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Sample records for rna-homology shows triplex

  1. 5. EXTERIOR OF TRIPLEX COTTAGE ROOF SHOWING MANVILLE COMPOSITION SHINGLES, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. EXTERIOR OF TRIPLEX COTTAGE ROOF SHOWING MANVILLE COMPOSITION SHINGLES, POURED CONCRETE CHIMNEYS, AND TRANSLUCENT PLASTIC COVERING OVER WALKWAY AT REAR OF HOUSE (PHOTO LEFT). VIEW TO NORTHWEST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  2. 25S ribosomal RNA homologies of basidiomycetous yeasts: taxonomic and phylogenetic implications

    NASA Technical Reports Server (NTRS)

    Baharaeen, S.; Vishniac, H. S.

    1984-01-01

    Genera, families, and possibly orders of basidiomycetous yeasts can be defined by 25S rRNA homology and correlated phenotypic characters. The teleomorphic genera Filobasidium, Leucosporidium, and Rhodosporidium have greater than 96 relative binding percent (rb%) intrageneric 25S rRNA homology and significant intergeneric separation from each other and from Filobasidiella. The anamorphic genus Cryptococcus can be defined by morphology (monopolar budding), colony color, and greater than 75 rb% intrageneric homology; Vanrija is heterogeneous. Agaricostilbum (Phragmobasidiomycetes, Auriculariales), Hansenula (Ascomycotera, Endomycota), Tremella (Phragmobasidiomycetes, Tremellales), and Ustilago (Ustomycota, Ustilaginales) appear equally unrelated to the Cryptococcus, Filobasidiella, and Rhodosporidium spp. used as probes. The Filobasidiaceae and Sporidiaceae, Filobasidiales and Sporidiales, form coherent homology groups which appear to have undergone convergent 25S rRNA evolution, since their relatedness is much greater than that indicated by 5S rRNA homology. Ribosomal RNA homologies do not appear to measure evolutionary distance.

  3. 2. OVERVIEW OF TRIPLEX COTTAGE IN POOLE POWERHOUSE SETTING. TRIPLEX ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. OVERVIEW OF TRIPLEX COTTAGE IN POOLE POWERHOUSE SETTING. TRIPLEX COTTAGE IS VISIBLE AT PHOTO CENTER LEFT. POOLE POWERHOUSE IS ADJACENT TRIPLEX COTTAGE AT PHOTO CENTER RIGHT. SWITCHRACKS ARE VISIBLE ADJACENT TO POWERHOUSE BUILDING. VIEW TO SOUTH. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  4. G-triplex structure and formation propensity

    PubMed Central

    Cerofolini, Linda; Amato, Jussara; Giachetti, Andrea; Limongelli, Vittorio; Novellino, Ettore; Parrinello, Michele; Fragai, Marco; Randazzo, Antonio; Luchinat, Claudio

    2014-01-01

    The occurrence of a G-triplex folding intermediate of thrombin binding aptamer (TBA) has been recently predicted by metadynamics calculations, and experimentally supported by Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) data collected on a 3′ end TBA-truncated 11-mer oligonucleotide (11-mer-3′-t-TBA). Here we present the solution structure of 11-mer-3′-t-TBA in the presence of potassium ions. This structure is the first experimental example of a G-triplex folding, where a network of Hoogsteen-like hydrogen bonds stabilizes six guanines to form two G:G:G triad planes. The G-triplex folding of 11-mer-3′-t-TBA is stabilized by the potassium ion and destabilized by increasing the temperature. The superimposition of the experimental structure with that predicted by metadynamics shows a great similarity, with only significant differences involving two loops. These new structural data show that 11-mer-3′-t-TBA assumes a G-triplex DNA conformation as its stable form, reinforcing the idea that G-triplex folding intermediates may occur in vivo in human guanine-rich sequences. NMR and CD screening of eight different constructs obtained by removing from one to four bases at either the 3′ and the 5′ ends show that only the 11-mer-3′-t-TBA yields a relatively stable G-triplex. PMID:25378342

  5. Accumulation of poly(3-hydroxybutyrate) from octanoate in different pseudomonas belonging to the rRNA homology group I.

    PubMed

    Diard, Stéphane; Carlier, Jean-Philippe; Ageron, Elisabeth; Grimont, Patrick A D; Langlois, Valérie; Guérin, Philippe; Bouvet, Odile M M

    2002-08-01

    It is admitted that one of the characteristics of pseudomonads is their inability to accumulate poly(3-hydroxybutyrate). In this paper, we show that poly(3-hydroxyoctanoate) synthesis is restricted to Pseudomonas rRNA homology group I, which includes both fluorescent and nonfluorescent species. However, within the genus Pseudomonas, the P. aeruginosa complex can be subdivided into two groups: the "P. aeruginosa group", which includes P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, produce poly(3-hydroxyoctanoate) from octanoate and the "P. oleovorans group" which includes the type strain of P. oleovorans, P. pseudoalcaligenes and two Pseudomonas sp., produce poly(3-hydroxybutyrate) during cultivation on octanoate. Strain GPo1 (ATCC 29347) formely identified as P. oleovorans and known to produce various medium-side-chain PHAs such as poly(3-hydroxyoctanoate) has been reclassified in the P. putida complex. PMID:12353870

  6. Quantitative analysis of the ion-dependent folding stability of DNA triplexes

    NASA Astrophysics Data System (ADS)

    Chen, Gengsheng; Chen, Shi-Jie

    2011-12-01

    A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-base DNA triplex and a pair of interacting 14-base triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann (PB) equation. The improvement is more significant for a triplex, which has a higher charge density than a duplex. This is possibly due to the higher ion concentration around the triplex and hence a stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induce an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.

  7. DNA Triplexes That Bind Several Cofactor Molecules.

    PubMed

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA. PMID:26561335

  8. Integrated hybrid silicon triplexer.

    PubMed

    Chang, Hsu-Hao; Kuo, Ying-hao; Jones, Richard; Barkai, Assia; Bowers, John E

    2010-11-01

    We demonstrate an integrated triplexer on silicon with a compact size of 1mm by 3.5mm by utilizing a selective area wafer bonding technique. The wavelength demultiplexer on the triplexer chip successfully separates signals at wavelengths of 1310 nm, 1490 nm and 1550 nm with more than 10 dB extinction ratio. The measured 3 dB bandwidth of the integrated laser and photodetectors are 2 GHz and 16 GHz, respectively. Open eye diagrams are also measured for the integrated photodetector up to 12.5 GHz PRBS inputs. PMID:21164734

  9. Solution structure of a dsDNA:LNA triplex

    PubMed Central

    Sørensen, Jesper J.; Nielsen, Jakob T.; Petersen, Michael

    2004-01-01

    We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5′-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19°C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1. PMID:15550567

  10. Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH.

