aim of this study was to compare the external apical root resorption (EARR) in patients receiving fixed orthodontic treatment with self-ligating or conventional brackets. Studies comparing the EARR between orthodontic patients using self-ligating or conventional brackets were identified through electronic search in databases including CENTRAL, PubMed, EMBASE, China National Knowledge Infrastructure (CNKI) and SIGLE, and manual search in relevant journals and reference lists of the included studies until Apr 2016. The extraction of data and risk of bias evaluation were conducted by two investigators independently. The original outcome underwent statistical pooling by using Review Manager 5. Seven studies were included in the systematic review, out of which, five studies were statistically pooled in meta-analysis. The value of EARR of maxillary central incisors in the self-ligating bracket group was significantly lower than that in the conventional bracket group (SMD -0.31; 95% CI: -0.60--0.01). No significant differences in other incisors were observed between self-ligating and conventional brackets. Current evidences suggest self-ligating brackets do not outperform conventional brackets in reducing the EARR in maxillary lateral incisors, mandible central incisors and mandible lateral incisors. However, self-ligating brackets appear to have an advantage in protecting maxillary central incisor from EARR, which still needs to be confirmed by more high-quality studies.

  3. Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

    Science.gov (United States)

    Alsop, Eric B; Raymond, Jason

    2013-01-01

    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.

  4. 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.

  5. Papillary Cystadenoma: An Incidental Finding in Tubal Ligation

    Directory of Open Access Journals (Sweden)

    Tabitha Lynn Ward

    2018-01-01

    Full Text Available von Hippel-Lindau disease (vHLD is a rare autosomal dominant disorder with multiple benign and malignant tumors of different organs. We report a papillary cystadenoma of the mesosalpinx found in close association with an adenomatoid tumor discovered incidentally following tubal ligation in a patient with vHLD.

  6. Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate nanocapsules in cells

    Directory of Open Access Journals (Sweden)

    Tomcin S

    2014-11-01

    Full Text Available Stephanie Tomcin,1 Grit Baier,1 Katharina Landfester,1 Volker Mailänder1,21Max Planck Institute for Polymer Research, 2University Medical Center of the Johannes Gutenberg University, III Medical Clinic, Mainz, GermanyAbstract: For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate (PBCA nanocapsules as a shell and separately an oligonucleotide (20 mer as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.Keywords: drug delivery, mitochondria, miniemulsion, colocalization

  7. Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

    Science.gov (United States)

    Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

    2012-01-13

    Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. How I do it: surgical ligation of craniocervical junction dural AV fistulas.

    Science.gov (United States)

    Sorenson, Thomas J; La Pira, Biagia; Hughes, Joshua; Lanzino, Giuseppe

    2017-08-01

    Dural arteriovenous fistulas (DAVFs) of the craniocervical junction are uncommon vascular lesions, which often require surgical treatment even in the endovascular era. Most commonly, the fistula is placed laterally, and surgical ligation is performed through a lateral suboccipital craniotomy. After dural opening, the area is inspected, and the arterialized vein is identified emerging from the dura, often adjacent to the entry point of the vertebral artery, and ligated. A far lateral craniotomy is the authors' preferred surgical approach for accessing and treating dural arteriovenous fistulas of the craniocervical junction that cannot be reached endovascularly.

  9. Role of lubricants on friction between self-ligating brackets and archwires.

    Science.gov (United States)

    Leal, Renata C; Amaral, Flávia L B; França, Fabiana M G; Basting, Roberta T; Turssi, Cecilia P

    2014-11-01

    To evaluate the effect of different lubricants on friction between orthodontic brackets and archwires. Active (Quick, Forestadent) and passive (Damon 3MX, Ormco) self-ligating brackets underwent friction tests in the presence of mucin- and carboxymethylcellulose (CMC)-based artificial saliva, distilled water, and whole human saliva (positive control). Dry friction (no lubricant) was used as the negative control. Bracket/wire samples (0.014 × 0.025 inch, CuNiTi, SDS Ormco) underwent friction tests eight times in a universal testing machine. Two-way analysis of variance showed no significant interaction between bracket type and lubricant (P  =  .324). Friction force obtained with passive self-ligating brackets was lower than that for active brackets (P Friction observed in the presence of artificial saliva did not differ from that generated under lubrication with natural human saliva, as shown by Tukey test. Higher friction forces were found with the use of distilled water or when the test was performed under dry condition (ie, with no lubricant). Lubrication plays a role in friction forces between self-ligating brackets and CuNiTi wires, with mucin- and CMC-based artificial saliva providing a reliable alternative to human natural saliva.

  10. Hypotension following patent ductus arteriosus ligation: the role of adrenal hormones.

    Science.gov (United States)

    Clyman, Ronald I; Wickremasinghe, Andrea; Merritt, T Allen; Solomon, Tabitha; McNamara, Patrick; Jain, Amish; Singh, Jaideep; Chu, Alison; Noori, Shahab; Sekar, Krishnamurthy; Lavoie, Pascal M; Attridge, Joshua T; Swanson, Jonathan R; Gillam-Krakauer, Maria; Reese, Jeff; DeMauro, Sara; Poindexter, Brenda; Aucott, Sue; Satpute, Monique; Fernandez, Erika; Auchus, Richard J

    2014-06-01

    To test the hypothesis that an impaired adrenal response to stress might play a role in the hypotension that follows patent ductus arteriosus (PDA) ligation. We performed a multicenter study of infants born at 15. Of 95 infants enrolled, 43 (45%) developed hypotension and 14 (15%) developed catecholamine-resistant hypotension. Low postoperative cortisol levels were not associated with the overall incidence of hypotension after ligation. However, low cortisol levels were associated with the refractoriness of the hypotension to catecholamine treatment. In a multivariate analysis: the OR for developing catecholamine-resistant hypotension was OR 36.6, 95% CI 2.8-476, P = .006. Low cortisol levels (in infants with catecholamine-resistant hypotension) were not attributable to adrenal immaturity or impairment; their cortisol precursor concentrations were either low or unchanged, and their response to cosyntropin was similar to infants without catecholamine-resistant hypotension. Infants with low cortisol concentrations after PDA ligation are likely to develop postoperative catecholamine-resistant hypotension. We speculate that decreased adrenal stimulation, rather than an impaired adrenal response to stimulation, may account for the decreased production. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The importance of the ligation of the inferior thyroid artery in parathyroid function after subtotal thyroidectomy

    Directory of Open Access Journals (Sweden)

    Araujo Filho Vergilius José Furtado de

    2000-01-01

    Full Text Available We prospectively studied the effects of the ligation of the inferior thyroid artery (ITA on postoperative hypoparathyroidism in 48 patients who underwent functional subtotal thyroidectomy. Patients were randomized into two groups: A, with bilateral ligation of the ITA and B, without ligation of the ITA. Parathyroid function was checked preoperatively and after surgery by clinical examination and measurement of total calcium, intact PTH, urinary calcium, and AMPc. RESULTS: A significant incidence of postoperative hypocalcemia occurred: 17% in group A and 13% in B on the 4th postoperative day. Six months later, the incidence was 5% in Group A and 0% in Group B. These differences were not statistically significant between the two groups, and neither were any of the other clinical and laboratory observations. CONCLUSION: The ligation of the ITA was not an important causal factor for the occurrence of postoperative hypocalcemia after subtotal thyroidectomy.

  12. Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II; FINAL

    International Nuclear Information System (INIS)

    None

    2000-01-01

    The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank(reg s ign) finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

  13. Banding ligation versus no intervention for primary prevention in adults with oesophageal varices

    DEFF Research Database (Denmark)

    Yong, Charles Wei Kit; Vadera, Sonam; Morgan, Marsha Y.

    2017-01-01

    This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess the beneficial and harmful effects of banding ligation versus no intervention in adults with cirrhosis and gastro-oesophageal varices that have not bled.......This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess the beneficial and harmful effects of banding ligation versus no intervention in adults with cirrhosis and gastro-oesophageal varices that have not bled....

  14. Development of a 11C-labeled tetrazine for rapid tetrazine–trans-cyclooctene ligation

    DEFF Research Database (Denmark)

    Herth, Matthias Manfred; Andersen, Valdemar L.; Lehel, Szabolcs

    2013-01-01

    Tetrazine–trans-cyclooctene ligations are remarkably fast and selective reactions even at low micro-molar concentrations. In bioorthogonal radiochemistry, tools that enable conjugation of radioactive probes to pre-targeted vectors are of great interest. Herein, we describe the successful developm......Tetrazine–trans-cyclooctene ligations are remarkably fast and selective reactions even at low micro-molar concentrations. In bioorthogonal radiochemistry, tools that enable conjugation of radioactive probes to pre-targeted vectors are of great interest. Herein, we describe the successful...

  15. 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......, and their data was retrieved from electronic patient records. RESULTS: In total, eight patients had this procedure performed for a total of ten inguinal regions. In all regions, leaking lymph vessels were easily found by the blue color and a median of 3 (range 1-5 vessels) vessels per region were ligated using...... and had the procedure performed again with immediate effect. CONCLUSIONS: Ligation of lymph vessels for the treatment of recurrent inguinal lymphoceles appears to be an appropriate treatment modality that is both quick and easy to perform with minimum risk, and in most cases, it results in immediate...

  16. Preoperative embolization for juvenile nasopharyngeal angiofibroma. Usefulness of IADSA and problems of external carotid ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saitoh, Ryuichi; Nakagawa, Nobuo; Kohrogi, Yukinori; Izunaga, Hiroshi; Bussaka, Hiromasa; Miyawaki, Masayuki; Takahashi, Mutsumasa; Ikawa, Tsutomu; Ishikawa, Takashi (Kumamoto Univ. (Japan). School of Medicine)

    1984-10-01

    Three male cases of juvenile nasopharyngeal angiofibroma are presented. Satisfactory results were obtained by performing preoperative embolization combined with intraarterial digital subtraction angiography (IADSA) in two of them. The other case had undergone external carotid ligation. Usefulness of IADSA in performing preoperative embolization for this disease and problems of external carotid ligation are discussed.

  17. An oral oligonucleotide delivery system based on a thiolated polymer: Development and in vitro evaluation.

    Science.gov (United States)

    Martien, Ronny; Hoyer, Herbert; Perera, Glen; Schnürch, Andreas Bernkop

    2011-08-01

    The purpose of this study was to develop and evaluate an oral oligonucleotide delivery system based on a thiolated polymer/reduced glutathione (GSH) system providing a protective effect toward nucleases and permeation enhancement. A polycarbophil-cysteine conjugate (PCP-Cys) was synthesized. Enzymatic degradation of a model oligonucleotide by DNase I and within freshly collected intestinal fluid was investigated in the absence and presence of PCP-Cys. Permeation studies with PCP-Cys/GSH versus control were performed in vitro on Caco-2 cell monolayers and ex vivo on rat intestinal mucosa. PCP-Cys displayed 223 ± 13.8 μmol thiol groups per gram polymer. After 4h, 61% of the free oligonucleotides were degraded by DNase I and 80% within intestinal fluid. In contrast, less than 41% (DNase I) and 60% (intestinal fluid) were degraded in the presence of 0.02% (m/v) PCP-Cys. Permeation studies revealed an 8-fold (Caco-2) and 10-fold (intestinal mucosa) increase in apparent permeability compared to buffer control. Hence, this PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotides as it allows a significant protection toward degrading enzymes and facilitates their transport across intestinal membranes. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

    Directory of Open Access Journals (Sweden)

    Moizza Mansoor

    2008-01-01

    Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

  19. Sequence-Dependent Mechanism of DNA Oligonucleotide Dehybridization Resolved through Infrared Spectroscopy.

    Science.gov (United States)

    Sanstead, Paul J; Stevenson, Paul; Tokmakoff, Andrei

    2016-09-14

    Despite its important role in biology and nanotechnology, many questions remain regarding the molecular mechanism and dynamics by which oligonucleotides recognize and hybridize to their complementary sequence. The thermodynamics and kinetics of DNA oligonucleotide hybridization and dehybridization are often assumed to involve an all-or-nothing two-state dissociation pathway, but deviations from this behavior can be considerable even for short sequences. We introduce a new strategy to characterize the base-pair-specific thermal dissociation mechanism of DNA oligonucleotides through steady-state and time-resolved infrared spectroscopy. Experiments are interpreted with a lattice model to provide a structure-specific interpretation. This method is applied to a model set of self-complementary 10-base-pair sequences in which the placement of GC base pairs is varied in an otherwise AT strand. Through a combination of Fourier transform infrared and two-dimensional infrared spectroscopy, experiments reveal varying degrees of deviation from simple two-state behavior. As the temperature is increased, duplexes dissociate through a path in which the terminal bases fray, without any significant contribution from loop configurations. Transient temperature jump experiments reveal time scales of 70-100 ns for fraying and 10-30 μs for complete dissociation near the melting temperature. Whether or not frayed states are metastable intermediates or short-lived configurations during the full dissociation of the duplex is dictated by the nucleobase sequence.

  20. Incorporation of selenomethionine into proteins through selenohomocysteine-mediated ligation

    NARCIS (Netherlands)

    Roelfes, G; Hilvert, D

    2003-01-01

    The chemical synthesis of moderate-sized proteins has benefited enormously from the development of chemoselective methods for the ligation of peptide fragments. In this regard, the reaction of peptide thioesters with peptides containing an N-terminal cysteine has proved particularly powerful. The

  1. End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

    Science.gov (United States)

    Jiang, Rui; Weingart, Jacob; Zhang, Hailong; Ma, Yong; Sun, Xue-Long

    2012-01-01

    We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4–6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and is rapid occurring in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein’s biological activity. PMID:22372933

  2. Venous surgery in erectile dysfunction. The role of dorsal-penile-vein ligation and spongiosolysis for impotence.

    Science.gov (United States)

    Gilbert, P; Sparwasser, C; Beckert, R; Treiber, U; Pust, R

    1992-01-01

    We report here on our surgical experience with venous leakage of the cavernous bodies. Out of 159 patients operated on, 134 were available for long-term follow-up. Depending on the cavernosographic findings, one of three different surgical procedures was carried out: ligation of the deep dorsal vein of the penis, spongiosolysis, or ligation of the crura. 18% of the patients undergoing ligation of the deep dorsal vein, postoperatively attained spontaneous erections, while 35% needed adjuvant corpus cavernosum autoinjection therapy. Spongiosolysis gave a more favourable result: spontaneous erections in 30% and vasoactive drug-dependent erections in 35%. Crural ligation did not prove successful. No serious complications were encountered postoperatively. Our data suggest that venous surgery should only be offered to a selected group of patients comprising young impotent men with venous leakage, maybe in combination with arterial disease, and patients suffering from distal venous leakage. Old age, neurogenic disorders causing erectile dysfunction, and diabetes mellitus should represent exclusion criteria for venous surgery.

  3. Management of Small Urethrocutaneous Fistula by Tight Ligation ...

    African Journals Online (AJOL)

    After identifying the fistulous opening, the fistula tract was circumferentially and meticulously dissected ,then the dissected tract was lifted up and the base was ligated tightly with 5/0 vicryl, the external epithelium of the dissected tract was fulgurated with the diathermy, then a second layer of local soft tissue was secured over ...

  4. 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 ...

  5. 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.

  6. 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.

  7. Evaluation of friction produced by self-ligating, conventional and Barbosa Versatile brackets

    Directory of Open Access Journals (Sweden)

    Jurandir Antonio BARBOSA

    Full Text Available Abstract Introduction The Barbosa Versatile bracket design may provide lower frictional force and greater sliding. However, no in vitro studies have shown its sliding mechanisms and frictional resistance, particularly in comparison with other self-ligating or conventional brackets. Objective To compare the frictional resistance among self-ligating brackets (EasyClip/ Aditek, Damon MX/ Ormco and In Ovation R/ GAC; conventional brackets (Balance Roth/ GAC, and Roth Monobloc/ Morelli; and Barbosa Versatile bracket (Barbosa Versatile/ GAC with different angles and arch wires. Material and method Brackets were tested with the 0.014", 0.018", 0.019"×0.025" and 0.021"×0.025" stainless steel wires, with 0, 5, 10, 15 and 20 degree angulations. Tying was performed with elastomeric ligature for conventional and Barbosa Versatile brackets, or with a built-in clip system of the self-ligating brackets. A universal testing machine was used to obtain sliding strength and friction value readouts between brackets and wires. Result Three-way factorial ANOVA 4×5×6 (brackets × angulation × wire and Tukey tests showed statistically significant differences for all factors and all interactions (p<0.0001. Static frictional resistance showed a lower rate for Barbosa Versatile bracket and higher rates for Roth Monobloc and Balance brackets. Conclusion The lowest frictional resistance was obtained with the Barbosa Versatile bracket and self-ligating brackets in comparison with the conventional type. Increasing the diameter of the wires increased the frictional resistance. Smaller angles produced less frictional resistance.