    PubMed

    Torigoe, Hidetaka; Nakagawa, Osamu; Imanishi, Takeshi; Obika, Satoshi; Sasaki, Kiyomi

    2012-04-01

    Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo. PMID:22245184

  11. Spectral and electrochemical detection of protonated triplex formation by a small-molecule anticancer agent

    NASA Astrophysics Data System (ADS)

    Feng, Lingyan; Li, Xi; Peng, Yinghua; Geng, Jie; Ren, Jinsong; Qu, Xiaogang

    2009-10-01

    Triplex helical formation has been the focus of considerable interest because of possible applications in developing new molecular biology tools as well as therapeutic agents and the possible relevance of H-DNA structures in biology system. We report here that a small-molecule anticancer agent, coralyne, has binding preference to the less stable protonated triplex d(C +-T) 6:d(A-G) 6·d(C-T) 6 over duplex d(A-G) 6·d(C-T) 6 and shows different spectral and electrochemical characteristics when binding to triplex and duplex DNA, indicating that electrochemical technique can detect the less stable protonated triplex formation.

  12. Studying RNA Homology and Conservation with Infernal: From Single Sequences to RNA Families.

    PubMed

    Barquist, Lars; Burge, Sarah W; Gardner, Paul P

    2016-01-01

    Emerging high-throughput technologies have led to a deluge of putative non-coding RNA (ncRNA) sequences identified in a wide variety of organisms. Systematic characterization of these transcripts will be a tremendous challenge. Homology detection is critical to making maximal use of functional information gathered about ncRNAs: identifying homologous sequence allows us to transfer information gathered in one organism to another quickly and with a high degree of confidence. ncRNA presents a challenge for homology detection, as the primary sequence is often poorly conserved and de novo secondary structure prediction and search remain difficult. This unit introduces methods developed by the Rfam database for identifying "families" of homologous ncRNAs starting from single "seed" sequences, using manually curated sequence alignments to build powerful statistical models of sequence and structure conservation known as covariance models (CMs), implemented in the Infernal software package. We provide a step-by-step iterative protocol for identifying ncRNA homologs and then constructing an alignment and corresponding CM. We also work through an example for the bacterial small RNA MicA, discovering a previously unreported family of divergent MicA homologs in genus Xenorhabdus in the process. © 2016 by John Wiley & Sons, Inc. PMID:27322404

  13. A novel fluorescent reagent for recognition of triplex DNA with high specificity and selectivity.

    PubMed

    Chen, Zongbao; Zhang, Huimi; Ma, Xiaoming; Lin, Zhenyu; Zhang, Lan; Chen, Guonan

    2015-11-21

    A fluorescent agent (DMT) was screened for recognizing triplex DNA with a specific and selective characteristic, which was embedded into the triplex DNA structure. The triplex DNA was firstly formed by a complementary target sequence through two distinct and sequential events. The conditions including pH and hybridization time, fluorescent agent concentration and embedding time were optimized in the experiment. Under the optimum conditions, the fluorescence signal was enhanced up to 9-fold in comparison with the DMT embedding into the ssDNA, dsDNA and G-quadruplexes. Under the same fluorescence conditions, the changes of the fluorescence signal were also investigated by several kinds of base mismatched DNAs in the experiment. The results showed that our biosensor provided excellent discrimination efficiency toward the perfectly mismatched target DNA with no formation of triplex DNA. We preliminarily deduced the mechanism of the fluorescent reagent for recognizing triplex DNA with high specificity and selectivity. PMID:26456316

  14. A new triplex STR system without irregular alleles by silver staining and its potential application to forensic analysis.

    PubMed

    Yoshimoto, T; Yamamoto, T; Uchihi, R; Tamaki, K; Huang, X L; Mizutani, M; Tanaka, M; Armour, J A; Katsumata, Y

    2001-05-01

    In order to increase the discriminating power of DNA analysis in forensic science, we devised a new triplex STR system using three novel STR loci we previously reported, D14S299 (wglc5), D15S233 (wgldl), and 9q2h2. We designated this system a CDH triplex system. The CDH triplex system showed a high discriminating power, especially in Caucasians. This system is composed of three STR loci showing only regular tetranucleotide repeat alleles. We easily enlarged the databases mainly of Japanese, using this system, and compared them with those of Caucasian and Chinese. This CDH triplex system therefore appears to be useful for forensic practice. PMID:11372981

  15. Triplex-forming twisted intercalating nucleic acids (TINAs): design rules, stabilization of antiparallel DNA triplexes and inhibition of G-quartet-dependent self-association.

    PubMed

    Doluca, Osman; Boutorine, Alexandre S; Filichev, Vyacheslav V

    2011-10-17

    The majority of studies on DNA triple helices have been focused on pH-sensitive parallel triplexes with Hoogsteen CT-containing third strands that require protonation of cytosines. Reverse Hoogsteen GT/GA-containing antiparallel triplex-forming oligonucleotides (TFOs) do not require an acidic pH but their applicability in triplex technology is limited because of their tendency to form undesired highly stable aggregates such as G-quadruplexes. In this study, G-rich oligonucleotides containing 2-4 insertions of twisted intercalating nucleic acid(TINA) monomers are demonstrated to disrupt the formation of G-quadruplexes and form stable antiparallel triplexes with target DNA duplexes. The structure of TINA-incorporated oligonucleotides was optimized, the rules of their design were established and the optimal triplex-forming oligonucleotides were selected. These oligonucleotides show high affinity towards a 16 bp homopurine model sequence from the HIV-1 genome; dissociation constants as low as 160 nM are observed whereas the unmodified TFO does not show any triplex formation and instead forms an intermolecular G-quadruplex with T(m) exceeding 90°C in the presence of 50 mM NaCl. Here we present a set of rules that help to reach the full potential of TINATFOs and demonstrate the effect of TINA on the formation and stability of triple helical DNA. PMID:23106083

  16. Triplex glue by synthesizing conjugated flexible intercalators.

    PubMed

    Pedersen, Erik B; Osman, Amany M A; Globisch, Daniel; Paramasivam, Manikandan; Cogoi, Susanna; Bomholt, Niels; Jørgensen, Per T; Xodo, Luigi E; Filichev, Vyacheslav V

    2008-01-01

    Bulge insertions of conjugated intercalators into the DNA triplex structure are found to give a dramatic contribution to the triplex stability. On the other hand insertions of conjugated intercalators are found to diminish quadruplex structures and in this way breaking down the self association of G-rich oligonucleotides under physiologically potassium ion conditions. A large number of intercalators are described here and they all result in dramatic increases of thermal stability of the corresponding triplexes. Another interesting aspect of conjugated intercalators is their use for assembling alternate strand triplexes. Targeting of neighbouring purine sequences on each their strand in the duplex DNA is a challenge for the 5'- 5' connectivity of the TFOs because of a large distance between the 5'-ends. The intercalator approach offers a linkage with the proper combination of flexibility and rigidity to produce alternate strand triplexes with higher stability than a similar wild type triplex of the same total length. PMID:18776241

  17. A study of the non-covalent interaction between flavonoids and DNA triplexes by electrospray ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Wan, Cuihong; Cui, Meng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2009-06-01

    The binding interactions of 22 flavonoids (9 aglycones and 13 glycosides) with DNA triplexes were investigated using electrospray ionization mass spectrometry (ESI-MS). The results revealed that the hydroxyl positions of aglycones, the locations and numbers of saccharide, as well as the aglycone skeletons play roles in the triplex-binding properties of flavonoids. The presence of 3-OH, or 3'-OH, or replacement of 4'-OH with methoxy group in aglycones decreased the fraction of bound DNA sharply. Flavonoid glycosides exhibit higher binding affinities towards the DNA triplexes than their aglycone counterparts. Glycosylations of flavones at the 8-C position and isoflavones at the 7-O position show higher binding affinities than those on the other positions of ring A of aglycones. Glycosylation with a disaccharide on C3 position of flavonol results in higher binding affinity than that with monosaccharide. Flexibility of the ring B is favorable for its interaction with DNA triplex. According to sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments, glycosylation and non-planarity of flavonoid aglycones lead to different dissociation pathways of the flavonoid/triplex complexes. The differences between dissociation patterns suggest different DNA-binding modes or DNA-binding affinities. Although the exact binding geometry of the flavonoid-triplex complexes cannot be specified, the results may be helpful for understanding the triplex-binding properties of flavonoids and give a clue to design of triplex-binding ligands.