  8. Intraabdominal laparoscopy-assisted "open" vessel ligation of testicular vessels: a potential treatment for varicocele.

    Science.gov (United States)

    Miyano, Go; Miyahara, Katsumi; Halibieke, Abudebieke; Lane, Geoffrey J; Okazaki, Tadaharu; Yamataka, Atsuyuki

    2011-10-01

    We tested our laparoscopy-assisted "open" ligation (LOL) technique on testicular vessels. We ligated the left testicular artery and vein (TAV) in 8-week-old male Wister rats using LOL (LOL group; n=10) or laparotomy (open group; n=10). In LOL, a 0-degree laparoscope was introduced through a 5-mm epigastric trocar. A 3-mm grasper was used to expose the left TAV. A lapa-her-closure (LHC) needle loaded with 3-0 SurgiPro was directly inserted into the left lower quadrant where the left TAV should be and advanced under the vessels, and the suture material was released leaving one end outside. The LHC was then withdrawn a little and advanced again over the vessels to grasp the end of the suture material just released to bring it outside. This was proximally repeated. The two ends of both sutures were conventionally tied outside, and the knot was passed through the insertion site and tightened around the vessels. In the open group, the left TAV were ligated using two 3-0 SurgiPro ties. In both groups, the right side was left intact. All rats were sacrificed 2 weeks postoperatively, and both testes were examined with hematoxylin and eosin. Treatment time was 5-7 minutes for LOL and 7-8 minutes for the open group. Postoperative recovery was uneventful. No adhesions were present between the ligated vessels and bowel in any rat. Histopathology of all left testes showed coagulative necrosis of germinal cells and seminiferous tubules; all right testes were normal. LOL appears to be as effective as open ligation and may find application for treating varicocele.

  9. Extrahepatic portal venous obstruction: The effects of early ligation of splenic artery during splenectomy

    Directory of Open Access Journals (Sweden)

    Gazula Suhasini

    2009-01-01

    Full Text Available Aim: To objectively demonstrate the gain in blood volume and blood components following early ligation of splenic artery during splenectomy and splenorenal shunts in children with extra hepatic portal venous obstruction (EHPVO. Methods: Twenty-eight children (20 males and 8 females, mean age: 9.9 (±3.2 years with EHPVO and hypersplenism were recruited. We followed a protocol of systematically locating and ligating the splenic artery first, followed by a 30-minute waiting period to allow the massive spleen to decongest via the splenic vein and venous collaterals and then completing the splenectomy by standard procedure. No intravenous fluid was administered during this 30-minute period. Blood samples were drawn just prior to splenic artery ligation and soon after splenectomy for the estimation of hematological and biochemical parameters. Results: We noticed a highly significant increase in the hemoglobin, hematocrit, leukocyte, platelet, and RBC counts by early ligation of the splenic artery (p < 0.0004. The gain in hemoglobin and hematocrit was equivalent to a transfusion of atleast 100-150 ml of packed RBC. The increase in platelet count was equivalent to a platelet transfusion of atleast 4 units of platelet concentrates in an adult. There is a positive correlation between the splenic weight and the platelet gain (p= 0.0568 and the splenic volume on preoperative imaging and the platelet gain (p= 0.0251. Conclusion: Early ligation of the splenic artery during splenectomy results in passive splenic decongestion and thereby a significant gain in blood components. This protocol appears to be a feasible blood conservation method to avoid blood transfusions in this group of hypersplenic EHPVO patients.

  10. Comparison of infrared coagulation and rubber band ligation for first and second degree haemorrhoids: a randomised prospective clinical trial.

    Science.gov (United States)

    Templeton, J L; Spence, R A; Kennedy, T L; Parks, T G; Mackenzie, G; Hanna, W A

    1983-01-01

    One hundred and thirty seven previously untreated out-patients with first and second degree haemorrhoids were allocated at random to treatment by infrared coagulation (n=66) or rubber band ligation (n=71). Complete follow up was obtained in 122 patients (60 who had undergone infrared coagulation (group 1), and 62 rubber band ligation (group 2)) at periods from three months to one year after completion of treatment. Infrared coagulation produced a satisfactory outcome in 51 patients (85%): 34 were rendered asymptomatic and 17 improved. Rubber band ligation produced a satisfactory outcome in 57 patients (92%): 33 were rendered asymptomatic and 24 improved. Both methods were equally effective in first and second degree haemorrhoids. The incidence of side effects, particularly discomfort, during and after treatment was significantly higher in those treated by rubber band ligation (p less than 0.001). This appeared to be an appreciable deterrent to future patient compliance. The number of patients losing more than 24 hours from work was higher after rubber band ligation than after infrared coagulation. The number of treatments necessary to cure symptoms did not differ significantly between the two methods. Infrared coagulation was significantly faster than rubber band ligation (p less than 0.001). Infrared coagulation is a simple, fast, and effective outpatient method for the treatment of first and second degree haemorrhoids with fewer troublesome side effects and higher patient acceptability than rubber band ligation. PMID:6404471

  11. Optimal primary surgical management of infected pseudoaneurysm in intravenous drug abusers: ligation or reconstruction?

    International Nuclear Information System (INIS)

    Jamil, M.; Usman, R.; Afzal, M.; Malik, N.I.

    2017-01-01

    Objective: To find out the optimal primary surgical treatment options for infected pseudoaneurysm in intravenous drug abusers. Study Design: Cross sectional descriptive study. Place and Duration of Study: Department of Vascular Surgery, Combined Military Hospital Lahore, from Jan 2010 to Jun 2015. Material and Methods: A total of 31 consecutive patients with a history of intravenous drug abuse and an infected pseudoaneurysm in the groin or elbow, presenting in emergency department; were included in this study. All patients were primarily treated with ligation of the artery, excision of infected pseudoaneurysm and debridement of necrotic tissues. Only one patient underwent additional revascularization procedure. Results: All patients who underwent ligation and excision procedures did well initially. One (3.2%) patient developed severe distal ischemia after ligation of femoral artery within first 24 hours, so extra anatomic revascularization procedure was performed. Five (16.1%) patients required revascularization procedure after 16 weeks due to disabling distal ischemia. No amputation was needed and mortality rate was zero. Conclusion: Primary ligation of the artery with excision of infected pseudoaneurysm and necrotic material was found the optimal initial management for infected pseudoaneurysm in intravenous drug addicts. Ischemic complications if develop should be treated with early or late revascularization.

  12. 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.

  13. Early or Late Surgical Ligation of Medical Refractory Patent Ductus Arteriosus in Premature Infants

    Directory of Open Access Journals (Sweden)

    Chien-Chou Hsiao

    2009-01-01

    Full Text Available Optimal time to surgical ligation of patent ductus arteriosus (PDA in very-low-birth-weight ( 14 days groups. Basic clinical features, major morbidity of prematurity and mortality were compared. Clinical features and major outcomes were similar. The early ligation group had earlier onset of symptomatic PDA (5.7 ± 1.6 days vs. 8.1 ± 3.6 days, p = 0.024, and fewer days of total parenteral nutrition (TPN (39.6 ± 13.9 days vs. 60.4 ± 31.4 days, p = 0.025 and ventilator use (11.1 ± 6.7 days vs. 18.6 ± 10.5 days, p = 0.019. Early ligation of medical refractory PDA in very-low-birth-weight premature infants improves enteral feeding tolerance and reduces TPN and ventilator use, but long-term benefits need further investigation.

  14. Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2'-S-cycloadenosine.

    Science.gov (United States)

    Ikehara, M; Tezuka, T

    1975-01-01

    A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9. PMID:170595

  15. Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Awsiuk, K., E-mail: kamil.awsiuk@uj.edu.pl [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Rysz, J. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Petrou, P. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Budkowski, A. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Bernasik, A. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Kakabakos, S. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Marzec, M.M. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Raptis, I. [Institute for Advanced Materials, Physicochemical Processes, Nanotechnology and Microsystems, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece)

    2014-01-30

    To immobilize effectively oligonucleotide probes on SiO{sub 2} modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m{sup 2}) and second (1.31(±0.22) mg/m{sup 2}) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m{sup 2}) and fourth (0.41(±0.11) mg/m{sup 2}) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

  16. Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

    International Nuclear Information System (INIS)

    Awsiuk, K.; Rysz, J.; Petrou, P.; Budkowski, A.; Bernasik, A.; Kakabakos, S.; Marzec, M.M.; Raptis, I.

    2014-01-01

    To immobilize effectively oligonucleotide probes on SiO 2 modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m 2 ) and second (1.31(±0.22) mg/m 2 ) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m 2 ) and fourth (0.41(±0.11) mg/m 2 ) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

  17. 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...

  18. 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

  19. 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.

  20. 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.

  1. Gingival response in orthodontic patients: Comparative study between self-ligating and conventional brackets.

    Science.gov (United States)

    Folco, Alejandra A; Benítez-Rogé, Sandra C; Iglesias, Marina; Calabrese, Diana; Pelizardi, Cristina; Rosa, Alcira; Brusca, Marisa I; Hecht, Pedro; Mateu, María E

    2014-01-01

    Orthodontic brackets contribute to the accumulation of bacterial plaque on tooth surfaces because they hinder oral hygiene. In contrast to conventional brackets, self-ligating brackets do not require additional parts to support the arches, thus improving dental hygiene. The aim of this study was to compare the gingival response in orthodontic patients wearing self-ligating or conventional brackets. A sample of 22 patients aged 16 to 30 years was divided into two groups: Group A, treated with selfligating brackets (Damon system) and Group B, treated with conventional brackets (Roth technique). The following were assessed during the treatment: Plaque Index (PI), Gingival Index (GI) and Probing Depth (PD), and sub-gingival samples were taken from teeth 14/24 for microbiological observation. No statistically significant difference was found between Groups A and B; p>0.05 (sign-ranked) or between PI, GI and PD at the different times (Friedman's Analysis of Variance), even though the indices were found to increase at 14 days, particularly for self-ligating brackets. The quantity and quality of microorganisms present were compatible with health on days 0, 28 and 56. As from day 14 there is a predominance of microbiota compatible with gingivitis in both groups. In the samples studied, orthodontic treatment increases bacterial plaque and inflammatory gingival response, but gingival-periodontal health can be maintained with adequate basic therapy. Self-ligating and conventional brackets produced similar gingival response.

  2. Change of hepatic volume after selective bile duct ligation: an experimental study in the rabbit

    International Nuclear Information System (INIS)

    Lee, Hye Won; Yoon, Yup; Ko, Young Tae; Choi, Woo Suk; Lim, Joo Won; Oh, Joo Hyeong; Rim, Hyeong Teck; Kim, Youn Wha; Lee, Seok Hwan

    1998-01-01

    To evaluate the role of bile duct obstuction in the development of atrophy of the liver, using an animal model. Seven rabbits were divided into two groups: group 1(n=3D5), in which there was selective bile duct ligation, and group 2(n=3D2), which underwent a sham operation. Each group was evaluated using CT for changes in hepatic volume after selective bile duct ligation or a sham operation. In group I, the diameter of dilated bile duct was measured 2, 4, 8, 12 and 16 weeks after bile duct ligation, while gross and histologic change were evaluated in all cases. In group 1, bile duct dilatation was seen on CT two weeks after selective bile duct ligation, and did not change significantly during follow-up. In four of five cases, CT revealed no evidence of significant atrophy of the involved segment. Pathologic specimens, however, revealed dilatation of the bile duct, periductal fibrosis, infiltration of chronic inflammatory cells, and periportal fibrosis. One of five cases showed segmental liver atrophy after selective bile duct ligation. In addion to the above pathologic findings, there was obstruction of the portal vein by foreign body reaction. In group 2, no evidence of dilated bile duct or liver atrophy was revealed by CT or pathologic specimen after a sham operation. During long-term follow-up of 16 weeks, obstruction of the bile duct did not play a major role in the development of lobar atrophy in the rabbit.=20

  3. 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.

  4. Does the number of veins ligated during varicococele surgery influence post-operative semen and hormone results?

    Science.gov (United States)

    Majzoub, A; Elbardisi, H; Arafa, M; Agarwal, A; Al Said, S; Al Rumaihi, K

    2016-09-01

    Varicocele is a well-established cause of male subfertility, which is directly proportional to its clinical grade. Although newer ultrasonic grading systems have taken into account the existence of pampiniform venous plexi, little is known about the clinical significance of the number of veins ligated during surgery. Very few undersized studies reported an influence which triggered the need to evaluate such association. This is a retrospective study of 378 patients who underwent left microsurgical subinguinal varicocoelectomy. Semen analyses and blood hormone studies performed pre-operatively were compared to those executed 6 months after surgery. Patients were divided into abnormal semen and normal semen groups based on their initial semen results. They were also subdivided according to the number of veins ligated intraoperatively into three groups: 10 veins. Sperm count, total motility, and progressive motility were significantly increased in 62, 60.3, and 53.3% of patients post-operatively (p = 0.001), respectively. No significant differences in hormone levels were detected overall. Of the 378 patients, 332 had an abnormal semen analysis, while the remaining 46 patients had a normal result. Sperm count, total motility, and progressive motility significantly increased after varicocoelectomy in patients with an abnormal initial semen analysis (p = 0.001). In 48.7% of patients, 5-10 veins were ligated during surgery, whereas 28.3% had >10 and 23% had <5 ligated veins. No statistically significant differences were noted in the initial or the follow-up results among the number of vein subgroups. Varicocele ligation improves patients' fertility potential. This improvement, however, is not influenced by the number of veins ligated intraoperatively. Clinical grading maintains its superiority in the evaluation of varicocele patients. © 2016 American Society of Andrology and European Academy of Andrology.

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

    African Journals Online (AJOL)

    Background: Oesophageal varices are common sequelae of cirrhosis, which when they bleed can be difficult to control. Oesophageal variceal band ligation, being the best modality for controlling variceal bleeding has not been common practice in West Africa, a region recognized to have a high prevalence of liver diseases.

  6. Development of oligonucleotide microarrays for simultaneous multi-species identification of Phellinus tree-pathogenic fungi.

    Science.gov (United States)

    Tzean, Yuh; Shu, Po-Yao; Liou, Ruey-Fen; Tzean, Shean-Shong

    2016-03-01

    Polyporoid Phellinus fungi are ubiquitously present in the environment and play an important role in shaping forest ecology. Several species of Phellinus are notorious pathogens that can affect a broad variety of tree species in forest, plantation, orchard and urban habitats; however, current detection methods are overly complex and lack the sensitivity required to identify these pathogens at the species level in a timely fashion for effective infestation control. Here, we describe eight oligonucleotide microarray platforms for the simultaneous and specific detection of 17 important Phellinus species, using probes generated from the internal transcribed spacer regions unique to each species. The sensitivity, robustness and efficiency of this Phellinus microarray system was subsequently confirmed against template DNA from two key Phellinus species, as well as field samples collected from tree roots, trunks and surrounding soil. This system can provide early, specific and convenient detection of Phellinus species for forestry, arboriculture and quarantine inspection, and could potentially help to mitigate the environmental and economic impact of Phellinus-related diseases. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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

    Directory of Open Access Journals (Sweden)

    Suzan M Hammond

    2014-01-01

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

  8. 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 brackets do not have an advantage over conventional brackets with respect to periodontal status and halitosis.

  9. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Science.gov (United States)

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  10. Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

    OpenAIRE

    Jensen, O N; Kulkarni, S; Aldrich, J V; Barofsky, D F

    1996-01-01

    Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research an...

  11. 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 ...

  12. Rapid Induction of Protective Immunity Against Biothreat Agents Using CPG-Based Oligonucleotides

    National Research Council Canada - National Science Library

    Klinman, Dennis

    2003-01-01

    This research project examines the ability of synthetic oligonucleotides (ODN) containing immunostimulatory "CpG motifs' to trigger the innate immune system, thereby improving the host's ability to survive infection by biowarfare agents...

  13. 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

    -life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non...... 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....

  14. Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

    International Nuclear Information System (INIS)

    Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.

    2002-01-01

    Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion

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

    Directory of Open Access Journals (Sweden)

    Boyang Cao

    Full Text Available 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.

  16. 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. PMID:25469776

  17. Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

    International Nuclear Information System (INIS)

    Muller, E.