  18. XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    PubMed Central

    Kaushik Tiwari, Meetu; Rogers, Faye A.

    2013-01-01

    DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation. PMID:23913414

  19. Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

    PubMed Central

    Willkomm, Dagmar K.; Minnerup, Jens; Hüttenhofer, Alexander; Hartmann, Roland K.

    2005-01-01

    By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites. PMID:15814812

  20. Novel design of bi-directional triplexer based on PLC

    NASA Astrophysics Data System (ADS)

    Xu, Chenglin; Shen, Linping; Zhou, Dong; Huang, Wei-Ping; Hong, Jin

    2005-09-01

    As the development of the technology, fiber-to-the-home (FTTH) becomes a feasible solution to meet the increasing demand on bandwidth. Due to the massive number of end users, cheap and reliable components become the bottleneck to deploy the new technology. Triplexer is one of the key components in the FTTH and is used by every end user. Currently, the available triplexers are either based on bulk optics or fiber optics with large size and high price due to manual labor involved. Planar lightwave circuit (PLC) is a possible technology for massive production and cost reduction. However, it is very challenging to design such bi-directional triplexer on PLC. The first challenge is that three channels, at λ=1310nm, 1490nm, and 1555nm, are separated unevenly over a very large wavelength range; Secondly, the bandwidths of the three channels, Δλ=100nm, 20nm, and 10nm, are very different. In the paper, we proposed a novel design by combining both coarse WDM and dense WDM. In the design, a multi-mode interference (MMI) device is used for coarse WDM to separate the 1310nm from the other two channels. The dense WDM for the remaining two channels is performed by an array waveguide gratings (AWG). The MMI and AWG are built on the same wafer with monolithic integration. Initial simulation results show it is a very promising device.

  1. DNA-triplex stabilizing properties of 8-aminoguanine

    PubMed Central

    Soliva, Robert; Güimil García, Ramón; Blas, J. Ramón; Eritja, Ramon; Asensio, Juan Luis; González, Carlos; Luque, F. Javier; Orozco, Modesto

    2000-01-01

    A DNA-triplex stabilizing purine (8-aminoguanine) is designed based on molecular modeling and synthesized. The substitution of guanine by 8-aminoguanine largely stabilizes the triplex both at neutral and acidic pH, as suggested by molecular dynamics and thermodynamic integration calculations, and demonstrated by melting experiments. NMR experiments confirm the triplex-stabilizing properties of 8-aminoguanine and demonstrate that few changes are found in the structure of the triplex due to the presence of this modified base. PMID:11071942

  2. Selectivity and affinity of DNA triplex forming oligonucleotides containing the nucleoside analogues 2'-O-methyl-5-(3-amino-1-propynyl)uridine and 2'-O-methyl-5-propynyluridine.

    PubMed

    Li, Hong; Miller, Paul S; Seidman, Michael M

    2008-11-21

    Triplex forming oligonucleotides (TFOs) containing the nucleoside analogues 2'-O-methyl-5-propynyluridine (1) and 2'-O-methyl-5-(3-amino-1-propynyl)uridine (2) were synthesized. The affinity and selectivity of triplex formation by these TFOs were studied by gel shift analysis, T(m) value measurement, and association rate assays. The results show that the introduction of 1 and 2 into TFOs can improve the stability of the triplexes under physiological conditions. Optimized distribution of 1 or 2 in the TFOs combined with a cluster of contiguous nucleosides with 2'-aminoethoxy sugars resulted in formation of triplexes with further enhanced stability and improved selectivity. PMID:18972052

  3. Effect of DNA target sequence on triplex formation by oligo-2′-deoxy- and 2′-O-methylribonucleotides

    PubMed Central

    Cassidy, Rachel A.; Puri, Nitin; Miller, Paul S.

    2003-01-01

    The interactions of pyrimidine deoxyribo- or 2′-O-methylribo-psoralen-conjugated, triplex-forming oligonucleotides, psTFOs, with a 17-bp env-DNA whose purine tract is 5′-AGAGAGAAAAAAGAG-3′, or an 18-bp gag-DNA whose purine tract is 5′-AGG GGGAAAGAAAAAA-3′, were studied over the pH range 6.0–7.5. The stability of the triplex formed by a deoxy-env-psTFO containing 5-methylcytosines and thymines decreased with increasing pH (Tm = 56°C at pH 6.0; 27°C at pH 7.5). Replacement of 5-methylcytosines with 8-oxo-adenines reduced the pH dependence, but lowered triplex stability. A 2′-O-methyl-env-psTFO containing uracil and cytosine did not form a triplex at pH 7.5. Surprisingly, replacement of the cytosines in this oligomer with 5-methylcytosines dramatically increased triplex stability (Tm = 25°C at pH 7.5), and even greater stability was achieved by selective replacement of uracils with thymines (Tm = 37°C at pH 7.5). Substitution of the contiguous 5-methylcytosines of the deoxy-gag-psTFO with 8-oxo-adenines significantly reduced pH dependence and increased triplex stability. In contrast to the behavior of env-specific TFOs, triplexes formed by 2′-O-methyl-gag-psTFOs did not show enhanced stability. Replacement of the 3′-terminal phosphodiester of the TFO with a methylphosphonate group significantly increased the resistance of both deoxy- and 2′-O-methyl-TFOs to degradation by 3′-exonucleases, while maintaining triplex stability. PMID:12853627