    2002-01-01

    Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

  18. Automated synthesis of an {sup 18}F-labelled pyridine-based alkylating agent for high yield oligonucleotide conjugation

    Energy Technology Data Exchange (ETDEWEB)

    Guggenberg, Elisabeth von; Sader, Jayden A.; Wilson, John S.; Shahhosseini, Soraya; Koslowsky, Ingrid; Wuest, Frank [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada); Mercer, John R. [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada)], E-mail: johnmerc@cancerboard.ab.ca

    2009-09-15

    Alkylating agents have been shown to be very promising for the radiolabelling of oligonucleotides with fluorine-18. In this report we describe the fully automated synthesis of 2-bromo-N-[3-(2-[{sup 18}F]fluoropyridin-3-yloxy)propyl]acetamide ([{sup 18}F]FPyBrA) utilizing a modular synthesis unit. Reaction conditions for the coupling of this pyridine-based alkylating agent at the 5' end of a fully phosphorothioated random 20-mer DNA sequence were optimized to achieve very high radiochemical yields (>90%) and a maximum specific activity of 5-6 GBq/{mu}moL. The potential for rapid purification by solid phase extraction without need of chromatographic isolation of the radiolabelled oligonucleotide presents an overall benefit for the application of oligonucleotides in preclinical studies and potential clinical applications.

  19. Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

    DEFF Research Database (Denmark)

    Astakhova, Irina; Kumar, Santhosh T.; Wengel, Jesper

    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...

  20. 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 ...

  1. Clinical and psychological repercussions of videolaparoscopic tubal ligation: observational, single cohort, retrospective study

    Directory of Open Access Journals (Sweden)

    Daniel Spadoto Dias

    Full Text Available CONTEXT AND OBJECTIVE: Tubal ligation is one of the most commonly used contraceptive methods worldwide. Since the controversy over the potential effects of tubal sterilization still continues, this study aimed to evaluate the clinical and psychological repercussions of videolaparoscopic tubal ligation.DESIGN AND SETTING: Observational, single cohort, retrospective study, conducted in a tertiary public hospital.METHODS: A questionnaire was applied to 130 women aged 21-46 years who underwent videolaparoscopic tubal ligation by means of tubal ring insertion or bipolar electrocoagulation and sectioning, between January 1999 and December 2007. Menstrual cycle interval, intensity and duration of bleeding, premenstrual symptoms, dysmenorrhea, dyspareunia, noncyclic pelvic pain and degree of sexual satisfaction were assessed in this questionnaire. Each woman served as her own control, and comparisons were made between before and after the surgical procedure and between the two techniques used.RESULTS: The clinical and psychological repercussions were significant, with increases in bleeding (P = 0.001, premenstrual symptoms (P < 0.001, dysmenorrhea (P = 0.019 and noncyclic pelvic pain (P = 0.001; and reductions in the number of sexual intercourse occurrences per week (P = 0.001 and in libido (P = 0.001. Women aged ≤ 35 years at the time of sterilization were more likely to develop menstrual abnormalities. The bipolar electrocoagulation method showed greater clinical and psychological repercussions.CONCLUSION: Regardless of the technique used, videolaparoscopic tubal ligation had repercussions consisting of increased menstrual flow and premenstrual symptoms, especially in women aged ≤ 35 years, and also had a negative influence on sexual activity.

  2. Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  3. 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 ...

  4. Alterations in plaque accumulation and gingival inflammation promoted by treatment with self-ligating and conventional orthodontic brackets.

    Science.gov (United States)

    Cardoso, Mauricio de Almeida; Saraiva, Patrícia Pinto; Maltagliati, Liliana Ávila; Rhoden, Fernando Kleinübing; Costa, Carla Cristina Alvarenga; Normando, David; Capelozza Filho, Leopoldino

    2015-01-01

    The aim of the present study was to evaluate, comparatively, the periodontal response during orthodontic treatment performed with self-ligating and conventional brackets. Sixteen Caucasian individuals of both sexes, aged between 12 and 16 years old and in permanent dentition were selected. Eight individuals were treated with conventional brackets installed on the lower dental arch and self-ligating brackets on the upper arch. Another eight individuals received self-ligating brackets in the lower arch and conventional brackets in the upper arch. The subjects received material and instructions for oral hygiene. Visible plaque index (VPI), gingival bleeding index (GBI) and clinical attachment level (CAL) were evaluated just after installation of orthodontic appliances, and 30, 60 and 180 days later. Mann-Whitney test was used to compare differences between groups (self-ligating and conventional), two-way ANOVA followed by Tukey's test was used to assess CAL at each site of each tooth. Significance level was set at 5%. No significant changes were found with regard to the assessed parameters (VPI, GBI and CAL) in either one of the systems. No significant changes were found with regard to the periodontal response to orthodontic treatment for the variables assessed and between subjects receiving passive self-ligating and conventional brackets. All individuals had received oral hygiene instructions and had their periodontal conditions monitored.

  5. Alterations in plaque accumulation and gingival inflammation promoted by treatment with self-ligating and conventional orthodontic brackets

    Directory of Open Access Journals (Sweden)

    Mauricio de Almeida Cardoso

    2015-04-01

    Full Text Available OBJECTIVE: The aim of the present study was to evaluate, comparatively, the periodontal response during orthodontic treatment performed with self-ligating and conventional brackets. METHODS: Sixteen Caucasian individuals of both sexes, aged between 12 and 16 years old and in permanent dentition were selected. Eight individuals were treated with conventional brackets installed on the lower dental arch and self-ligating brackets on the upper arch. Another eight individuals received self-ligating brackets in the lower arch and conventional brackets in the upper arch. The subjects received material and instructions for oral hygiene. Visible plaque index (VPI, gingival bleeding index (GBI and clinical attachment level (CAL were evaluated just after installation of orthodontic appliances, and 30, 60 and 180 days later. Mann-Whitney test was used to compare differences between groups (self-ligating and conventional, two-way ANOVA followed by Tukey's test was used to assess CAL at each site of each tooth. Significance level was set at 5%. RESULTS: No significant changes were found with regard to the assessed parameters (VPI, GBI and CAL in either one of the systems. CONCLUSION: No significant changes were found with regard to the periodontal response to orthodontic treatment for the variables assessed and between subjects receiving passive self-ligating and conventional brackets. All individuals had received oral hygiene instructions and had their periodontal conditions monitored.

  6. Meta-analysis: banding ligation and medical interventions for the prevention of rebleeding from oesophageal varices

    DEFF Research Database (Denmark)

    Thiele, Maja; Krag, A; Rohde, Ulrich

    2012-01-01

    In patients with oesophageal varices, the combination of endoscopic variceal ligation (EVL) and medical therapy is recommended as standard of care for prevention of rebleeding. The results of previous meta-analyses on this topic are equivocal.......In patients with oesophageal varices, the combination of endoscopic variceal ligation (EVL) and medical therapy is recommended as standard of care for prevention of rebleeding. The results of previous meta-analyses on this topic are equivocal....

  7. Management of Fistula‑In‑Ano with Special Reference to Ligation of ...

    African Journals Online (AJOL)

    Department of General Surgery, MKCG Medical College, Berhampur, Odisha, India. ABSTRACT ... Management of fistula‑in‑ano through ligation of intersphincteric fistula tract. 2 ... At the time of admission, a detailed history and the clinical.

  8. 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...... 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...

  9. 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...

  10. 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.

  11. Evaluation of low ligation and high proceedings of varicocele

    International Nuclear Information System (INIS)

    Khan, M.; Khan, S.; Pervez, A.; Nawaz, H.; Ahmed, S.; Tareen, S.

    2003-01-01

    Objective: To describe the mode of presentation of varicocele and to compare the low ligation (Inguinal approach/ ivanissevich s procedure) and high ligation (Retroperitoneal approach/modified Palomo's procedure) of varicocele repair regarding efficacy and postoperative complications. Subjects and Methods: A total of 213 patients of varicocele who underwent treatment in the department and completed 6 months follow-up were included in the study. The mode of presentation of varicocele was recorded for all patients. For treatment purpose patients were randomly divided into two groups: in group I (n:115) varicocele was ligated by inguinal approach (Ivanissevich s procedure) while in group II (n: 98) by retroopertional approach (modified Palomo s procedure). The efficacy and postoperative complications of the two procedures were carefully recorded and compared between the two groups. Results: The mean age of the patients of group-I and group-II was 27.2 and 27.5 years respectively. More than 50% patients of both groups presented with feeling of heaviness/ dragging sensation on ipsilateral side. Presentation with infertility/sub fertility in group-I and group-II was 16.5% and 15.3% respectively. Majority of patients of both groups had grade III varicocele with visible deformity. The rate of postoperative complications was very low in both groups of patients. Recurrence of varicocele and postoperative hydrocele formation were slightly more common in group-II as compared to group-I but the difference was statistically not significant (p>0.05). An extremely significant improvement occurred postoperatively in both sperms density and motility in infertile/sub fertile patients of both groups. Conclusion: Both procedures of varicocele repair are equally equally effective and have a low rate of complications. There is no significant difference in postoperatively complications. An extremely significant improvement occurs in semen parameters of infertile/sub fertile patients

  12. Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

    DEFF Research Database (Denmark)

    Lamberth, K; Claesson, M H

    2001-01-01

    Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti-apoptotic......Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP......-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP...

  13. External root resorption with the self-ligating Damon system—a retrospective study

    Directory of Open Access Journals (Sweden)

    Roberta Heiffig Handem

    2016-07-01

    Full Text Available Abstract Background The aim of this study was to compare the degree of external apical root resorption (EARR in patients treated with self-ligating Damon appliances and with conventional preadjusted appliances. Methods The sample comprised 52 patients, divided into two groups. Group 1 consisted of 25 patients treated with self-ligating Damon appliances, with an initial age of 16.04 years, final age of 18.06 years, and treatment time of 2.02 years. Group 2 consisted of 27 patients, treated with conventional preadjusted appliances, with an initial age of 16.77 years, final age of 18.47 years and treatment time of 1.70 years. The groups were matched regarding the initial and final ages, treatment time, type of malocclusion, and treatment protocol without extractions. Root resorption was evaluated on periapical radiographs of the maxillary and mandibular incisors at the end of orthodontic treatment with the scores of Levander and Malmgren. Intergroup comparisons of root resorption were performed with Mann-Whitney tests. Results No significant difference in the degree of root resorption between the two groups was found. Conclusions Similar degrees of resorption can be expected after non-extraction treatment with Damon self-ligating or conventional preadjusted appliances.

  14. External root resorption with the self-ligating Damon system-a retrospective study.

    Science.gov (United States)

    Handem, Roberta Heiffig; Janson, Guilherme; Matias, Murilo; de Freitas, Karina Maria Salvatore; de Lima, Darwin Vaz; Garib, Daniela Gamba; de Freitas, Marcos Roberto

    2016-12-01

    The aim of this study was to compare the degree of external apical root resorption (EARR) in patients treated with self-ligating Damon appliances and with conventional preadjusted appliances. The sample comprised 52 patients, divided into two groups. Group 1 consisted of 25 patients treated with self-ligating Damon appliances, with an initial age of 16.04 years, final age of 18.06 years, and treatment time of 2.02 years. Group 2 consisted of 27 patients, treated with conventional preadjusted appliances, with an initial age of 16.77 years, final age of 18.47 years and treatment time of 1.70 years. The groups were matched regarding the initial and final ages, treatment time, type of malocclusion, and treatment protocol without extractions. Root resorption was evaluated on periapical radiographs of the maxillary and mandibular incisors at the end of orthodontic treatment with the scores of Levander and Malmgren. Intergroup comparisons of root resorption were performed with Mann-Whitney tests. No significant difference in the degree of root resorption between the two groups was found. Similar degrees of resorption can be expected after non-extraction treatment with Damon self-ligating or conventional preadjusted appliances.

  15. Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

    Science.gov (United States)

    Carlsson, Nils; Winge, Ann-Sofie; Engström, Sven; Akerman, Björn

    2005-10-06

    We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

  16. Enzyme Treatment-Free and Ligation-Independent Cloning Using Caged Primers in Polymerase Chain Reactions

    Directory of Open Access Journals (Sweden)

    Akinori Kuzuya

    2011-12-01

    Full Text Available A new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment has been developed. By using caged primers in PCR, unnatural sticky-ends of any sequence, which are sufficiently long for ligation-independent cloning (LIC, are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE PCR join together in the desired arrangement in a simple mixture of them, tightly enough to be repaired and ligated in competent cells.

  17. Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

    Science.gov (United States)

    Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

    2000-05-01

    Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

  18. 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.

  19. Compliance with Papanicolaou smear screening following tubal ligation in women with cervical cancer.

    Science.gov (United States)

    Winkler, H A; Anderson, P S; Fields, A L; Runowicz, C D; DeVictoria, C; Goldberg, G L

    1999-01-01

    To evaluate patient compliance with Papanicolaou (Pap) smear screening after tubal ligation compared with other methods of birth control in patients who develop cervical cancer, a retrospective review of 262 women with cervical cancer diagnosed at age Albert Einstein College of Medicine from January 1987 to December 1995. Demographic data, stage of the disease, histologic type, history of smoking, history of sexually transmitted disease (STD), and birth control use were recorded. The Pap screening history was obtained from all the patients. Women who had a bilateral tubal ligation (BTL) were compared with those who did not have this form of birth control. The date and result of their last Pap test prior to their diagnosis of cervical cancer was noted. Two hundred fourteen women with cervical cancer were evaluable. The clinical stage, mean age, history of smoking, and history of STD were similar for both groups. Gravidity among the BTL group was higher than in the non-BTL group (p < 0.01). Forty-eight (22.4%) women had a previous BTL. Twenty-seven of these 48 patients (56.3%) did not have a Pap smear within 3 years prior to the diagnosis of cervical cancer. Of the 166 patients, 61 (36.7%) did not have a Pap test within 3 years (p < 0.05). Fourteen women (29.2%) in the tubal ligation group never returned for a Pap test following the BTL. An average of 6.2+/-5.9 years elapsed since the last Pap test in the BTL group, with 4.0+/-5.1 years in the nontubal ligation group (p < 0.05). There was a correlation between the number of years since BTL (14.2+/-7.7) to the number of years since the last Pap test (6.2+/-5.9) (p < 0.05). Women who have had a BTL should be considered high risk because of poor screening compliance. A Pap test every 3 years is not adequate in this high-risk population group. We advocate improved counseling regarding the importance of continued annual Pap screening for women who are considering tubal ligation.

  20. Preparation of C-terminally modified chemokines by expressed protein ligation.

    Science.gov (United States)

    Baumann, Lars; Steinhagen, Max; Beck-Sickinger, Annette G

    2013-01-01

    In order to link structural features on a molecular level to the function of chemokines, site-specific modification strategies are strongly required. These can be used to incorporate fluorescent dyes and/or physical probes to allow investigations in a wide range of biological and physical techniques, e.g., nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, fluorescence resonance energy transfer (FRET), or fluorescence correlation spectroscopy (FCS). Only a limited number of functional groups within the 20 canonical amino acids allow ligation strategies that can be helpful to introduce novel functionalities, which in turn expand the scope of chemoselective and orthogonal reactivity of (semi)synthetic chemokines. In the present chapter we mainly focus on the fabulous history of native chemical ligation (NCL) and provide a general protocol for the preparation of C-terminally modified SDF-1α including tips and tricks for practical work. We believe that this protocol can be easily adapted to other chemokines and many proteins in general.

  1. Whole genome DNA copy number changes identified by high density oligonucleotide arrays

    Directory of Open Access Journals (Sweden)

    Huang Jing

    2004-05-01

    Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

  2. 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.

  3. miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy.

    Science.gov (United States)

    Gao, Shiqian; Tian, Huayu; Guo, Ye; Li, Yuce; Guo, Zhaopei; Zhu, Xiaojuan; Chen, Xuesi

    2015-10-01

    MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer combines the high transfection efficiency of polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post transfection. Compared with the negative control, PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4 (PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. This work was a combination of the high transfection efficiency of polyethylenimine (PEI), the good biodegradability of polyllysine (PLL) and the breast cancer-killing effect of miR-21 inhibitors. The poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer was employed as the vector of miR-21 sponge plasmid DNA (Sponge) or

  4. Development of a modified model of spinal cord ischemia injury by selective ligation of lumbar arteries in rabbits.

    Science.gov (United States)

    Xiao, W; Wen, J; Huang, Y-C; Yu, B-S

    2017-11-01

    Experimental study. The aim of this study is to develop a modified model of spinal cord ischemia in rabbits. Shenzhen Key Laboratory of Spine Surgery, Shenzhen, China. In total, 20 New Zealand rabbits were divided into the following four groups according to the level of ligation of bilateral lumbar arteries: (1) group A, sham group, no ligation, n=5; (2) group B, ligation of bilateral lumbar arteries at three levels (L2-L4, n=5); (3) group C, ligation of bilateral lumbar arteries at four levels (L2-L5, n=5); and (4) group D, ligation of bilateral lumbar arteries at five levels (L1-L5, n=5). The latency of motor-evoked potentials was measured intraoperatively and the modified Tarlov grades were scored, followed by a histological observation of spinal cord, on the seventh day after surgery. All 10 rabbits in Group A and Group B were electrophysiologically, neurologically and histologically normal. In Group C, moderate spinal cord ischemia injury was found in three of five rabbits: they had prolonged latency of motor-evoked potentials and neuronal karyopyknosis in the anterior horn of spinal cord, and the average Tarlov score was 4.2±0.8. In Group D, severe spinal cord ischemia injury was recorded in all the five rabbits: the latency of motor-evoked potential prolonged in one rabbit, whereas the waveform disappeared in four rabbits; loss of neurons and vacuolation of gray matter were seen in spinal cord sections, and the average Tarlov score was 0.6±0.9. Selective ligation of lumbar arteries was a modified method to induce feasible and reproducible model of spinal cord ischemia in rabbits.