  4. RNA triplexes: from structural principles to biological and biotech applications.

    PubMed

    Devi, Gitali; Zhou, Yuan; Zhong, Zhensheng; Toh, Desiree-Faye Kaixin; Chen, Gang

    2015-01-01

    The diverse biological functions of RNA are determined by the complex structures of RNA stabilized by both secondary and tertiary interactions. An RNA triplex is an important tertiary structure motif that is found in many pseudoknots and other structured RNAs. A triplex structure usually forms through tertiary interactions in the major or minor groove of a Watson-Crick base-paired stem. A major-groove RNA triplex structure is stable in isolation by forming consecutive major-groove base triples such as U·A-U and C(+) ·G-C. Minor-groove RNA triplexes, e.g., A-minor motif triplexes, are found in almost all large structured RNAs. As double-stranded RNA stem regions are often involved in biologically important tertiary triplex structure formation and protein binding, the ability to sequence specifically target any desired RNA duplexes by triplex formation would have great potential for biomedical applications. Programmable chemically modified triplex-forming oligonucleotides (TFOs) and triplex-forming peptide nucleic acids (PNAs) have been developed to form TFO·RNA2 and PNA·RNA2 triplexes, respectively, with enhanced binding affinity and sequence specificity at physiological conditions. Here, we (1) provide an overview of naturally occurring RNA triplexes, (2) summarize the experimental methods for studying triplexes, and (3) review the development of TFOs and triplex-forming PNAs for targeting an HIV-1 ribosomal frameshift-inducing RNA, a bacterial ribosomal A-site RNA, and a human microRNA hairpin precursor, and for inhibiting the RNA-protein interactions involving human RNA-dependent protein kinase and HIV-1 viral protein Rev. PMID:25146348

  5. Triplex-mediated genome targeting and editing.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2014-01-01

    Genome targeting and editing in vitro and in vivo can be achieved through an interplay of exogenously introduced molecules and the induction of endogenous recombination machinery. The former includes a repertoire of sequence-specific binding molecules for targeted induction and appropriation of this machinery, such as by triplex-forming oligonucleotides (TFOs) or triplex-forming peptide nucleic acids (PNAs) and recombinagenic donor DNA, respectively. This versatile targeting and editing via recombination approach facilitates high-fidelity and low-off-target genome mutagenesis, repair, expression, and regulation. Herein, we describe the current state-of-the-art in triplex-mediated genome targeting and editing with a perspective towards potential translational and therapeutic applications. We detail several materials and methods for the design, delivery, and use of triplex-forming and recombinagenic molecules for mediating and introducing specific, heritable, and safe genomic modifications. Furthermore we denote some guidelines for endogenous genome targeting and editing site identification and techniques to test targeting and editing efficiency. PMID:24557900

  6. Selective Preference of Parallel DNA Triplexes Is Due to the Disruption of Hoogsteen Hydrogen Bonds Caused by the Severe Nonisostericity between the G*GC and T*AT Triplets.

    PubMed

    Goldsmith, Gunaseelan; Rathinavelan, Thenmalarchelvi; Yathindra, Narayanarao

    2016-01-01

    Implications of DNA, RNA and RNA.DNA hybrid triplexes in diverse biological functions, diseases and therapeutic applications call for a thorough understanding of their structure-function relationships. Despite exhaustive studies mechanistic rationale for the discriminatory preference of parallel DNA triplexes with G*GC & T*AT triplets still remains elusive. Here, we show that the highest nonisostericity between the G*GC & T*AT triplets imposes extensive stereochemical rearrangements contributing to context dependent triplex destabilisation through selective disruption of Hoogsteen scheme of hydrogen bonds. MD simulations of nineteen DNA triplexes with an assortment of sequence milieu reveal for the first time fresh insights into the nature and extent of destabilization from a single (non-overlapping), double (overlapping) and multiple pairs of nonisosteric base triplets (NIBTs). It is found that a solitary pair of NIBTs, feasible either at a G*GC/T*AT or T*AT/G*GC triplex junction, does not impinge significantly on triplex stability. But two overlapping pairs of NIBTs resulting from either a T*AT or a G*GC interruption disrupt Hoogsteen pair to a noncanonical mismatch destabilizing the triplex by ~10 to 14 kcal/mol, implying that their frequent incidence in multiples, especially, in short sequences could even hinder triplex formation. The results provide (i) an unambiguous and generalised mechanistic rationale for the discriminatory trait of parallel triplexes, including those studied experimentally (ii) clarity for the prevalence of antiparallel triplexes and (iii) comprehensive perspectives on the sequence dependent influence of nonisosteric base triplets useful in the rational design of TFO's against potential triplex target sites. PMID:27010368

  7. Selective Preference of Parallel DNA Triplexes Is Due to the Disruption of Hoogsteen Hydrogen Bonds Caused by the Severe Nonisostericity between the G*GC and T*AT Triplets

    PubMed Central

    Goldsmith, Gunaseelan; Rathinavelan, Thenmalarchelvi; Yathindra, Narayanarao

    2016-01-01

    Implications of DNA, RNA and RNA.DNA hybrid triplexes in diverse biological functions, diseases and therapeutic applications call for a thorough understanding of their structure-function relationships. Despite exhaustive studies mechanistic rationale for the discriminatory preference of parallel DNA triplexes with G*GC & T*AT triplets still remains elusive. Here, we show that the highest nonisostericity between the G*GC & T*AT triplets imposes extensive stereochemical rearrangements contributing to context dependent triplex destabilisation through selective disruption of Hoogsteen scheme of hydrogen bonds. MD simulations of nineteen DNA triplexes with an assortment of sequence milieu reveal for the first time fresh insights into the nature and extent of destabilization from a single (non-overlapping), double (overlapping) and multiple pairs of nonisosteric base triplets (NIBTs). It is found that a solitary pair of NIBTs, feasible either at a G*GC/T*AT or T*AT/G*GC triplex junction, does not impinge significantly on triplex stability. But two overlapping pairs of NIBTs resulting from either a T*AT or a G*GC interruption disrupt Hoogsteen pair to a noncanonical mismatch destabilizing the triplex by ~10 to 14 kcal/mol, implying that their frequent incidence in multiples, especially, in short sequences could even hinder triplex formation. The results provide (i) an unambiguous and generalised mechanistic rationale for the discriminatory trait of parallel triplexes, including those studied experimentally (ii) clarity for the prevalence of antiparallel triplexes and (iii) comprehensive perspectives on the sequence dependent influence of nonisosteric base triplets useful in the rational design of TFO’s against potential triplex target sites. PMID:27010368

  8. Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

    PubMed Central

    Devi, Gitali; Yuan, Zhen; Lu, Yunpeng; Zhao, Yanli; Chen, Gang

    2014-01-01

    Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA2–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA2 triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA2–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA2–PNA triplex is significantly more stable than a DNA2–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications. PMID:24423869

  9. Spectroscopic and calorimetric studies on the binding of an indoloquinoline drug to parallel and antiparallel DNA triplexes.

    PubMed

    Riechert-Krause, Fanny; Autenrieth, Karolin; Eick, Andrea; Weisz, Klaus

    2013-01-01

    11-Phenyl-substituted indoloquinolines have been found to exhibit significant antiproliferative potency in cancer cells but to show only moderate affinity toward genomic double-helical DNA. In this study, parallel as well as antiparallel triple-helical DNA targets are employed to evaluate the triplex binding of these ligands. UV melting experiments with parallel triplexes indicate considerable interactions with the drug and a strong preference for TAT-rich triplexes in line with an increasing number of potential intercalation sites of similar binding strength between two TAT base triads. Via substitution of a singly charged aminoethylamine side chain by a longer and doubly charged bis(aminopropyl)amine substituent at the ligand, binding affinities increase and also start to exhibit long-range effects as indicated by a strong correlation between the binding affinity and the overall length of the TAT tract within the triplex stem. Compared to parallel triplexes, an antiparallel triplex with a GT-containing third strand constitutes a preferred target for the indoloquinoline drug. On the basis of pH-dependent titration experiments and corroborated by a Job analysis of continuous variation, binding of the drug to the GT triplex not only is strongly enhanced when the solution pH is lowered from 7 to 5 but also reveals a pH-dependent stoichiometry upon formation of the complex. Calorimetric data demonstrate that stronger binding of a protonated drug at acidic pH is associated with a more exothermic binding process. However, at pH 7 and 5, binding is enthalpically driven with additional favorable entropic contributions. PMID:23234257

  10. Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides.

    PubMed

    Timofeev, Edward N; Goryaeva, Baira V; Florentiev, Vladimir L

    2006-10-01

    DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes. PMID:16928141

  11. Analysis of various sequence-specific triplexes by electron and atomic force microscopies.

    PubMed Central

    Cherny, D I; Fourcade, A; Svinarchuk, F; Nielsen, P E; Malvy, C; Delain, E

    1998-01-01

    Sequence-specific interactions of 20-mer G,A-containing triple helix-forming oligonucleotides (TFOs) and bis-PNAs (peptide nucleic acids) with double-stranded DNA was visualized by electron (EM) and atomic force (AFM) microscopies. Triplexes formed by biotinylated TFOs are easily detected by both EM and AFM in which streptavidin is a marker. AFM images of the unlabeled triplex within a long plasmid DNA show a approximately 0.4-nm height increment of the double helix within the target site position. TFOs conjugated to a 74-nt-long oligonucleotide forming a 33-bp-long hairpin form extremely stable triplexes with the target site that are readily imaged by both EM and AFM as protruding DNA. The short duplex protrudes in a perpendicular direction relative to the double helix axis, either in the plane of the support or out of it. In the latter case, the apparent height of the protrusion is approximately 1.5 nm, when that of the triplex site is increased by 0.3-0.4 nm. Triplex formation by bis-PNA, in which two decamers of PNA are connected via a flexible linker, causes deformations of the double helix at the target site, which is readily detected as kinks by both EM and AFM. Moreover, AFM shows that these kinks are often accompanied by an increase in the DNA apparent height of approximately 35%. This work shows the first direct visualization of sequence-specific interaction of TFOs and PNAs, with their target sequences within long plasmid DNAs, through the measurements of the apparent height of the DNA double helix by AFM. PMID:9533714

  12. Development and validation of a triplex real-time PCR assay for the simultaneous detection of three mustard species and three celery varieties in food.

    PubMed

    Palle-Reisch, Monika; Hochegger, Rupert; Cichna-Markl, Margit

    2015-10-01

    The paper presents a triplex real-time PCR assay allowing the simultaneous detection of three mustard species (white, black and brown mustard) and three celery varieties (celery roots, celery stalks and leaf celery) in foodstuffs. The triplex assay does not show cross-reactivity with other Brassicaceae. Low cross-reactivities were observed with fenugreek, cumin, ginger, caraway, turmeric, lovage and rye, the ΔCt values were, however, ⩾ 12 compared to positive controls. The triplex assay allows the detection of traces of DNA of the allergenic components in spite of an excess of the other DNA templates. Analysis of extracts from model sausages containing defined concentrations of mustard and celery showed that the triplex assay is applicable to both raw and processed foods. It was found to allow the detection of 1 ppm black/brown mustard and 50 ppm white mustard and celery in raw and brewed sausages with a probability ⩾ 95%. PMID:25872425

  13. DNA Triplexes That Bind Several Cofactor Molecules.

    PubMed

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Invited for the cover of this issue are Sven Vollmer and Clemens Richert of the University of Stuttgart. The cover image hints at the analogy between a honey comb, as a macroscopic storage device, and DNA triplexes with designed binding sites, as molecular storage motifs that can release ATP to fuel a bioluminescence reaction. Read the full text of the article at 10.1002/chem.201503220. PMID:26534779

  14. Triplex-Induced DNA Damage Response

    PubMed Central

    Rogers, Faye A.; Tiwari, Meetu Kaushik

    2013-01-01

    Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage. PMID:24348211

  15. 2',4'-BNA bearing a 2-pyridine nucleobase for CG base pair recognition in the parallel motif triplex DNA.

    PubMed

    Hari, Yoshiyuki; Matsugu, Sachiko; Inohara, Hiroyasu; Hatanaka, Yuri; Akabane, Masaaki; Imanishi, Takeshi; Obika, Satoshi

    2010-09-21

    We succeeded in the synthesis of triplex-forming oligonucleotides (TFOs) that contain a deoxyribonucleotide (Py) bearing a 2-pyridine nucleobase or the 2',4'-BNA congener (Py(B)). By UV melting experiments, it was found that 2-pyridine was a very promising nucleobase for the sequence-selective recognition of a CG base pair within double-stranded DNA (dsDNA) in a parallel motif triplex. Moreover, Py(B) in TFOs showed stronger affinity to a CG base pair than Py with further increase in the selectivity. Using TFO including multiple Py(B) units, triplex formation with dsDNA containing three CG base pairs was observed. PMID:20648389

  16. Monitoring triplex DNA formation with fluorescence resonance energy transfer between a fluorophore-labeled probe and intercalating dyes.

    PubMed

    Chiou, Chiuan-Chian; Chen, Shiau-Wen; Luo, Ji-Dung; Chien, Yu-Tzu

    2011-09-01

    Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction. PMID:21609711

  17. 2'-Aminoethoxy-2-amino-3-methylpyridine in triplex-forming oligonucleotides: high affinity, selectivity and resistance to enzymatic degradation.

    PubMed

    Lou, Chenguang; Shelbourne, Montserrat; Fox, Keith R; Brown, Tom

    2011-12-23

    The phosphoramidite monomer of the C-nucleoside 2'-aminoethoxy-2-amino-3-methylpyridine (AE-MAP) has been synthesized for the first time and incorporated into triplex-forming oligonucleotides (TFOs). Ultraviolet melting and DNase I footprinting studies show that AE-MAP is a potent triplex-stabilizing monomer that is selective for GC base pairs. TFOs containing AE-MAP bind with high affinity to duplexes but only weakly to single stranded DNA. In addition, AE-MAP confers high nuclease resistance on oligonucleotides. TFOs containing AE-MAP have potential for gene knock-out and gene expression studies. PMID:22127905

  18. DNA duplexes and triplex-forming oligodeoxynucleotides incorporating modified nucleosides forming stable and selective triplexes.