  5. New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

    Science.gov (United States)

    Gbaj, A; Bichenkova, Ev; Walsh, L; Savage, He; Sardarian, Ar; Etchells, Ll; Gulati, A; Hawisa, S; Douglas, Kt

    2009-12-01

    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.

  6. Distribution of extracellular potassium and electrophysiologic changes during two-stage coronary ligation in the isolated, perfused canine heart

    NARCIS (Netherlands)

    Coronel, R.; Fiolet, J. W.; Wilms-Schopman, J. G.; Opthof, T.; Schaapherder, A. F.; Janse, M. J.

    1989-01-01

    We studied the relation between [K+]o and the electrophysiologic changes during a "Harris two-stage ligation," which is an occlusion of a coronary artery, preceded by a 30-minute period of 50% reduction of flow through the artery. This two-stage ligation has been reported to be antiarrhythmic. Local

  7. Hepatic extraction of the aminoterminal propeptide of type III procollagen before and after bile duct ligation in pigs

    DEFF Research Database (Denmark)

    Bentsen, K D; Henriksen, Jens Henrik Sahl; Boesby, S

    1991-01-01

    before and during a 4-h period after ligation of the common bile duct was assessed from serum PIIINP concentrations in a systemic artery, the portal vein and a hepatic vein of seven healthy anaesthetized pigs. Seven sham-operated anaesthetized pigs served as controls. Ligation of the bile duct did...

  8. Robot-assisted ligation of bronchial artery could be an alternative to embolization.

    Science.gov (United States)

    Pochulu, Bruno; Sarsam, Omar; Peillon, Christophe; Baste, Jean-Marc

    2018-03-01

    A 37-year-old patient presented with a self-limiting episode of moderate haemoptysis. Contrast-enhanced chest computed tomography showed a tortuous and dilated right bronchial artery arising from the descending aorta at the level of T6. Therapeutic angiography was attempted, but in the presence of spinal artery arising from the bronchial artery in question, selective embolization was contraindicated due to risk of spinal cord ischaemia. After a multidisciplinary meeting, it was decided to attempt surgery to ligate this pathological artery. We performed minimally invasive robot-assisted ligation of this pathological artery. The postoperative course was uneventful with good clinical and radiological outcome at 3-month follow-up. A minimally invasive approach provides a real alternative to embolization and could be a therapeutic option.

  9. Effective intracellular delivery of oligonucleotides in order to make sense of antisense

    NARCIS (Netherlands)

    Shi, FX; Hoekstra, D

    2004-01-01

    For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the

  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. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    Science.gov (United States)

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  12. 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.

  13. Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane.

    Science.gov (United States)

    Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

    2017-01-01

    Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Significant statistical differences (pbrackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film.

  14. Investigation into the effects of stainless steel ligature ties on the mechanical characteristics of conventional and self-ligated brackets subjected to torque.

    Science.gov (United States)

    Al Fakir, Hussam; Carey, Jason P; Melenka, Garrett W; Nobes, David S; Heo, Giseon; Major, Paul W

    2014-09-01

    Torque is applied to orthodontic brackets in order to alter the buccal-lingual angulation of a tooth. One factor that can affect torque is the ligation mode used to retain the archwire in the bracket slot. The objective of this study was to investigate the effects of stainless steel ligation on torque expression and bracket deformation. This study utilized 60 upper right central incisor Damon Q brackets and 60 Ormco Orthos Twin brackets. The brackets used in this study were subdivided into four groups: (1) Damon Q ligated with SS ligature; (2) Damon Q with the sliding bracket door; (3) Orthos Twin bracket ligated with SS wire; and (4) Orthos Twin ligated with elastic ties. All brackets were tested using an orthodontic torque simulating device that applied archwire rotation from 0° to 45°. All brackets ligated with stainless steel ties exhibited greater torque expression and less deformation than brackets without stainless steel ties. As well, Damon Q brackets exhibit less bracket deformation than Orthos Twin brackets. Stainless steel ties can reduce the amount of plastic deformation for both types of brackets used in this study. © 2014 British Orthodontic Society.

  15. Superantigen and HLA-DR ligation induce phospholipase-C gamma 1 activation in class II+ T cells

    DEFF Research Database (Denmark)

    Kanner, S B; Odum, Niels; Grosmaire, L

    1992-01-01

    Bacterial enterotoxin superantigens bind directly to HLA class II molecules (HLA-DR) expressed on both APC and activated human T cells, and simultaneously bind to certain V beta chains of the TCR. In this report, we compared early T cell signaling events in human alloantigen-stimulated T cells when...... activated by HLA-DR ligation through antibody cross-linking or by direct enterotoxin superantigen binding. Both types of stimuli induced tyrosine phosphorylation of phosphatidylinositol-specific phospholipase C gamma 1 (PLC gamma 1) and an increase in intracellular calcium concentration; however......, superantigen-induced signaling was stronger than class II ligation alone. Antibody-mediated ligation of HLA-DR with CD3 resulted in augmented PLC gamma 1 activation and increased calcium mobilization, consistent with a mechanism of superantigen activity through a combination of class II and CD3/Ti signals...

  16. Hetero-oligonucleotide Nanoscale Tiles Capable of Two-Dimensional Lattice Formation as Testbeds for a Rapid, Affordable Purification Methodology

    Science.gov (United States)

    2013-01-01

    devices; however, DNA is not the only polymer that can take advantage of the specicity of the Watson – Crick base-pair to achieve these goals. Central to...14. ABSTRACT 16. SECURITY CLASSIFICATION OF: New nanoscale hetero-oligonucleotide tiles are assembled from DNA , RNA and morpholino oligos and...purified using size exclusion filtration. Homo-oligonucleotide tiles assembled from RP-cartridge processed DNA oligos are purified by nondenaturing gel

  17. Synthesis of water-soluble scaffolds for peptide cyclization, labeling, and ligation

    NARCIS (Netherlands)

    Smeenk, L.E.J.; Dailly, N.; Hiemstra, H.; van Maarseveen, J.H.; Timmerman, P.

    2012-01-01

    The synthesis and applications of water-soluble scaffolds that conformationally constrain side chain unprotected linear peptides containing two cysteines are described. These scaffolds contain a functionality with orthogonal reactivity to be used for labeling and ligation. This is illustrated by the

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

    KAUST Repository

    El-Batta, Amer; Waltman, Andrew W.; Grubbs, Robert H.

    2011-01-01

    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

  19. Testicular reperfusion injury, post ligation of the spermatic cord in ...

    African Journals Online (AJOL)

    This experiment was aimed at investigating the level of tissue regeneration following sanguineous reperfusion post ligation of the spermatic cord in dogs. A total of 5 adult Nigerian local dogs conditioned for 3 weeks, laboratorily evaluated twice weekly prior to surgery were maintained for 1month. Dogs were aseptically ...

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

    International Nuclear Information System (INIS)

    Suriano, Raffaella; Biella, Serena; Cesura, Federico; Levi, Marinella; Turri, Stefano

    2013-01-01

    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. 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

  2. Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

    Science.gov (United States)

    Jin, Lian-Qun; Li, Jun-Wen; Wang, Sheng-Qi; Chao, Fu-Huan; Wang, Xin-Wei; Yuan, Zheng-Quan

    2005-01-01

    AIM: To detect the common intestinal pathogenic bacteria quickly and accurately. METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays. RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified. CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost. PMID:16437687

  3. Evaluation of the force generated by gradual deflection of orthodontic wires in conventional metallic, esthetic, and self-ligating brackets

    Directory of Open Access Journals (Sweden)

    Manoela Fávaro Francisconi

    Full Text Available ABSTRACT Objective: The purpose of this study was to evaluate the deflection forces of Nitinol orthodontic wires placed in different types of brackets: metallic, reinforced polycarbonate with metallic slots, sapphire, passive and active self-ligating, by assessing strength values variation according to gradual increase in wire diameter and deflection and comparing different combinations in the different deflections. Material and Methods: Specimens were set in a clinical simulation model and evaluated in a Universal Testing Machine (INSTRON 3342, using the ISO 15841 protocol. Data were subjected to One-way ANOVA, followed by Tukey tests (p<0.05. Results: Self-ligating brackets presented the most similar behavior to each other. For conventional brackets there was no consistent behavior for any of the deflections studied. Conclusions: Self-ligating brackets presented the most consistent and predictable results while conventional brackets, as esthetic brackets, showed very different patterns of forces. Self-ligating brackets showed higher strength in all deflections when compared with the others, in 0.020-inch wires.

  4. 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 o...

  5. Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

    Science.gov (United States)

    Chen, Wen; Djama, Zeinab Robleh; Coffey, Michael D; Martin, Frank N; Bilodeau, Guillaume J; Radmer, Lorien; Denton, Geoff; Lévesque, C André

    2013-01-01

    Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

  6. PCR amplification on microarrays of gel immobilized oligonucleotides

    Science.gov (United States)

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  7. 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...

  8. 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.

  9. 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.

  10. Formation and rearrangement of homoserine depsipeptides and depsiproteins in the α-ketoacid-hydroxylamine ligation with 5-oxaproline.

    Science.gov (United States)

    Wucherpfennig, Thomas G; Rohrbacher, Florian; Pattabiraman, Vijaya R; Bode, Jeffrey W

    2014-11-03

    The primary products of the chemical ligation of α-ketoacids and 5-oxaproline peptides are esters, rather than the previously reported amides. The depsipeptide product rapidly rearranges to the amide in basic buffers. The formation of esters sheds light on possible mechanisms for the type II KAHA ligations and opens an avenue for the chemical synthesis of depsiproteins. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

    DEFF Research Database (Denmark)

    Lamberth, K; Claesson, M H

    2001-01-01

    Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP......-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP...

  12. Angiographic findings of collateral vessels in cervicofacial vascular lesions with previously ligated carotid artery

    International Nuclear Information System (INIS)

    Na, Dong Gyu; Han, Moon Hee; Chang, Kee Hyun; Han, Gi Seok; Yeon, Kung Mo

    1995-01-01

    The purpose of this study is to describe the angiographic findings of collateral vessels in cervicofacial vascular lesions with previously ligated carotid arteries and to evaluate the extent of angiographic assessment needed before embolization. We retrospectively reviewed 10 cervicofacial vascular lesions with previously ligated carotid artery, which were 6 cases of arteriovenous malformation, 2 cases of carotid cavernous fistula, 1 case of hemangioma and 1 case of arteriovenous malformation with carotid cavernous fistula. The previously ligated arteries are proximal external carotid artery (n = 5), branches of external carotid artery (n = 2) and common carotid artery (n = 3). Common carotid artery or internal carotid artery (n = 9), vertebral artery (n = 5), ipsilateral external carotid artery (n = 4), contralateral external carotid artery (n = 5), costocervical trunk (n = 2), thyrocervical trunk (n = 2) were assessed by conventional angiography. Angiography of both carotid and vertebral arteries was performed in 5 cases. The collateral vascular channels were inferolateral trunk of internal carotid artery (n = 8), vertebral artery (n = 5), contralateral external carotid artery (n = 5), ipsilateral external carotid artery (n = 4), deep cervical artery (n = 2) and ascending cervical artery (n = 1). Embolization were performed in 9 cases with operative cannulation (n = 4), embolization via collateral branches of ipsilateral external carotid artery (n = 1), embolization via collateral branches of contralateral external carotid artery (n = 3) and balloon occlusion via direct puncture (n = 1). The collateral channels in cervicofacial vascular lesions with previously ligated carotid artery were inferolateral trunk of internal carotid artery, contralateral or ipsilateral external carotid artery, vertebral artery, deep cervical artery and ascending cervical artery on angiography. Complete angiographic assessment of possible collateral channels is mandatory for the

  13. Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides.

    Science.gov (United States)

    de Bruin, Donny; Bossert, Nelli; Aartsma-Rus, Annemieke; Bouwmeester, Dirk

    2018-04-06

    Short nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson-Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided. We employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results. The primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data.

  14. Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation.

    Science.gov (United States)

    He, Chunmao; Kulkarni, Sameer S; Thuaud, Frédéric; Bode, Jeffrey W

    2015-10-26

    The chemical synthesis of the 184-residue ferric heme-binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α-ketoacid-hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild-type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well-suited for the preparation of hydrophobic protein targets. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. An in vitro study into the efficacy of complex tooth alignment with conventional and self-ligating brackets.

    Science.gov (United States)

    Montasser, M A; Keilig, L; Bourauel, C

    2015-02-01

    To evaluate the efficacy of tooth alignment achieved by various small cross-section archwire/bracket combinations using the orthodontic measurement and simulation system. The study comprised three types of orthodontic brackets 1) conventional ligating (Victory Series and Mini-Taurus), 2) self-ligating (SmartClip a passive self-ligating bracket and Time3 an active self-ligating bracket), and 3) a conventional low-friction bracket (Synergy). All brackets had a nominal 0.022″ slot size. Brackets were combined with 1) 0.012″ stainless steel, 2) 0.012″ Orthonol, 3) 0.012″ Thermalloy, and 4) 0.0155″ coaxial archwires. Archwires were tied to the conventional brackets with stainless steel ligatures and elastomeric rings. The malocclusion simulated represented a central upper incisor displaced 2 mm gingivally (x-axis) and 2 mm labially (z-axis). The inciso-gingival correction achieved by the different archwire/bracket combinations ranged from 15 to 95%, while the labio-lingual correction ranged from 10 to 95%. The smallest correction was achieved by coaxial, Orthonol, and thermally archwires when ligated with the elastomeric rings to conventional brackets. Stainless steel archwires achieved from 65 to 90% of inciso-gingival correction and from 60 to 90% of labio-lingual correction. The resultant tooth alignment was the product of interaction between the archwire type, bracket type, and bracket design including ligature type. Small cross-sectional archwires might produce up to 95% correction if combined properly with the bracket system. Elastomeric rings when used with conventional brackets limit the efficacy of malalignment correction. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Consecutive native chemical ligation-route to synthetic mouse prion protein

    Czech Academy of Sciences Publication Activity Database

    Zawada, Zbigniew; Šebestík, Jaroslav; Šafařík, Martin; Březinová, Anna; Bouř, Petr; Hlaváček, Jan; Stibor, Ivan

    2009-01-01

    Roč. 103, č. 11 (2009), s. 1001-1001 ISSN 0009-2770. [Pokroky v organické, bioorganické a farmaceutické chemii /44./. 27.11.2009-29.11.2009, Liblice] R&D Projects: GA ČR GA203/07/1517 Institutional research plan: CEZ:AV0Z40550506 Keywords : prion * chemical ligation Subject RIV: CC - Organic Chemistry

  17. 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.

  18. The use of oligonucleotide aptamers in cancer therapy

    Directory of Open Access Journals (Sweden)

    Adrian Odrzywolski

    2016-05-01

    Full Text Available Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers – particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug.

  19. A bipyridine-ligated zinc(II) complex with bridging flavonolate ligation: synthesis, characterization, and visible-light-induced CO release reactivity.

    Science.gov (United States)

    Sorenson, Shayne; Popova, Marina; Arif, Atta M; Berreau, Lisa M

    2017-09-01

    Metal-flavonolate compounds are of significant current interest as synthetic models for quercetinase enzymes and as bioactive compounds of importance to human health. Zinc-3-hydroxyflavonolate compounds, including those of quercetin, kampferol, and morin, generally exhibit bidentate coordination to a single Zn II center. The bipyridine-ligated zinc-flavonolate compound reported herein, namely bis(μ-4-oxo-2-phenyl-4H-chromen-3-olato)-κ 3 O 3 :O 3 ,O 4 ;κ 3 O 3 ,O 4 :O 3 -bis[(2,2'-bipyridine-κ 2 N,N')zinc(II)] bis(perchlorate), {[Zn 2 (C 15 H 9 O 3 ) 2 (C 10 H 8 N 2 ) 2 ](ClO 4 ) 2 } n , (1), provides an unusual example of bridging 3-hydroxyflavonolate ligation in a dinuclear metal complex. The symmetry-related Zn II centers of (1) exhibit a distorted octahedral geometry, with weak coordination of a perchlorate anion trans to the bridging deprotonated O atom of the flavonolate ligand. Variable-concentration conductivity measurements provide evidence that, when (1) is dissolved in CH 3 CN, the complex dissociates into monomers. 1 H NMR resonances for (1) dissolved in d 6 -DMSO were assigned via HMQC to the H atoms of the flavonolate and bipyridine ligands. In CH 3 CN, (1) undergoes quantitative visible-light-induced CO release with a quantum yield [0.004 (1)] similar to that exhibited by other mononuclear zinc-3-hydroxyflavonolate complexes. Mass spectroscopic identification of the [(bpy) 2 Zn(O-benzoylsalicylate)] + ion provides evidence of CO release from the flavonol and of ligand exchange at the Zn II center.