    PubMed

    Kanamori, Takashi; Masaki, Yoshiaki; Mizuta, Masahiro; Tsunoda, Hirosuke; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2012-02-01

    We have previously reported DNA triplexes containing the unnatural base triad G-PPI·C3, in which PPI is an indole-fused cytosine derivative incorporated into DNA duplexes and C3 is an abasic site in triplex-forming oligonucleotides (TFOs) introduced by a propylene linker. In this study, we developed a new unnatural base triad A-ψ·C(R1) where ψ and C(R1) are base moieties 2'-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (C(Br)) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPI(Me), having a methyl group on the indole ring in order to achieve selective triplex formation between DNA duplexes incorporating various Watson-Crick base pairs, such as T-A, C-G, A-ψ, and G-PPI(Me), and TFOs containing T, C, C(Br), and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays. PMID:22146807

  19. An efficient antigene activity and antiproliferative effect by targeting the Bcl-2 or survivin gene with triplex forming oligonucleotides containing a W-shaped nucleoside analogue (WNA-βT).

    PubMed

    Taniguchi, Yosuke; Sasaki, Shigeki

    2012-10-01

    Triplex forming oligonucleotides (TFOs) are some of the most promising tools in the antigene strategy for the development of gene targeting therapeutics. However, the stable triplex formation is restricted to the homopurine sequences consisting of purine nucleosides, dG and dA. Therefore, the T or dC nucleoside in the homopurine strand inhibits the stable triplex formation. We have developed W-shaped nucleoside analogues (WNAs) for the formation of the unnatural type triplex DNA, with sequences containing the interrupting site in an antiparallel triplex formation. In the present study, we tested the antigene effect of TFOs having WNA-βT, which increased the stability of the triplex formation with a target sequence including the TA interrupting site. We designed the GU TFO (WNA) and GU TFO (natural) for targeting sequences of the Bcl-2 or survivin oncogene. The gel shift assay showed that the TFO (WNA) formed more stable triplexes than the natural TFO. Remarkably, the Bcl-2- or survivin-targeted TFO (WNA) inhibited the cell proliferation and induced a caspase-dependent apoptosis. It was confirmed that the survivin-targeted TFO (WNA) more effectively decreased the number of survivin products in the A549 cell than the natural TFOs. PMID:22987068

  20. Evidence for the conformational rigidity of triplex d(C +T) 8-d(AG) 8·d(CT) 8 on silver electrode revealed by Fourier transform Raman scattering studies

    NASA Astrophysics Data System (ADS)

    Fang, Ye; Bai, Chunli; Wang, Ting; Zhong, Faping; Tang, Youqi; Lin, S. B.; Kan, Lou-sing

    1996-03-01

    Fourier transform surface enhanced Raman scattering spectroscopy (FT-SERS) has been first applied to characterize the triple stranded helix d(C +T) 8-d(AG) 8·d(CT) 8 (pH 4.5) (triplex) and its corresponding double-stranded helix d(AG) 8·d(CT) 8 (pH 7.0) (duplex) at an ex situ roughened silver electrode polarized at between 0.0 and -1.0 V vs. {Ag}/{AgCl}. The triplex adsorbed on the silver electrode yields five intense and one weak SERS bands located at 241, 839, 1189, 1293, 1643, and 1530 cm -1, respectively. These bands are not seen in the FT-SERS spectrum of the duplex, which showed a pattern similar to that of an oligo-DNA duplex observed by Koglin and Sequaries (Top. Curr. Chem., 134 (1989) 1). In the case of the triplex, the occurrence of the 839 cm -1 band with concomitant disappearance of the adsorbed adenine ring-breathing band at 734 cm -1 of the duplex indicates that the helical structure of the triplex near the surface of the silver electrode is well preserved and exhibits conformational rigidity. We observed a desorption process of the triplex from the silver electrode when the electrode approached -0.9 V (potential of zero charge of the triplex) vs. {Ag}/{AgCl}. This indicates that the highly negatively charged triplex shows a more favorable absorption on a highly positively charged silver surface. The structure of the triplex has also been studied by Fourier transform infrared (FTIR) and Raman spectroscopy. The vibrational spectra obtained clearly revealed that the conformation of the sugar moiety of the purine strand and one pyrimidine strand in the triplex is C 2'-endo/anti, whereas that of the other pyrimidine strand is C 3'-endo/anti.

  1. A gold nanoparticle based approach for screening triplex DNA binders.

    PubMed

    Han, Min Su; Lytton-Jean, Abigail K R; Mirkin, Chad A

    2006-04-19

    Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change do not occur in the presence of duplex DNA binders and therefore provide a significantly better screening process for triplex DNA binding molecules compared to standard methods. PMID:16608320

  2. A Gold Nanoparticle Based Approach for Screening Triplex DNA Binders

    PubMed Central

    Han, Min Su; Lytton-Jean, Abigail K. R.; Mirkin, Chad A.

    2011-01-01

    Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change does not occur in the presence of duplex DNA binders and therefore provides a significantly better screening process for triplex DNA binding molecules compared to standard methods. PMID:16608320

  3. DNA triplex-mediated inhibition of MET leads to cell death and tumor regression in hepatoma

    PubMed Central

    Singhal, G; Akhter, MZ; Stern, DF; Gupta, SD; Ahuja, A; Sharma, U; Jagannathan, NR; Rajeswari, MR

    2016-01-01

    Mesenchymal epithelial transition factor (MET) is one of the critical cell signaling molecules whose aberrant expression is reported in several human cancers. The aim of the study is to investigate the antigene and antiproliferative effect of short triplex forming oligonucleotides, TFO-1 (part of the positive regulatory element) and TFO-2 (away from the transcription start site) on MET expression. HepG2 cells transfected only with TFO-1 (but not with TFO-2 and non-specific TFO) significantly decreased MET levels, which is accompanied by decrease in antiapoptotic proteins and increase in pro-apoptotic proteins. Phosphoproteome-array analysis of 46 intracellular kinases revealed hypophosphorylation of about 15 kinases including ERK, AKT, Src and MEK, suggesting the growth inhibitory effect of TFO-1. Further, the efficacy of TFO-1 was tested on diethylnitrosamine-induced liver tumors in wistar rats. T2-weighted magnetic resonance imaging showed decrease in liver tumor volume up to 90% after treatment with TFO-1. Decreased MET expression and elevated apoptotic activity further indicate that TFO-1 targeted to c-met leads to cell death and tumor regression in hepatoma. Formation of stable DNA triplex between TFO-1 and targeted gene sequence was confirmed by circular dichroic spectroscopy and gel retardation assay. Therefore, it can be concluded that DNA triplex-based therapeutic approaches hold promise in the treatment of malignancies associated with MET overexpression. PMID:21660063

  4. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    PubMed

    Paugh, Steven W; Coss, David R; Bao, Ju; Laudermilk, Lucas T; Grace, Christy R; Ferreira, Antonio M; Waddell, M Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F; Panetta, John C; Wilkinson, Mark R; Pui, Ching-Hon; Naeve, Clayton W; Uberbacher, Edward C; Bonten, Erik J; Evans, William E

    2016-02-01

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16)) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

  5. MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

    DOE PAGESBeta

    Paugh, Steven W.; Coss, David R.; Bao, Ju; Laudermilk, Lucas T.; Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael Rex; et al

    2016-02-04

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10-16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

  6. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

    PubMed Central

    Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F.; Panetta, John C.; Wilkinson, Mark R.; Pui, Ching-Hon; Naeve, Clayton W.; Uberbacher, Edward C.; Bonten, Erik J.; Evans, William E.