  20. Nucleoside-O-Methyl-(H)-Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H-Phosphonate Chemistry.

    Science.gov (United States)

    Kostov, Ondřej; Páv, Ondřej; Rosenberg, Ivan

    2017-09-18

    This unit comprises the straightforward synthesis of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in both 3'- and 5'-series. These compounds represent a new class of monomers compatible with the solid-phase synthesis of oligonucleotides using H-phosphonate chemistry and are suitable for the preparation of both 3'- and 5'-O-methylphosphonate oligonucleotides. The synthesis of 4-toluenesulfonyloxymethyl-(H)-phosphinic acid as a new reagent for the preparation of O-methyl-(H)-phosphinic acid derivatives is described. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. Ligation of MHC class I molecules on peripheral blood T lymphocytes induces new phenotypes and functions

    DEFF Research Database (Denmark)

    Bregenholt, S; Röpke, M; Skov, S

    1996-01-01

    of T cell-mediated cytotoxicity. Immediately following MHC-I ligation, the T cells responded with increased protein tyrosine phosphorylation, with new bands appearing in the SDS-PAGE. Exposure of T cells to immobilized anti-MHC-I Ab for 24 h induced an increased surface expression of the TCR/CD3 and CD......28 molecules. MHC-I-induced proliferation of purified T cells was dependent on cellular interactions with non-T cells. Under certain conditions, in which MHC-I was ligated by picogram concentrations of immobilized anti-MHC-I Ab, anti-TCR/CD3 Ab-induced proliferation of T cells was strongly inhibited....... These data clearly demonstrate that ligation of the MHC-I complex on T cells may induce both positive and negative signals. Since the physiologic ligands for MHC-I molecules are TCR and the CD8 molecules, our data may suggest that MHC-I molecules are instrumental in cellular interactions between T cells....

  2. Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

    Science.gov (United States)

    Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

    2007-01-01

    RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

  3. 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.

  4. 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...

  5. Intravesical ligation as a new technique to manage a refluxing native ureter without simultaneous nephrectomy in renal transplantation.

    Science.gov (United States)

    Guzmán, J A

    2012-12-01

    This article aims to describe an original technique to correct refluxing native ureters observed during a prerenal transplantation study. The correction is performed by intravesical ligation of the native refluxing ureters at the same time as renal transplantation without simultaneous nephrectomy. Between January 2004 and December 2010 we performed intravesical ligation of a refluxing ureter simultaneous with a transplantation procedure without a concomittant native nephrectomy in 12 of 345 subjects (3.47%). The 8 bilateral and 4 unilateral ligations were performed on 11 cadaveric and 1 living-related nonidentical donor transplantations. The implantation of the kidney donor ureter was performed anatomically in the bladder trigone through a transvesical ureteroneocystostomy with a transmural, submucosal antireflux tunnel. Early and late postoperative recovery was satisfactory in all patients. There was no documented kidney area pain, proven urinary tract infection, morbidity or mortality attributed to the procedure. Intravesical ligation is a practical technique to manage vesicoureteral reflux into the native ureters simultaneously with the ureteral implantation of the kidney donor in a single surgical renal transplant procedure without native kidney nephrectomy. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

    Science.gov (United States)

    Tian, Lingyang; Sato, Takuichi; Niwa, Kousuke; Kawase, Mitsuo; Tanner, Anne C R; Takahashi, Nobuhiro

    2014-01-01

    A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

  7. Rapid and Sensitive PCR-Dipstick DNA Chromatography for Multiplex Analysis of the Oral Microbiota

    Directory of Open Access Journals (Sweden)

    Lingyang Tian

    2014-01-01

    Full Text Available A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

  8. Are self-ligating brackets related to less formation of Streptococcus mutans colonies? A systematic review

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence.

    NARCIS (Netherlands)

    Pellegrini, P.; Sauerwein, R.W.; Finlayson, T.; McLeod, J.; Covell, D.A.; Maier, T.; Machida, C.A.

    2009-01-01

    INTRODUCTION: Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate

  11. Level of arterial ligation in sigmoid colon and rectal cancer surgery.

    Science.gov (United States)

    Yasuda, Koji; Kawai, Kazushige; Ishihara, Soichiro; Murono, Koji; Otani, Kensuke; Nishikawa, Takeshi; Tanaka, Toshiaki; Kiyomatsu, Tomomichi; Hata, Keisuke; Nozawa, Hiroaki; Yamaguchi, Hironori; Aoki, Shigeo; Mishima, Hideyuki; Maruyama, Tsunehiko; Sako, Akihiro; Watanabe, Toshiaki

    2016-04-01

    Curative resection of sigmoid colon and rectal cancer includes "high tie" of the inferior mesenteric artery (IMA). However, IMA ligation compromises blood flow to the anastomosis, which may increase the leakage rate, and it is unclear whether this confers a survival advantage. Accordingly, the IMA may be ligated at a point just below the origin of the left colic artery (LCA) "low tie" combined with lymph node dissection (LND) around the origin of the IMA (low tie with LND). However, no study has investigated the detailed prognostic results between "high tie" and "low tie with LND." The aim of this study was to assess the utility of "low tie with LND" on survival in patients with sigmoid colon or rectal cancer. A total of 189 sigmoid colon or rectal cancer patients who underwent curative operation from 1997 to 2007 were enrolled in this study. The patient's medical records were reviewed to obtain clinicopathological information. Overall survival (OS) and relapse-free survival (RFS) rates were calculated using the Kaplan-Meier method, with differences assessed using log-rank test. Forty-two and 147 patients were ligated at the origin of the IMA (high tie) and just below the origin of the LCA combined with LND around the origin of the IMA (low tie with LND), respectively. No significant differences were observed in the complication rate and OS and RFS rates in the two groups. Further, no significant difference was observed in the OS and RFS rates in the lymph node-positive cases in the two groups. "Low tie with LND" is anatomically less invasive and is not inferior to "high tie" with prognostic point of view.

  12. Chemically modified oligonucleotides with efficient RNase H response

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  13. 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

    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...

  14. Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography.

    Science.gov (United States)

    Dang, Bobo; Kubota, Tomoya; Mandal, Kalyaneswar; Bezanilla, Francisco; Kent, Stephen B H

    2013-08-14

    We have re-examined the utility of native chemical ligation at -Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites without side reactions. After optimization, ligation at a -Glu-Cys- site could also be used as a ligation site, with minimal levels of byproduct formation. However, -Asp-Cys- is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of β-linked byproduct. The feasibility of native chemical ligation at -Gln-Cys- enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high-resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.

  15. E-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions

    DEFF Research Database (Denmark)

    Perrais, Michaël; Chen, Xiao; Perez-Moreno, Mirna

    2007-01-01

    growth inhibitory signals. To address this question, we have selectively formed E-cadherin homophilic bonds at the cell surface of isolated epithelial cells by using functionally active recombinant E-cadherin protein attached to microspheres. We find that E-cadherin ligation alone reduces the frequency...... of cells entering the S phase, demonstrating that E-cadherin ligation directly transduces growth inhibitory signals. E-cadherin binding to beta-catenin is required for cell growth inhibition, but beta-catenin/T-cell factor transcriptional activity is not involved in growth inhibition resulting from...... homophilic binding. Neither E-cadherin binding to p120-catenin nor beta-catenin binding to alpha-catenin, and thereby the actin cytoskeleton, is required for growth inhibition. E-cadherin ligation also inhibits epidermal growth factor (EGF) receptor-mediated growth signaling by a beta...

  16. Dose-Dependent Lowering of Mutant Huntingtin Using Antisense Oligonucleotides in Huntington Disease Patients.

    Science.gov (United States)

    van Roon-Mom, Willeke M C; Roos, Raymund A C; de Bot, Susanne T

    2018-04-01

    On December 11 of 2017, Ionis Pharmaceuticals published a press release announcing dose-dependent reductions of mutant huntingtin protein in their HTTRx Phase 1/2a study in Huntington disease (HD) patients. The results from this Ionis trial have gained much attention from the patient community and the oligonucleotide therapeutics field, since it is the first trial targeting the cause of HD, namely the mutant huntingtin protein, using antisense oligonucleotides (ASOs). The press release also states that the primary endpoints of the study (safety and tolerability) were met, but does not contain data. This news follows the approval of another therapeutic ASO nusinersen (trade name Spinraza) for a neurological disease, spinal muscular atrophy, by the U.S. Food and Drug Administration and European Medicines Agency, in 2016 and 2017, respectively. Combined, this offers hope for the development of the HTTRx therapy for HD patients.

  17. Comparison of frictional resistance between self-ligating and conventional brackets tied with elastomeric and metal ligature in orthodontic archwires.

    Science.gov (United States)

    Leite, Vanessa Vieira; Lopes, Murilo Baena; Gonini Júnior, Alcides; Almeida, Marcio Rodrigues de; Moura, Sandra Kiss; Almeida, Renato Rodrigues de

    2014-01-01

    To compare the frictional resistance between self-ligating and conventional brackets tied to different types of wire. Abzil Kirium Capelozza (Pattern I) and Easy Clip (Roth prescription) incisor brackets were used. An elastomeric ligature or a 0.10-in ligating wire was used to ligate the wire to the Abzil bracket. Three types of orthodontic archwire alloys were assessed: 0.016-in NiTi wire, 0.016 x 0.021-in NiTi wire and 0.019 x 0.025-in steel wire. Ten observations were carried out for each bracket-archwire angulation combination. Brackets were mounted in a special appliance, positioned at 90 degrees in relation to the wire and tested in two angulations. Frictional test was performed in a Universal Testing Machine at 5 mm/min and 10 mm of displacement. The means (MPa) were submitted to ANOVA and Tukey's test set at 5% of significance. The surfaces of wires and brackets were observed at SEM. Steel-tied brackets (16.48 ± 8.31) showed higher means of frictional resistance than elastomeric-tied brackets (4.29 ± 2.16 ) and self-ligating brackets (1.66 ± 1.57) (P 0.05). No statistical differences (P > 0.05) were found between zero (7.76 ± 8.46) and five-degree (7.19 ± 7.93) angulations. Friction was influenced not only by the type of bracket, but also by the ligating systems. Different morphological aspects were observed for the brackets and wires studied.

  18. Comparative study of friction between metallic and conventional interactive self-ligating brackets in different alignment conditions.

    Science.gov (United States)

    Jakob, Sérgio Ricardo; Matheus, Davison; Jimenez-Pellegrin, Maria Cristina; Turssi, Cecília Pedroso; Amaral, Flávia Lucisano Botelho

    2014-01-01

    The aim of this study was to compare the friction between three bracket models: conventional stainless steel (Ovation, Dentsply GAC), self-ligating ceramic (In-Ovation, Denstply GAC) and self-ligating stainless steel brackets (In-Ovation R, Dentsply GAC). Five brackets were used for each model. They were bonded to an aluminum prototype that allowed the simulation of four misalignment situations (n = 10). Three of these situations occured at the initial phase (in which a 0.016-in nickel-titanium wire was used): 1. horizontal; 2. vertical; and 3. simultaneous horizontal/vertical. One of the situations occurred at the final treatment phase: 4. no misalignment (in which a 0.019 x 0.025-inch stainless steel rectangular wire was used). The wires slipped through the brackets and friction was measured by a Universal Testing Machine. Analysis of variance followed by Tukey's Test for multiple comparisons (α = 0.05) were applied to assess the results. Significant interaction (p friction. The two self-ligating models resulted in lower and similar values, except for the horizontal situation, in which In-Ovation C® showed lower friction, which was similar to the In-Ovation R® metallic model. For the no misalignment situation, the same results were observed. The self-ligating system was superior to the conventional one due to producing less friction. With regard to the material used for manufacturing the brackets, the In-Ovation C® ceramic model showed less friction than the metallic ones.

  19. In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

    Directory of Open Access Journals (Sweden)

    A Sesilja Aranko

    Full Text Available BACKGROUND: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. PRINCIPAL FINDINGS: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. CONCLUSION: The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

  20. Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

    Science.gov (United States)

    Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

  1. Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

    Directory of Open Access Journals (Sweden)

    Laia Ramos

    Full Text Available Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb. Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14(q10;q10. Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

  2. Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

    Science.gov (United States)

    Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers. PMID:25415307

  3. CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

    DEFF Research Database (Denmark)

    Zaghloul, Eman M; Gissberg, Olof; Moreno, Pedro M D

    2017-01-01

    Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion....... Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT...

  4. Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays

    Directory of Open Access Journals (Sweden)

    Jee Justin

    2008-12-01

    Full Text Available Abstract Background Mismatched oligonucleotides are widely used on microarrays to differentiate specific from nonspecific hybridization. While many experiments rely on such oligos, the hybridization behavior of various degrees of mismatch (MM structure has not been extensively studied. Here, we present the results of two large-scale microarray experiments on S. cerevisiae and H. sapiens genomic DNA, to explore MM oligonucleotide behavior with real sample mixtures under tiling-array conditions. Results We examined all possible nucleotide substitutions at the central position of 36-nucleotide probes, and found that nonspecific binding by MM oligos depends upon the individual nucleotide substitutions they incorporate: C→A, C→G and T→A (yielding purine-purine mispairs are most disruptive, whereas A→X were least disruptive. We also quantify a marked GC skew effect: substitutions raising probe GC content exhibit higher intensity (and vice versa. This skew is small in highly-expressed regions (± 0.5% of total intensity range and large (± 2% or more elsewhere. Multiple mismatches per oligo are largely additive in effect: each MM added in a distributed fashion causes an additional 21% intensity drop relative to PM, three-fold more disruptive than adding adjacent mispairs (7% drop per MM. Conclusion We investigate several parameters for oligonucleotide design, including the effects of each central nucleotide substitution on array signal intensity and of multiple MM per oligo. To avoid GC skew, individual substitutions should not alter probe GC content. RNA sample mixture complexity may increase the amount of nonspecific hybridization, magnify GC skew and boost the intensity of MM oligos at all levels.

  5. A comparative study of frictional force in self-ligating brackets according to the bracket-archwire angulation, bracket material, and wire type

    Science.gov (United States)

    Lee, Souk Min

    2015-01-01

    Objective This study aimed to compare the frictional force (FR) in self-ligating brackets among different bracket-archwire angles, bracket materials, and archwire types. Methods Passive and active metal self-ligating brackets and active ceramic self-ligating brackets were included as experimental groups, while conventional twin metal brackets served as a control group. All brackets were maxillary premolar brackets with 0.022 inch [in] slots and a -7° torque. The orthodontic wires used included 0.018 round and 0.019 × 0.025 in rectangular stainless steel wires. The FR was measured at 0°, 5°, and 10° angulations as the wire was drawn through the bracket slots after attaching brackets from each group to the universal testing machine. Static and kinetic FRs were also measured. Results The passive self-ligating brackets generated a lower FR than all the other brackets. Static and kinetic FRs generally increased with an increase in the bracket-archwire angulation, and the rectangular wire caused significantly higher static and kinetic FRs than the round wire (p brackets exhibited the lowest static FR at the 0° angulation and a lower increase in static and kinetic FRs with an increase in bracket-archwire angulation than the other brackets, while the conventional twin brackets showed a greater increase than all three experimental brackets. Conclusions The passive self-ligating brackets showed the lowest FR in this study. Self-ligating brackets can generate varying FRs in vitro according to the wire size, surface characteristics, and bracket-archwire angulation. PMID:25667913

  6. 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...

  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. A Fast, Sensitive and Label Free Electrochemical DNA Sensor

    International Nuclear Information System (INIS)

    Chen Yu; Elling; Lee Yokeling; Chong Serchoong

    2006-01-01

    A label free and sensitive DNA/RNA silicon based electrochemical microsensor array was developed by using thin film of the conducting polymer polypyrrole doped with an oligonucleotide probe. The electrochemical potential pulse amperometry technique was used for a biowarfare pathogen target DNA detection. The electrical potential assistanted DNA hybridisation method was applied. The sensor signal was increased by increasing the electrical potential assistanted DNA hybridisation time. It was possible to detect 0.34pmol and 0.072fmol of complementary oligonucleotide target in 0.1ml in seconds by using unpolished and polished gold electrode respectively. The probe preparation was also in seconds time, comparing indirect electrochemical DNA sensor, it has a fast sensor preparation as well as sensor response and label free advantages. The silicon microfabrication technique was used for this sensor array fabrication, which holds the potential to integrate with sensor electrical circuits. The conducting polymer polypyrrole was electrochemically deposited on each electrode respectively which has a possibility to dope the different DNA probe into the individual electrode to form a sensor array

  9. Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

    Energy Technology Data Exchange (ETDEWEB)

    Gonçalves, J.O.; Tannuri, A.C.A.; Coelho, M.C.M.; Bendit, I.; Tannuri, U. [Laboratório de Pesquisa em Cirurgia Pediátrica (LIM-30), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

    2014-08-15

    We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.

  10. Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

    International Nuclear Information System (INIS)

    Gonçalves, J.O.; Tannuri, A.C.A.; Coelho, M.C.M.; Bendit, I.; Tannuri, U.

    2014-01-01

    We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method

  11. Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 Increases Neovascularization and Blood Flow Recovery After Ischemia

    DEFF Research Database (Denmark)

    Welten, S. M. J.; Bastiaansen, Ajnm; de Jong, R. C. M.

    2014-01-01

    in mice after single femoral artery ligation. Methods and Results: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4...... GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased...

  12. A multiplex ligation detection assay for the characterization of Salmonella enterica strains

    NARCIS (Netherlands)

    Aarts, H.J.M.; Vos, P.; Larsson, J.T.; Hoek, van A.H.A.M.; Huehn, S.; Weijers, T.; Gronlund, H.A.; Malorny, B.

    2011-01-01

    A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube (R) microarray detection. The

  13. Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides

    International Nuclear Information System (INIS)

    Qin Guangming; Zhang Yongxue; Cao Wei; An Rui; Gao Zairong; Xu Wendai; Zhang Kaijun; Li Guiling; Li Shuren

    2005-01-01

    The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq 99m Tc-labelled ASON or 99m Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-mycASON was evaluated in vivo in atherosclerotic rabbits. Planar imaging revealed accumulation of 99m Tc-labelled c-mycASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage. (orig.)

  14. Streptococcus sanguinis meningitis following endoscopic ligation for oesophageal variceal haemorrhage.

    Science.gov (United States)

    Liu, Yu-Ting; Lin, Chin-Fu; Lee, Ya-Ling

    2013-05-01

    We report a case of acute purulent meningitis caused by Streptococcus sanguinis after endoscopic ligation for oesophageal variceal haemorrhage in a cirrhotic patient without preceding symptoms of meningitis. Initial treatment with flomoxef failed. The patient was cured after 20 days of intravenous penicillin G. This uncommon infection due to S. sanguinis adds to the long list of infectious complications among patients with oesophageal variceal haemorrhage.

  15. A Simple Suturing Technique for Laparoscopic Ligation of Vascular Pedicles

    Directory of Open Access Journals (Sweden)

    Keith A. Aqua

    1996-01-01

    Full Text Available We report on the performance of 348 adnexectomies and 35 uterine artery ligations for both benign and malignant disease using a simple laparoscopic suturing technique. Only 5-mm ports are required, and there was no morbidity directly associated with this approach. The procedure can be performed quickly, is relatively inexpensive, and allows hysterectomy and oophorectomy to be performed without bipolar electrocautery.

  16. Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation

    Directory of Open Access Journals (Sweden)

    Takashi Kubota

    2015-08-01

    Full Text Available The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.

  17. A novel setup for the determination of absolute cross sections for low-energy electron induced strand breaks in oligonucleotides - The effect of the radiosensitizer 5-fluorouracil

    International Nuclear Information System (INIS)

    Rackwitz, J.; Rankovic, M.L.; Milosavljevic, A.R.; Bald, I.

    2017-01-01

    Low-energy electrons (LEEs) play an important role in DNA radiation damage. Here we present a method to quantify LEE induced strand breakage in well-defined oligonucleotide single strands in terms of absolute cross sections. An LEE irradiation setup covering electron energies <500 eV is constructed and optimized to irradiate DNA origami triangles carrying well-defined oligonucleotide target strands. Measurements are presented for 10.0 and 5.5 eV for different oligonucleotide targets. The determination of absolute strand break cross sections is performed by atomic force microscopy analysis. An accurate fluence determination ensures small margins of error of the determined absolute single strand break cross sections σ SSB . In this way, the influence of sequence modification with the radiosensitive 5-Fluorouracil ( 5F U) is studied using an absolute and relative data analysis. We demonstrate an increase in the strand break yields of 5F U containing oligonucleotides by a factor of 1.5 to 1.6 compared with non-modified oligonucleotide sequences when irradiated with 10 eV electrons. (authors)

  18. Biomechanical characteristics of self-ligating brackets in a vertically displaced canine model: a finite element analysis.

    Science.gov (United States)

    Kim, S-J; Kwon, Y-H; Hwang, C-J

    2016-05-01

    The objective of this study was to compare the biomechanical characteristics between two types of self-ligating brackets and conventional metal brackets using finite element analysis of a vertically displaced canine model focusing on the desired force on the canine and undesirable forces on adjacent teeth. Three-dimensional finite element models of the maxillary dentition with 1-mm, 2-mm, and 3-mm vertically displaced canines were constructed. Two different self-ligating brackets (In-Ovation C and Smart clip) and a conventional metal bracket (Micro-arch) were modeled. After a 0.016-inch NiTi (0.40 mm, round) wire was engaged, the displacement of each tooth was calculated using x-, y-, and z-coordinates, and the tensile and compressive stresses were calculated. The extrusion and maximal tensile stress of the canine differed little between the three brackets, but the intrusion and minimal compressive stress values of the adjacent teeth differed considerably and were highest in the Smart clip and least in the In-Ovation C. The extrusion and maximal tensile stress of the canine in the 3-mm displacement model was less than that in the 2-mm displacement model, and the intrusion and minimal compressive stress of the adjacent teeth increased with the degree of displacement. Self-ligating brackets were not superior to conventional brackets in leveling a vertically displaced canine. A continuous arch wire may not be recommended for leveling of severely displaced canines whether using self-ligating or conventional brackets. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. 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.

  20. Dimensional accuracy of ceramic self-ligating brackets and estimates of theoretical torsional play.

    Science.gov (United States)

    Lee, Youngran; Lee, Dong-Yul; Kim, Yoon-Ji R

    2016-09-01

    To ascertain the dimensional accuracies of some commonly used ceramic self-ligation brackets and the amount of torsional play in various bracket-archwire combinations. Four types of 0.022-inch slot ceramic self-ligating brackets (upper right central incisor), three types of 0.018-inch ceramic self-ligating brackets (upper right central incisor), and three types of rectangular archwires (0.016 × 0.022-inch beta-titanium [TMA] (Ormco, Orange, Calif), 0.016 × 0.022-inch stainless steel [SS] (Ortho Technology, Tampa, Fla), and 0.019 × 0.025-inch SS (Ortho Technology)) were measured using a stereomicroscope to determine slot widths and wire cross-sectional dimensions. The mean acquired dimensions of the brackets and wires were applied to an equation devised by Meling to estimate torsional play angle (γ). In all bracket systems, the slot tops were significantly wider than the slot bases (P brackets, and Clippy-Cs (Tomy, Futaba, Fukushima, Japan) among the 0.018-inch brackets. The Damon Clear (Ormco) bracket had the smallest dimensional error (0.542%), whereas the 0.022-inch Empower Clear (American Orthodontics, Sheboygan, Wis) bracket had the largest (3.585%). The largest amount of theoretical play is observed using the Empower Clear (American Orthodontics) 0.022-inch bracket combined with the 0.016 × 0.022-inch TMA wire (Ormco), whereas the least amount occurs using the 0.018 Clippy-C (Tomy) combined with 0.016 × 0.022-inch SS wire (Ortho Technology).

  1. Actual versus theoretical torsional play in conventional and self-ligating bracket systems

    DEFF Research Database (Denmark)

    Dalstra, Michel; Eriksen, Henrik; Bergamini, Chiara

    2015-01-01

    Abstract Objective: The aim of this study was to assess the amount of torsional play in 32 commercially available self-ligating and conventional 0·018-inch and 0·022-inch bracket systems in relation to 0·017×0·022-inch and 0·019×0·025-inch stainless steel wires, respectively, and compare...... the results with the theoretical amount of play for the given bracket/wire combinations. Methods: Torque moments were measured in a mechanical force testing system by twisting straight pieces of stainless steel wire seated in the bracket slot in increments of 0·5° until a full torsional expression...... and the inability of self-ligation brackets to press the archwire into the bottom of the slot. In conventional brackets, the initial torque moment is generated by the steel ligatures pressing the arch wire against the bottom of the slot. The oversize of the slot is thus less critical in relation to the conventional...

  2. 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.

  3. Optical properties and electronic transitions of DNA oligonucleotides as a function of composition and stacking sequence.

    Science.gov (United States)

    Schimelman, Jacob B; Dryden, Daniel M; Poudel, Lokendra; Krawiec, Katherine E; Ma, Yingfang; Podgornik, Rudolf; Parsegian, V Adrian; Denoyer, Linda K; Ching, Wai-Yim; Steinmetz, Nicole F; French, Roger H

    2015-02-14

    The role of base pair composition and stacking sequence in the optical properties and electronic transitions of DNA is of fundamental interest. We present and compare the optical properties of DNA oligonucleotides (AT)10, (AT)5(GC)5, and (AT-GC)5 using both ab initio methods and UV-vis molar absorbance measurements. Our data indicate a strong dependence of both the position and intensity of UV absorbance features on oligonucleotide composition and stacking sequence. The partial densities of states for each oligonucleotide indicate that the valence band edge arises from a feature associated with the PO4(3-) complex anion, and the conduction band edge arises from anti-bonding states in DNA base pairs. The results show a strong correspondence between the ab initio and experimentally determined optical properties. These results highlight the benefit of full spectral analysis of DNA, as opposed to reductive methods that consider only the 260 nm absorbance (A260) or simple purity ratios, such as A260/A230 or A260/A280, and suggest that the slope of the absorption edge onset may provide a useful metric for the degree of base pair stacking in DNA. These insights may prove useful for applications in biology, bioelectronics, and mesoscale self-assembly.

  4. Chemical Ligation: A Versatile Method for Nucleoside Modification with Boron Clusters

    Czech Academy of Sciences Publication Activity Database

    Wojtczak, B. A.; Andrysiak, A.; Grüner, Bohumír; Lesnikowski, Z. J.

    -, č. 14 (2008), s. 10675-10682 ISSN 0947-6539 R&D Projects: GA MŠk LC523 Grant - others:MSHE(PL) N405 051 32/3592; MSHE(PL) K152/H03/2007/10 Institutional research plan: CEZ:AV0Z40320502 Keywords : alkynes * azides * chemical ligation Subject RIV: CA - Inorganic Chemistry Impact factor: 5.454, year: 2008

  5. Multiplex Ligation-Dependent Probe Amplification Technique for Copy Number Analysis on Small Amounts of DNA Material

    DEFF Research Database (Denmark)

    Sørensen, Karina; Andersen, Paal; Larsen, Lars

    2008-01-01

    The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim...... of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for MLPA...... analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total...

  6. Outcome of band ligation in oesophageal varices

    International Nuclear Information System (INIS)

    Abbasi, A.; Bhutto, A.R.; Bhatti, K.I.; Mahmood, K.; Lal, K.

    2013-01-01

    Objective: To find out the outcome og band ligation of oesophageal varices in decompensated chronic liver disease patients. Methods: The quasi experimental study was conducted at the Jinnah Postgraduate Medical Centre, Karachi, and Civil Hospital, Karachi, unit from September 2007 to August 2011. Subjects were eligible if they had a diagnosis of cirrhosis based on history, physical examination, biochemical parameters and liver biopsy in some cases. Patients with advanced cirrhosis (Child-Pugh class C), antibodies against human immunodeficiency virus, hepatocellular carcinoma, portal vein thrombosis evident on ultrasonography, parenteral drug addiction, current alcohol abuse, previous or current treatment with β-blockers were excluded from the study. All patients were asked about alcohol intake and tested to determine the cause of liver cirrhosis. Tests for other causes of cirrhosis were carried out only if there was a suggestive clue. All patients under-went upper gastrointestinal endoscopy after consent. SPSS 15 was used for statistical analysis. Results: The age of the 173 patients who met the inclusion criteria ranged from 15 to 85 years, with a mean of 48.39+-13.38 years. There were 112 (64.7%) males. High-grade varices were seen in 130 (75.1%) patients, while low-grade varices were observed in 43 (24.9%) on first endoscopy. At initial endoscopy, 111 (64.2%) patients had portal hypertensive gastropathy. The patients were followed up for a mean period of 5.20+-2.67 months. Variceal obliteration was achieved in 138 (79.8%), while 33 (19.1%) cases developed re-bleeding. Mean number of endoscopy sessions for these patients were 2.28+-.918 with a maximum of 4. Conclusion: Band ligation eradicated oesophageal varices with less complications and a lower re-bleeding rate, but at the same time eradication was associated with more frequent development of portal hypertensive gastropathy. (author)

  7. Alignment efficiency of standard versus tandem wire mechanics using conventional and self-ligating brackets: A pilot study

    Directory of Open Access Journals (Sweden)

    Prarthana Bhardwaj

    2017-01-01

    Full Text Available Objective: The objective of this study is to evaluate the clinical efficiency of 0.018″/0.022″ slot self-ligating (SL bracket system (standard and tandem mechanics in terms of rate of alignment by comparing it with a 0.022″ slot conventional ligating appliance system (MBT. Settings and Sample Population: The Department of Orthodontics. Materials and Methods: The pilot study was carried out using randomized controlled trial design. Forty patients having Little's irregularity index (II of 6–15 mm, treated by all first premolars extractions, were randomly allocated to 0.022″ slot conventional ligating bracket system, 0.018″ slot SL bracket system, 0.018″ slot SL bracket system (tandem archwires, 0.022″ slot SL bracket system, and 0.022″ slot SL bracket system (tandem archwires. The rate of alignment for each bracket system was measured from the difference in the II of serial casts taken at pretreatment and at the end of alignment, divided by the number of days between the two measurements. A one-way ANOVA model with post hoc Bonferroni multiple comparison procedures was used to identify intergroup differences. Results: The mean value of alignment efficiency was not found to be statistically significant in any of the five groups using digital models (P = 0.104. Conclusions: Alignment efficiency was not different between SL versus conventional ligating group, the 0.018″ slot versus 0.022″ slot and tandem versus standard mechanics.

  8. Comparison of effectiveness and cost of patent ductus arteriosus device occlusion versus surgical ligation of patent ductus arteriosus.

    Science.gov (United States)

    Zulqarnain, Arif; Younas, Muhammad; Waqar, Tariq; Beg, Ahsan; Asma, Touseef; Baig, Mirza Ahmad Raza

    2016-01-01

    Comparison of effectiveness and cost of transcatheter occlusion of patent ductus arteriosus (PDA) with surgical ligation of PDA. This retrospective comparative study was conducted in the pediatric cardiology department of Ch. Pervaiz Elahi Institute of Cardiology Multan, Pakistan. Data of 250 patients who underwent patent ductus arteriosus (PDA) closure either surgical or trans-catheter closure using SHSMA Occluder having weight >5 kg from April 2012 to October 2015 were included in this study. SPSS version 20 was used for data analysis. Quantitative variables were compared using independent sample t-test. Chi-square test and fishers exact was used for qualitative variables. P-value <0.05 was considered statistically significant. There were one hundred and twenty (120) patients who underwent transcatheter occlusion of PDA using SHSMA occluder (PDA Device Group) and one hundred and thirty (130) patients who underwent surgical ligation of PDA (Surgical Group). Incidence of residual shunting was two (1.5%) in surgical group and 0 (0.0%) in PDA Device group for one month follow up period. There were 4 (3.1%) major complications in surgical group. The rate of blood transfusions were high in surgical group (p-value 0.04). Hospital stay time was significantly less in PDA Device group (P-value <0.001). Total procedural cost was 110695+1054 Pakistani rupees in PDA Device group and 92414+3512 in surgical group (p-value <0.001). The cost of PDA device closure was 16.52% higher than the surgical ligation of PDA. There was no operative mortality. The transcatheter closure of PDA is an effective and less invasive method as compared to the surgical ligation. There is a lower rate of complications and the cost is not much high as compared to surgical PDA ligation.