    2016-01-01

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10−16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

  7. Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

    PubMed Central

    Thomas, T J; Faaland, C A; Gallo, M A; Thomas, T

    1995-01-01

    Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer. Images PMID:7567474

  8. Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase.

    PubMed

    Uchida, Yuko; Taniguchi, Yosuke; Aoki, Eriko; Togo, Mieko; Sasaki, Shigeki

    2008-01-01

    Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-beta T and WNA-beta C, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-beta 3AP, having 3-aminopyrazole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-beta 3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-beta C did not show the stabilizing effect. PMID:18776291

  9. Triplex-Inspector: an analysis tool for triplex-mediated targeting of genomic loci

    PubMed Central

    Buske, Fabian A.; Bauer, Denis C.; Mattick, John S.; Bailey, Timothy L.

    2013-01-01

    Summary: At the heart of many modern biotechnological and therapeutic applications lies the need to target specific genomic loci with pinpoint accuracy. Although landmark experiments demonstrate technological maturity in manufacturing and delivering genetic material, the genomic sequence analysis to find suitable targets lags behind. We provide a computational aid for the sophisticated design of sequence-specific ligands and selection of appropriate targets, taking gene location and genomic architecture into account. Availability: Source code and binaries are downloadable from www.bioinformatics.org.au/triplexator/inspector. Contact: t.bailey@uq.edu.au Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23740745

  10. Fluorescently Sensing of DNA Triplex Assembly Using an Isoquinoline Alkaloid as Selector, Stabilizer, Inducer, and Switch-On Emitter.

    PubMed

    Hu, Yuehua; Lin, Fan; Wu, Tao; Wang, Ying; Zhou, Xiao-Shun; Shao, Yong

    2016-07-20

    DNA triplex assembly has attracted a variety of interest in the regulation of genetic expression, drug screening, molecular switches, and sensors. However, these achievements are essentially dependent on the formation and stability of the triplex assembly. Herein, the recognition of DNA triplex assembly with various isoquinoline alkaloids was investigated. We found that natural chelerythrine (CHE) exhibits the highest selectivity in recognizing the triplex structure. The DNA triplex stability is substantially increased upon CHE binding, as opposed to the invariance in the stability of the duplex counterpart. CHE also favors the assembly of the triplex-forming oligonucleotide (TFO) with its duplex counterpart. The triplex binding switches CHE to a strong fluorescent emitter, which suggests CHE as a useful probe in following triplex assembly. As a unique triplex selector, inducer, and emitter, CHE successfully reports the wide pH- and metal-ion-dependent tunability of the triplex nanoswitch in a label-free manner. PMID:27252050

  11. Rupture force changes between the third strand and the double strand within an oligonucleotide-directed triplex in the presence of intercalative molecules

    NASA Astrophysics Data System (ADS)

    Ling, Liansheng; Butt, Hans-Jürgen; Berger, Rüdiger

    2006-09-01

    The rupture force changes to separate the third strand from the duplex within an oligonucleotide-directed triplex (triplex DNA) were measured in the presence of small DNA binding molecules by means of atomic force spectroscopy. Rupture force histograms revealed a rupture force of 44.3±0.4pN to separate the third strand from duplex DNA in pure buffer solution at a rupture velocity of 400nm/s. In 30μM of Ru(bipy)2(dppz)2+ buffer solution an increase of rupture force to 57±2.2pN was observed. For 30μM Ru(phen)2(dppz)2+ a rupture force of 65.1±3.9pN was determined. These results show that both intercalative molecules increase the stability of triplex DNA significantly.

  12. Triplex formation on DNA targets: how to choose the oligonucleotide.

    PubMed

    Vekhoff, Pierre; Ceccaldi, Alexandre; Polverari, David; Pylouster, Jean; Pisano, Claudio; Arimondo, Paola B

    2008-11-25

    Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA binders. TFOs provide a tool for controlling gene expression or, when attached to an appropriate chemical reagent, for directing DNA damage. Here, we report a set of rules for predicting the best out of five different triple-helical binding motifs (TM, UM, GA, GT, and GU, where M is 5-methyldeoxycytidine and U is deoxyuridine) by taking into consideration the sequence composition of the underlying duplex target. We tested 11 different triplex targets present in genes having an oncogenic role. The rules have predictive power and are very useful in the design of TFOs for antigene applications. Briefly, we retained motifs GU and TM, and when they do form a triplex, TFOs containing G and U are preferred over those containing T and M. In the case of the G-rich TFOs, triplex formation is principally dependent on the percentage of G and the length of the TFO. In the case of the pyrimidine motif, replacement of T with U is destabilizing; triplex formation is dependent on the percentage of T and destabilized by the presence of several contiguous M residues. An equation to choose between a GU and TM motif is given. PMID:18954091

  13. Detailed study of sequence-specific DNA cleavage of triplex-forming oligonucleotides linked to 1,10-phenanthroline.

    PubMed

    Shimizu, M; Inoue, H; Ohtsuka, E

    1994-01-18

    We introduced eight bases, including four base analogs, into 15-mer triplex-forming oligonucleotides (TFOs) [d-psTTTCTTTNTTTTCTT; ps = thiophosphate; N = A, G, C, T, 2'-deoxyinosine (I), 2'-deoxyxanthosine (X), 5-methyl-2'-deoxycytidine (m5C), or 5-bromo-2'-deoxyuridine(br5U)] to investigate the Hoogsteen-like hydrogen bonding to the base in the target 34-mer strand (d-TGAGTGAGTAAAGAAARAAAAGAATGAGTGCCAA.d-TTGGCACTCATTCTTTTYTTTCT TTACTCACTCA; RY = AT, GC, TA, or CG). We examined the thermal stability of 15-mer triplexes in buffer containing 100 mM sodium acetate and 1 M NaCl at pH 5.0. The triplexes with typical triplets of T.AT (51.3 degrees C), br5U.AT (52.4 degrees C), C+.GC (66.7 degrees C), and m5C+.GC (66.8 degrees C) at the central position showed relatively higher Tm values, as expected. The relatively high stability of the X.AT triplex (39.8 degrees C) was observed. Among the N.TA triplets, G.TA (44.8 degrees C) was thermally the most stable, and moreover, the data showed that the N.TA triplet was also stabilized by I in the N position (40.7 degrees C). Furthermore, the TFOs were converted to DNA-cleaving molecules by introducing a newly synthesized 1,10-phenanthroline (OP) derivative on the thiophosphate group at the 5' end. Cleavage reactions of the 32P-labeled DNA (34-mer) were carried out. The cleavage efficiencies were compared to the Tm values of triplexes with or without an OP derivative. Results showed that the increased cleavage yields reflect the higher thermal stability of the triplex formed in most cases, but a few exceptional cases existed. Especially, the G-containing TFO did not show the above correlation between thermal stability and cleavage yield.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8286392

  14. 22. FIRST FLOOR APARTMENT SOUTH BEDROOM INTERIOR SHOWING PAIRED 6LIGHT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. FIRST FLOOR APARTMENT SOUTH BEDROOM INTERIOR SHOWING PAIRED 6-LIGHT OVER 6-LIGHT DOUBLE-HUNG, WOOD-FRAMED WINDOWS. VIEW TO SOUTH. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  15. Secondary binding sites for heavily modified triplex forming oligonucleotides

    PubMed Central

    Cardew, Antonia S.; Brown, Tom; Fox, Keith R.