  9. 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

  10. Efficacy of carvedilol versus propranolol versus variceal band ligation for primary prevention of variceal bleeding.

    Science.gov (United States)

    Abd ElRahim, Ayman Yosry; Fouad, Rabab; Khairy, Marwa; Elsharkawy, Aisha; Fathalah, Waleed; Khatamish, Haytham; Khorshid, Omayma; Moussa, Mona; Seyam, Moataz

    2018-01-01

    Band ligation and propranolol are the current therapies for primary prevention of variceal bleeding. Carvedilol is a rising nonselective beta-blocker used for reducing portal pressure with favorable outcome. The aim of this study to assess the efficacy of carvedilol, propranolol, and band ligation for primary prevention of variceal bleeding based on the effect of each regimen on progression of Child score and portal hypertensive gastropathy after 1 year. The study included 264 cirrhotic patients with medium/large-sized varices who were candidates for primary prophylaxis of variceal bleeding. Patients were randomly divided into three groups: group I: band ligation; group II: propranolol; group III: carvedilol. Group I showed higher success rate of 75 %, followed by group III with 70.2 % and group II with 65.2 %. Risk of bleeding was comparable between the three groups, with group II carrying the highest rate of complications (34.7 %) followed by group III (14.2 %) and finally group I (5.7 %). After 1 year of follow-up, Child score did not improve in any of the studied groups, while portal hypertensive gastropathy significantly increased in group I but decreased in groups II and III. Band ligation is the best treatment option for primary prevention of variceal bleeding with minimal complications. Carvedilol is a good pharmaceutical alternative medicine to propranolol with lesser side-effects. Progress of liver disease as represented by Child score is not affected by any of the primary variceal prophylactic regimens, although medical treatment reduces portal hypertensive gastropathy. Choice of treatment depends on patient will, compliance with treatment, and endoscopist competence.

  11. Fluorogenic dansyl-ligated gold nanoparticles for the detection of sulfur mustard by displacement assay.

    Science.gov (United States)

    Knighton, Richard C; Sambrook, Mark R; Vincent, Jack C; Smith, Simon A; Serpell, Christopher J; Cookson, James; Vickers, Matthew S; Beer, Paul D

    2013-03-21

    The dansyl fluorophore ligated to gold nanoparticles via imidazole and amine groups affords conjugates capable of detecting micromolar concentrations of the chemical warfare agent sulfur mustard by a fluorescence switching 'ON' displacement assay.

  12. 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

  13. Dissecting the hybridization of oligonucleotides to structured complementary sequences.

    Science.gov (United States)

    Peracchi, Alessio

    2016-06-01

    When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Evaluation of force released by deflection of orthodontic wires in conventional and self-ligating brackets.

    Science.gov (United States)

    Higa, Rodrigo Hitoshi; Semenara, Nayara Thiago; Henriques, José Fernando Castanha; Janson, Guilherme; Sathler, Renata; Fernandes, Thais Maria Freire

    2016-01-01

    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. 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 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. Bracket systems provide different degrees of deflection force, with self-ligating brackets showing the highest forces.

  15. A multiplex ligation detection assay for the characterization of Salmonella enterica strains

    DEFF Research Database (Denmark)

    Aarts, Henk J.M.; Vos, Pieter; Larsson, Jonas T.

    2011-01-01

    A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection. The fea...... assessors that support bio-traceability models....

  16. 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

  17. The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

    Science.gov (United States)

    Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

    2017-05-01

    Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Difficulty with cuff deflation of reinforced tracheal tube caused by inflation line occlusion with silk thread ligation and fixation.

    Science.gov (United States)

    Gotoh, Sayoko; Sugiyama, Daisuke; Imai, Eriko; Kawamata, Mikito

    2015-03-05

    A reinforced tracheal tube, ligated with silk threads, was inserted into a tracheostomy orifice and fixed to the skin. The cuff inflation line of the reinforced tracheal tube became occluded. Reinforced 'armoured' tracheal tubes have a spiral of wire embedded into the wall of the tube to give strength and flexibility, and may be sharply bent without compromising the tube lumen. The tracheal cuff attached to the tube is inflated by injecting air through a narrow-diameter tube welded to the outside of the tracheal tube. When a reinforced tracheal tube is ligated and fixed with silk threads, it should be confirmed whether the tracheal tube cuff can be deflated and inflated after fixation. Moreover, because occlusion can be eliminated by removing all silk threads used to ligate a tracheal tube, they should be removed before extubation. 2015 BMJ Publishing Group Ltd.

  19. Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

    Science.gov (United States)

    Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

    2010-01-01

    Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

  20. N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Rosenbergová, Šárka; Ménová, Petra; Páv, Ondřej; Pohl, Radek; Novák, Pavel; Rosenberg, Ivan

    2015-01-01

    Roč. 13, č. 15 (2015), s. 4449-4458 ISSN 1477-0520 R&D Projects: GA ČR GAP207/11/0108; GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * oligonucleotides * solid phase synthesis Subject RIV: CC - Organic Chemistry Impact factor: 3.559, year: 2015

  1. Evaluation of fluorine-18-labeled alkylating agents as potential synthons for the labeling of oligonucleotides

    NARCIS (Netherlands)

    de Vries, EFJ; Vroegh, J; Elsinga, PH; Vaalburg, W

    Six fluorine-18-labeled alkylating agents were selected as potentially suitable synthons for the labeling of antisense oligonucleotides. The selected synthons were evaluated in a model reaction with the monomer adenosine 5'-O-thiomonophosphate. Of these synthons,

  2. Comparative study of friction between metallic and conventional interactive self-ligating brackets in different alignment conditions

    Directory of Open Access Journals (Sweden)

    Sérgio Ricardo Jakob

    2014-06-01

    Full Text Available OBJECTIVE: The aim of this study was to compare the friction between three bracket models: conventional stainless steel (Ovation, Dentsply GAC, self-ligating ceramic (In-Ovation, Denstply GAC and self-ligating stainless steel brackets (In-Ovation R, Dentsply GAC. METHODS: Five brackets were used for each model. They were bonded to an aluminum prototype that allowed the simulation of four misalignment situations (n = 10. Three of these situations occurred at the initial phase (in which a 0.016-in nickel-titanium wire was used: 1. horizontal; 2. vertical; and 3. simultaneous horizontal/vertical. One of the situations occurred at the final treatment phase: 4. no misalignment (in which a 0.019 x 0.025-inch stainless steel rectangular wire was used. The wires slipped through the brackets and friction was measured by a Universal Testing Machine. RESULTS: Analysis of variance followed by Tukey's Test for multiple comparisons (α = 0.05 were applied to assess the results. Significant interaction (p < 0.01 among groups was found. For the tests that simulated initial alignment, Ovation(r bracket produced the highest friction. The two self-ligating models resulted in lower and similar values, except for the horizontal situation, in which In-Ovation C(r showed lower friction, which was similar to the In-Ovation R(r metallic model. For the no misalignment situation, the same results were observed. CONCLUSION: The self-ligating system was superior to the conventional one due to producing less friction. With regard to the material used for manufacturing the brackets, the In-Ovation C(r ceramic model showed less friction than the metallic ones.

  3. PPAR-alpha agonist treatment increases trefoil factor family-3 expression and attenuates apoptosis in the liver tissue of bile duct-ligated rats.

    Science.gov (United States)

    Karakan, Tarkan; Kerem, Mustafa; Cindoruk, Mehmet; Engin, Doruk; Alper, Murat; Akın, Okan

    2013-01-01

    Peroxisome proliferators-activated receptor alpha activation modulates cholesterol metabolism and suppresses bile acid synthesis. The trefoil factor family comprises mucin-associated proteins that increase the viscosity of mucins and help protect epithelial linings from insults. We evaluated the effect of short-term administration of fenofibrate, a peroxisome proliferators activated receptor alpha agonist, on trefoil factor family-3 expression, degree of apoptosis, generation of free radicals, and levels of proinflammatory cytokines in the liver tissue of bile duct-ligated rats. Forty male Wistar rats were randomly divided into four groups: 1 = sham operated, 2 = bile duct ligation, 3 = bile duct-ligated + vehicle (gum Arabic), and 4 = bile duct-ligated + fenofibrate (100 mg/kg/day). All rats were sacrificed on the 7 th day after obtaining blood samples and liver tissue. Liver function tests, tumor necrosis factor-alpha and interleukin 1 beta in serum, and trefoil factor family-3 mRNA expression, degree of apoptosis (TUNEL) and tissue malondialdehyde (malondialdehyde, end-product of lipid peroxidation by reactive oxygen species) in liver tissue were evaluated. Fenofibrate administration significantly reduced serum total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and tumor necrosis factor-alpha and interleukin-1β levels. Apoptosis and malondialdehyde were significantly reduced in the fenofibrate group. Trefoil factor family-3 expression increased with fenofibrate treatment in bile duct-ligated rats. The peroxisome proliferators-activated receptor alpha agonist fenofibrate significantly increased trefoil factor family-3 expression and decreased apoptosis and lipid peroxidation in the liver and attenuated serum levels of proinflammatory cytokines in bile duct-ligated rats. Further studies are needed to determine the protective role of fenofibrate in human cholestatic disorders.

  4. Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Linfeng, E-mail: zhenglinfeng04@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Li Yujie, E-mail: yujieli01@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Han, E-mail: bingowh@hotmail.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhao Jinglong, E-mail: jinglongz@yahoo.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Xifu, E-mail: wangxiechen001@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Hu Yunsheng, E-mail: springmorninghu@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhang Guixiang, E-mail: guixiangzhang@sina.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China)

    2011-05-15

    Purpose: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. Methods: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 {mu}l 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. Results: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups (P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively (P < 0.05). Conclusion: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve

  5. Oligonucleotides with 1,4-dioxane-based nucleotide monomers

    DEFF Research Database (Denmark)

    Madsen, Andreas S; Wengel, Jesper

    2012-01-01

    An epimeric mixture of H-phosphonates 5R and 5S has been synthesized in three steps from known secouridine 1. Separation of the epimers has been accomplished by RP-HPLC, allowing full characterization and incorporation of monomers X and Y into 9-mer oligonucleotides using H-phosphonates building...... blocks 5R and 5S, respectively. A single incorporation of either monomer X or monomer Y in the central position of a DNA 9-mer results in decreased thermal affinity toward both DNA and RNA complements (ΔT(m) = -3.5 °C/-3.5 °C for monomer X and ΔT(m) = -11.0 °C/-6.5 °C for monomer Y). CD measurements do...

  6. 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 ...

  7. 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...

  8. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    OpenAIRE

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-01-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed prelim...

  9. Comparison of infrared coagulation and rubber band ligation for first and second degree haemorrhoids: a randomised prospective clinical trial.

    OpenAIRE

    Templeton, J L; Spence, R A; Kennedy, T L; Parks, T G; Mackenzie, G; Hanna, W A

    1983-01-01

    One hundred and thirty seven previously untreated out-patients with first and second degree haemorrhoids were allocated at random to treatment by infrared coagulation (n=66) or rubber band ligation (n=71). Complete follow up was obtained in 122 patients (60 who had undergone infrared coagulation (group 1), and 62 rubber band ligation (group 2)) at periods from three months to one year after completion of treatment. Infrared coagulation produced a satisfactory outcome in 51 patients (85%): 34 ...

  10. 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

    OBJECTIVE: According to the recent theories on the ovarian cancer origin, any protective effect of tubal ligation may vary with histologic subtype of ovarian cancer. Furthermore, bilateral salpingectomy may represent an opportunity for surgical prevention of serous ovarian cancer. DESIGN: Nationw......OBJECTIVE: According to the recent theories on the ovarian cancer origin, any protective effect of tubal ligation may vary with histologic subtype of ovarian cancer. Furthermore, bilateral salpingectomy may represent an opportunity for surgical prevention of serous ovarian cancer. DESIGN...... 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...

  11. Short Oligonucleotides Aligned in Stretched Humid Matrix: Secondary DNA Structure in Poly(vinyl alcohol) Environment

    KAUST Repository

    Hanczyc, Piotr; Å kerman, Bjö rn; Nordé n, Bengt

    2012-01-01

    ) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong

  12. Bilateral tubal ligation in a rural hospital in the Niger Delta, Nigeria ...

    African Journals Online (AJOL)

    Background and Objective: To document bilateral tubal ligation (BTL) rate and highlight the need to improve on the rates. Materials and Methods: A retrospective review of BTLs done in a five-year period from January 2000 to December 2004 constituted the study group. Results: There were a total of 103 BTLs, 58 were ...

  13. Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore.

    Science.gov (United States)

    Kühnemund, M; Nilsson, M

    2015-05-15

    Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Effects of CD44 Ligation on Signaling and Metabolic Pathways in Acute Myeloid Leukemia

    KAUST Repository

    Madhoun, Nour Y.

    2017-01-01

    Acute myeloid leukemia (AML) is characterized by a blockage in the differentiation of myeloid cells at different stages. CD44-ligation using anti-CD44 monoclonal antibodies (mAbs) has been shown to reverse the blockage of differentiation

  15. Evaluation of the Friction of Self-Ligating and Conventional Bracket Systems

    Science.gov (United States)

    Tecco, Simona; Di Iorio, Donato; Nucera, Riccardo; Di Bisceglie, Beatrice; Cordasco, Giancarlo; Festa, Felice

    2011-01-01

    Objectives: This in vitro study evaluated the friction (F) generated by aligned stainless steel (SS) conventional brackets, self-ligating Damon MX© brackets (SDS Ormco, Glendora, California, USA), Time3© brackets (American Orthodontics, Sheboygan, Wisconsin, USA), Vision LP© brackets (American Orthodontics), and low-friction Slide© ligatures (Leone, Firenze, Italy) coupled with various SS, nickel-titanium (NiTi), and beta-titanium (TMA) archwires. Methods: All brackets had a 0.022-inch slot, and the orthodontic archwires were 0.014-inch, 0.016-inch, 0.014×0.025-inch, 0.018×0.025-inch, and 0.019×0.025-inch NiTi; 0.017×0.025-inch TMA; and 0.019×0.025-inch SS. Each bracket-archwire combination was tested 10 times. In the test, 10 brackets of the same group were mounted in alignment on a metal bar. The archwires moved through all the 10 brackets at a crosshead speed of 0.5 mm/min (each run lasted approximately 5 min). The differences among 5 groups of brackets were analyzed through the Kruskal-Wallis test, and a Mann-Whitney test was calculated as post hoc analysis. The P value was set at 0.05. Results: Coupled with 0.014-inch NiTi and 0.016-inch NiTi, Victory Series© brackets generated the greatest F, while Damon MX© and Vision LP© brackets generated the lowest (Pbrackets and Slide© ligatures. Coupled with all the rectangular archwires, Victory Series© brackets, Slide© ligatures, and Vision LP© self-ligating brackets generated significantly lower F than did Time3© and Damon MX© self-ligating brackets (Pbrackets are a family of brackets that, in vitro, can generate different levels of F when coupled with thin or thick, rectangular, or round archwires. Clinical conclusions based on our results are not possible due to the limitations of the experimental conditions. PMID:21769273

  16. Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation

    Science.gov (United States)

    Ziegler, Mary E.; Souda, Puneet; Jin, Yi-Ping; Whitelegge, Julian P.; Reed, Elaine F.

    2012-01-01

    Background Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood. Methodology and Principal Findings The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin. Conclusions/Significance Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types

  17. 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...

  18. 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

  19. Biphasic activation of liver regeneration-associated signals in an early stage after portal vein branch ligation

    International Nuclear Information System (INIS)

    Yokoyama, Shinya; Yokoyama, Yukihiro; Kawai, Toru; Kobayashi, Satoshi; Nagino, Masato; Oda, Koji; Nimura, Yuji; Sokabe, Masahiro

    2006-01-01

    At an early stage in liver regeneration, a variety of factors including transcriptional factors, proinflammatory cytokines, and proto-oncogenes are activated or expressed. However, these responses are affected by surgical stress in the conventional portal vein branch ligation model (PVL). We sought to determine the net activation of early regeneration-associated signals after PVL using a newly developed non-surgical stress PVL model. Male Wistar rats were assigned into two groups, a stress-PVL (sPVL) model with laparotomy and portal vein branch ligation, and a non-stress-PVL (nsPVL) one subjected to portal vein branch ligation 1 week after laparotomy in which the effects of surgical stress were subsided. The activation of transcriptional factors and expression of immediate early genes were analyzed at an early time point (within 24 h) by Western blotting and RT-PCR, respectively. A monophasic upregulation of nuclear factor-κB (NF-κB) and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3) peaked at 3 h after sPVL was observed. In contrast, the expression pattern of these factors was biphasic (first peak at 0.5-1 h, second peak at 3-6 h) in nsPVL group. The expression pattern of immediate early genes showed a similar trend between sPVL and nsPVL. cDNA array analysis for the non-ligated lobe at 2 h after PVL revealed a much higher gene activation in sPVL than in nsPVL. These results indicate that previously observed activation pattern of regeneration-associated signals after PVL is significantly affected by the effect of laparotomy and our results using nsPVL model may more accurately represent liver regeneration-associated signal pattern

  20. 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.