    2012-01-01

    In order to enhance DNA triple helix stability synthetic oligonucleotides have been developed that bear amino groups on the sugar or base. One of the most effective of these is bis-amino-U (B), which possesses 5-propargylamino and 2′-aminoethoxy modifications. Inclusion of this modified nucleotide not only greatly enhances triplex stability, but also increases the affinity for related sequences. We have used a restriction enzyme protection, selection and amplification assay (REPSA) to isolate sequences that are bound by the heavily modified 9-mer triplex-forming oligonucleotide B6CBT. The isolated sequences contain An tracts (n = 6), suggesting that the 5′-end of this TFO was responsible for successful triplex formation. DNase I footprinting with these sequences confirmed triple helix formation at these secondary targets and demonstrated no interaction with similar oligonucleotides containing T or 5-propargylamino-dU. PMID:22180535

  16. Kinetics of the Triplex-Duplex Transition in DNA

    PubMed Central

    Lee, Il-Buem; Hong, Seok-Cheol; Lee, Nam-Kyung; Johner, Albert

    2012-01-01

    The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic. PMID:23260051

  17. Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

    PubMed Central

    Kukreti, S; Sun, J S; Garestier, T; Hélène, C

    1997-01-01

    Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates. PMID:9336456

  18. Repair of DNA lesions associated with triplex-forming oligonucleotides.

    PubMed

    Chin, Joanna Y; Glazer, Peter M

    2009-04-01

    Triplex-forming oligonucleotides (TFOs) are gene targeting tools that can bind in the major groove of duplex DNA in a sequence-specific manner. When bound to DNA, TFOs can inhibit gene expression, can position DNA-reactive agents to specific locations in the genome, or can induce targeted mutagenesis and recombination. There is evidence that third strand binding, alone or with an associated cross-link, is recognized and metabolized by DNA repair factors, particularly the nucleotide excision repair pathway. This review examines the evidence for DNA repair of triplex-associated lesions. PMID:19072762

  19. Binding of oligonucleotides to a viral hairpin forming RNA triplexes with parallel G*G•C triplets

    PubMed Central

    Carmona, Pedro; Molina, Marina

    2002-01-01

    Infrared and UV spectroscopies have been used to study the assembly of a hairpin nucleotide sequence (nucleotides 3–30) of the 5′ non-coding region of the hepatitis C virus RNA (5′-GGCGGGGAUUAUCCCCGCUGUGAGGCGG-3′) with a RNA 20mer ligand (5′-CCGCCUCACAAAGGUGGGGU-3′) in the presence of magnesium ion and spermidine. The resulting complex involves two helical structural domains: the first one is an intermolecular duplex stem at the bottom of the target hairpin and the second one is a parallel triplex generated by the intramolecular hairpin duplex and the ligand. Infrared spectroscopy shows that N-type sugars are exclusively present in the complex. This is the first case of formation of a RNA parallel triplex with purine motif and shows that this type of targeting RNA strands to viral RNA duplexes can be used as an alternative to antisense oligonucleotides or ribozymes. PMID:11884630

  20. Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation—a case study using triplex-forming LNAs

    PubMed Central

    Sau, Sujay P.; Kumar, Pawan; Sharma, Pawan K.; Hrdlicka, Patrick J.

    2012-01-01

    Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (Keq) and association/dissociation rate constants (kon and koff). The ‘fluorescent intercalator displacement replacement’ (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson–Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine. PMID:22855561

  1. Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation--a case study using triplex-forming LNAs.

    PubMed

    Sau, Sujay P; Kumar, Pawan; Sharma, Pawan K; Hrdlicka, Patrick J

    2012-11-01

    Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (K(eq)) and association/dissociation rate constants (k(on) and k(off)). The 'fluorescent intercalator displacement replacement' (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson-Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine. PMID:22855561

  2. Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

    PubMed Central

    Kragelund, L.; Leopold, K.; Nybroe, O.

    1996-01-01

    The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system. PMID:16535235

  3. Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads.

    PubMed

    Kragelund, L; Leopold, K; Nybroe, O

    1996-02-01

    The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system. PMID:16535235

  4. MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures.

    PubMed

    Mondal, Tanmoy; Subhash, Santhilal; Vaid, Roshan; Enroth, Stefan; Uday, Sireesha; Reinius, Björn; Mitra, Sanhita; Mohammed, Arif; James, Alva Rani; Hoberg, Emily; Moustakas, Aristidis; Gyllensten, Ulf; Jones, Steven J M; Gustafsson, Claes M; Sims, Andrew H; Westerlund, Fredrik; Gorab, Eduardo; Kanduri, Chandrasekhar

    2015-01-01

    Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-β pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-β genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation. We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes. Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs. PMID:26205790

  5. MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA–DNA triplex structures

    PubMed Central

    Mondal, Tanmoy; Subhash, Santhilal; Vaid, Roshan; Enroth, Stefan; Uday, Sireesha; Reinius, Björn; Mitra, Sanhita; Mohammed, Arif; James, Alva Rani; Hoberg, Emily; Moustakas, Aristidis; Gyllensten, Ulf; Jones, Steven J.M.; Gustafsson, Claes M; Sims, Andrew H; Westerlund, Fredrik; Gorab, Eduardo; Kanduri, Chandrasekhar

    2015-01-01

    Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-β pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-β genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA–DNA triplex formation. We have found that RNA–DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes. Our findings suggest that RNA–DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs. PMID:26205790

  6. Enhancement of TFO Triplex Formation by Conjugation with Pyrene via Click Chemistry.

    PubMed

    Taniguchi, Yosuke; Tomizaki, Akira; Matsueda, Nozomu; Okamura, Hidenori; Sasaki, Shigeki

    2015-01-01

    This paper reports the preparation of 14-mer triplex-forming oligonucleotides (TFOs) containing a 2-O-methyl-1-β-phenyl-α-propargyl-ribose unit, which was conjugated with azide-modified molecules via a click reaction. Modification of these TFOs with pyrene assisted triplex formation, improving the stability of the triplex DNA and the anti-proliferative effects against A549 cells. PMID:26521856

  7. Compact design of high-temperature superconducting duplexer and triplexer for satellite communications

    NASA Astrophysics Data System (ADS)

    Zhang, Xu; He, Ming; Ji, Lu; Zhao, Xinjie; Fang, Lan; Yan, Shaolin

    2012-10-01

    This paper presents a compact C-band high-temperature superconducting (HTS) input module duplexer, which can be scaled to a triplexer. The duplexer includes a common node as the input port, which is an extensible and effective structure for integrating a HTS multiplexer on a single superconducting film. The input node is realized by an open-loop microstrip line, which resonates at the frequency in the guard band between the two channels. In addition two four-pole bandpass filters consisting of rectangular spiral line resonators are used as output channels of the device. The duplexer is designed at 3