  1. Design and analysis of effects of triplet repeat oligonucleotides in cell models for myotonic dystrophy

    NARCIS (Netherlands)

    Gonzalez-Barriga, A.; Mulders, S.A.M.; Giessen, J. van der; Hooijer, J.D.; Bijl, S.; Kessel, I.D.G. van; Beers, J. van; Deutekom, J.C. van; Fransen, J.A.M.; Wieringa, B.; Wansink, D.G.

    2013-01-01

    Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a

  2. Synthetic study on prion protein fragments using a SPPS and native chemical ligation

    Czech Academy of Sciences Publication Activity Database

    Zawada, Z.; Šebestík, Jaroslav; Bednárová, Lucie; Bouř, Petr; Hlaváček, Jan; Stibor, Ivan

    2009-01-01

    Roč. 37, Suppl. 1 (2009), s. 44-44 ISSN 0939-4451. [International Congress on Amino Acids, Peptides and Proteins /11./. 03.08.2009-07.08.2009, Vienna] Institutional research plan: CEZ:AV0Z40550506 Keywords : prion protein * SPPS * native chemical ligation * fragments Subject RIV: CC - Organic Chemistry

  3. Treatment of grade 2 and 3 hemorrhoids with Doppler-guided hemorrhoidal artery ligation

    NARCIS (Netherlands)

    Wallis de Vries, B. M.; van der Beek, E. S. J.; de Wijkerslooth, L. R. H.; van der Zwet, W. C.; van der Hoeven, J. A. B.; Eeftinck Schattenkerk, M.; Eddes, E. H.

    2007-01-01

    We evaluated the results of the Doppler-guided hemorrhoidal arterial ligation (DG-HAL) method in the management of symptomatic grade 2 and 3 hemorrhoids. Between June 2005 and March 2006, 110 consecutive patients with symptomatic grade 2 and 3 hemorrhoids according to the DG-HAL method were treated.

  4. The role of prophylactic internal iliac artery ligation in abnormally invasive placenta undergoing caesarean hysterectomy: a randomized control trial.

    Science.gov (United States)

    Hussein, Ahmed M; Dakhly, Dina Mohamed Refaat; Raslan, Ayman N; Kamel, Ahmed; Abdel Hafeez, Ali; Moussa, Manal; Hosny, Ahmed Samir; Momtaz, Mohamed

    2018-04-25

    To identify the role of bilateral internal iliac artery (IIA) ligation on reducing blood loss in abnormally invasive placenta (AIP) undergoing caesarean hysterectomy. In this parallel-randomized control trial, 57 pregnant females with ultrasound features suggestive of AIP were enrolled. They were randomized into two groups; IIA group (n = 29 cases) performed bilateral IIA ligation followed by caesarean hysterectomies, while Control group (n = 28 cases) underwent caesarean hysterectomy only. The main outcome was the difference in the estimated intraoperative blood loss between the two groups. There was no significant difference between the two groups regarding the intraoperative estimated blood loss (1632 ± 804 versus 1698 ± 1251, p value .83). The operative procedure duration (minutes) (223 ± 66 versus 171 ± 41.4, p value .001) varied significantly between the two groups. Bilateral internal iliac artery ligation, in cases of AIP undergoing caesarean hysterectomy, is not recommended for routine practice to minimize blood loss intraoperatively.

  5. Swimming performance, venous oxygen tension and cardiac performance of coronary-ligated rainbow trout, Oncorhynchus mykiss, exposed to progressive hypoxia

    DEFF Research Database (Denmark)

    Steffensen, J F; Farrell, A P

    1998-01-01

    We performed in vivo studies to examine the idea that cardiac work is impaired in rainbow trout (Oncorhynchus mykiss) below a certain venous PO2 threshold. We hypothesized that coronary-ligated fish, swimming continuously at a reasonably high water velocity (1.5 body lengths x s(-1)) and exposed...... to progressive hypoxia, would fatigue at higher venous PO2 and ambient water PO2 compared with sham-operated fish. However, we found that both the lowest venous PO2 that supported hypoxic swimming (9.9 torr for coronary-ligated fish and 11.1 torr for sham-operated fish) and the venous PO2 at fatigue (7.8 torr...... and 8.6 torr, respectively) were the same for coronary-ligated and sham-operated fish. Also, both groups quit swimming at the same water PO2 heart rate and hematocrit. Nevertheless, significant differences in cardiac performance did exist between the two groups. Whereas ventral aortic blood pressure...

  6. Evaluation of the force generated by gradual deflection of orthodontic wires in conventional metallic, esthetic, and self-ligating brackets.

    Science.gov (United States)

    Francisconi, Manoela Fávaro; Janson, Guilherme; Henriques, José Fernando Castanha; Freitas, Karina Maria Salvatore de

    2016-01-01

    The purpose of this study was to evaluate the deflection forces of Nitinol orthodontic wires placed in different types of brackets: metallic, reinforced polycarbonate with metallic slots, sapphire, passive and active self-ligating, by assessing strength values variation according to gradual increase in wire diameter and deflection and comparing different combinations in the different deflections. Specimens were set in a clinical simulation model and evaluated in a Universal Testing Machine (INSTRON 3342), using the ISO 15841 protocol. Data were subjected to One-way ANOVA, followed by Tukey tests (pbrackets presented the most similar behavior to each other. For conventional brackets there was no consistent behavior for any of the deflections studied. Self-ligating brackets presented the most consistent and predictable results while conventional brackets, as esthetic brackets, showed very different patterns of forces. Self-ligating brackets showed higher strength in all deflections when compared with the others, in 0.020-inch wires.

  7. Surgical Ligation of Patent Ductus Arteriosus in Very-low-birth-weight Premature Infants in the Neonatal Intensive Care Unit

    Directory of Open Access Journals (Sweden)

    Yu-Chen Ko

    2009-01-01

    Full Text Available This study reported our experience of bedside patent ductus arteriosus (PDA ligation for prematurity in the neonatal intensive care unit (NICU. Between April 1992 and March 2006, 41 very-low-birth-weight premature infants underwent PDA ligation in the NICU. There were 18 male and 23 female infants. The mean gestational age and birth weight were 26.9 weeks and 900.9 g, respectively. Preoperatively, 25 infants were ventilator-dependent. After operation, there were five deaths caused by complications of prematurity. Surgical complications occurred in four and all recovered well after treatment. Twenty preoperatively intubated babies survived and were extubated at 21.6 ± 12.7 days postoperatively. In conclusion, bedside PDA ligation in the NICU is safe and effective. It can avoid transportation of critically ill, very small infants. We suggest surgical closure as the primary treatment in very-low-birth-weight infants who are ventilator-dependent to avoid the possible complications of indomethacin and prolonged intubation.

  8. Ligation of the Rectum with an Extracorporeal Sliding Knot Facilitating Laparoscopic Cross-Stapling

    DEFF Research Database (Denmark)

    Bulut, Orhan

    2013-01-01

    : The extracorporeal ligation of the rectum just proximal to the cut end of the rectum before applying the linear stapling stapler facilitates the procedure and requires only a few firings of the stapler during the laparoscopic rectal resections. Results: Ten patients with a median age of 72 years underwent rectal...

  9. Microbial complexes levels in conventional and self-ligating brackets.

    Science.gov (United States)

    Bergamo, Ana Zilda Nazar; Nelson-Filho, Paulo; Andrucioli, Marcela Cristina Damião; do Nascimento, Cássio; Pedrazzi, Vinícius; Matsumoto, Mírian Aiko Nakane

    2017-05-01

    The aims were to evaluate the levels of bacterial species in saliva and in situ and to assess whether the design of brackets influences the risk of developing periodontal disease. Twenty patients (13.3 mean age) were bonded with self-ligating brackets and a conventional bracket. Saliva was collected before bonding and 30 and 60 days after bonding. One sample of each bracket was removed 30 and 60 days after bonding. The analysis was determined by checkerboard DNA-DNA hybridization. The data was evaluated by the non-parametric test. A significant increase in the levels of bacterial species in the saliva occurred in 15 of the 22 analyzed species. The self-ligating brackets presented the highest incidence percentages for the orange and red complexes 60 days after bonding. In situ analyses showed different patterns according to the bracket design. The levels of Campylobacter rectus showed significant differences (p = 0.011) 60 days after bonding among the three brackets; the highest values were observed in the In-Ovation®R bracket. The bracket design seems to influence the levels of bacterial species involved in periodontal disease. Considering the wide variety of bacterial species, additional studies are needed to aid in the establishment of effective protocols to prevent the development of periodontal disease during orthodontic treatment. A dynamic alteration in the oral microbiota may lead to inflammatory reactions in the supporting soft and hard tissues. The different types of brackets interfere with bacterial adherence. Bracket design should be considered in orthodontic treatment.

  10. Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges.

    Science.gov (United States)

    Jensen, Michael A; Davis, Ronald W

    2018-03-27

    There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

  11. Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Rull, Jordi [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France); CEA, iRTSV, LCBM, Grenoble 38054 (France); CNRS, UMR 5249, Grenoble (France); Nonglaton, Guillaume, E-mail: guillaume.nonglaton@cea.fr [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France); Costa, Guillaume; Fontelaye, Caroline [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France); Marchi-Delapierre, Caroline; Ménage, Stéphane [Université Grenoble Alpes, Grenoble F38000 (France); CEA, iRTSV, LCBM, Grenoble 38054 (France); CNRS, UMR 5249, Grenoble (France); Marchand, Gilles [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France)

    2015-11-01

    Graphical abstract: - Highlights: • First example of grafting of 3,4-epoxybutyltrimethoxysilane (EBTMOS) onto silicon oxide by supercritical fluid deposition. • Extraordinary efficiency of the supercritical fluid deposition for the grafting of the EBTMOS compared with the conventional solution or vapor phase methodologies. • Demonstration of the efficiency of this functionalization process for the immobilization of amino-modified oligonucleotides. - Abstract: The functionalization of silicon oxide based substrates using silanes is generally performed through liquid phase methodologies. These processes involve a huge quantity of potentially toxic solvents and present some important disadvantages for the functionalization of microdevices or porous materials, for example the low diffusion. To overcome this drawback, solvent-free methodologies like molecular vapor deposition (MVD) or supercritical fluid deposition (SFD) have been developed. In this paper, the deposition process of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon oxide using supercritical carbon dioxide (scCO{sub 2}) as a solvent is studied for the first time. The oxirane ring of epoxy silanes readily reacts with amine group and is of particular interest for the grafting of amino-modified oligonucleotides or antibodies for diagnostic application. Then the ability of this specific EBTMOS layer to react with amine functions has been evaluated using the immobilization of amino-modified oligonucleotide probes. The presence of the probes is revealed by fluorescence using hybridization with a fluorescent target oligonucleotide. The performances of SFD of EBTMOS have been optimized and then compared with the dip coating and molecular vapor deposition methods, evidencing a better grafting efficiency and homogeneity, a lower reaction time in addition to the eco-friendly properties of the supercritical carbon dioxide. The epoxysilane layers have been characterized by surface enhanced ellipsometric

  12. Colorimetry and SERS dual-mode detection of telomerase activity: combining rapid screening with high sensitivity.

    Science.gov (United States)

    Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Hu, Guohua; Liu, Min; Chen, Peng; Cui, Yiping

    2014-01-01

    As an important biomarker and therapeutic target, telomerase has attracted considerable attention concerning its detection and monitoring. Here, we present a colorimetry and surface enhanced Raman scattering (SERS) dual-mode telomerase activity detection method, which has several distinctive advantages. First, colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Second, the employment of SERS technique results in greatly improved detection sensitivity. Third, the combination of colorimetry and SERS into one detection system can ensure highly efficacious and sensitive screening of numerous samples. Besides, the avoidance of polymerase chain reaction (PCR) procedures further guarantees fine reliability and simplicity. Generally, the presented method is realized by an "elongate and capture" procedure. To be specific, gold nanoparticles modified with Raman molecules and telomeric repeat complementary oligonucleotide are employed as the colorimetric-SERS bifunctional reporting nanotag, while magnetic nanoparticles functionalized with telomerase substrate oligonucleotide are used as the capturing substrate. Telomerase can synthesize and elongate telomeric repeats onto the capturing substrate. The elongated telomeric repeats subsequently facilitate capturing of the reporting nanotag via hybridization between telomeric repeat and its complementary strand. The captured nanotags can cause a significant difference in the color and SERS intensity of the magnetically separated sediments. Thus both the color and SERS can be used as indicators of the telomerase activity. With fast screening ability and outstanding sensitivity, we anticipate that this method would greatly promote practical application of telomerase-based early-stage cancer diagnosis.

  13. TIA-1 RRM23 binding and recognition of target oligonucleotides.

    Science.gov (United States)

    Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

    2017-05-05

    TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Application of hierarchical oligonucleotide primer extension (HOPE) to assess relative abundances of ammonia- and nitrite-oxidizing bacteria

    KAUST Repository

    Scarascia, Giantommaso; Cheng, Hong; Harb, Moustapha; Hong, Pei-Ying

    2017-01-01

    for ensuring the efficiency of nitrification in water treatment systems. Hierarchical oligonucleotide primer extension (HOPE), previously developed to rapidly quantify relative abundances of specific microbial groups of interest, was applied in this study

  15. Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals

    Directory of Open Access Journals (Sweden)

    Punit P Seth

    2012-01-01

    Full Text Available We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs modified with α-L-locked nucleic acid (LNA and related modifications targeting phosphatase and tensin homologue (PTEN messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3′- and 5′-flanks with R-5′-Me-α-L-LNA but not R-6′-Me- or 3′-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5′-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.

  16. 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.

  17. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  18. Antiproliferation effects of an androgen receptor triple-helix forming oligonucleotide on prostate cancer cells

    International Nuclear Information System (INIS)

    Zhang Yong; Chen Weizhen; Xie Yao; Gao Jinhui

    2005-01-01

    Objective: To provide experimental basis for antigene radiation therapy through exploring the effects of antigene strategy on androgen receptor (AR) expression and proliferation of prostate cancer cells. Methods: The triple-helix forming oligonucleotide (TFO) targeting 2447-2461nt of AR cDNA was designed and transfected LNCaP prostate cancer cells with liposome. 24-72 h after transfection, the cellular proliferation was detected by 3 H-thymidine (TdR) incorporation test, the expression of AR gene was examined by reverse transcription-polymerase chain reaction (RT-PCR) and expression of AR protein was performed by radioligand binding assay. The results of TFO were compared with antisense oligonucleotide (ASON). Results: At all time points, the AR expression levels in TFO group were markedly lower than that of ASON group (P<0.05). The inhibitory rate of TFO for cellular proliferation was significantly higher than that of ASON (P<0.05). Conclusion: The TFO was a potent inhibitor for AR expression and cell proliferation of LNCaP cells , and could be used in antigene radiotherapy. (authors)

  19. Usefulness of real-time elastography strain ratio in the assessment of bile duct ligation-induced liver injury and the hepatoprotective effect of chitosan: an experimental animal study.

    Science.gov (United States)

    Dudea, Marina; Clichici, Simona; Olteanu, Diana Elena; Nagy, Andras; Cucoş, Maria; Dudea, Sorin

    2015-01-01

    The purpose of the study described here was to evaluate the usefulness of the elastographic strain ratio in the assessment of liver changes in an experimental animal setting and the hepatoprotective effects of chitosan. Ultrasonography and Strain Ratio calculation were performed before and after bile duct ligation (BDL) in three groups of Wistar albino rats (n = 10 animals per group): (i) rats subjected to bile duct ligation only; (ii) rats subjected to bile duct ligation and administered chitosan for 14 d; (iii) rats subjected to bile duct ligation and administered chitosan for 7 d. The results were compared with the laboratory data and pathologic findings. Strain ratios revealed an increase in liver stiffness after bile duct ligation (p liver response to injury. To the best of our knowledge, this is the first study reporting on the usefulness of the sonoelastographic liver-to-kidney strain ratio in assessing the effects of experimentally induced liver lesions. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  20. Distribution of Sulfate-Reducing Bacteria, O2, and H2s in Photosynthetic Biofilms Determined by Oligonucleotide Probes and Microelectrodes Rid A-1977-2009

    DEFF Research Database (Denmark)

    RAMSING, NB; KUHL, M.; JØRGENSEN, BB

    1993-01-01

    The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen....... Fluorescent-dye-conjugated oligonucleotides were used as ''phylogenetic'' probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled...... with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed...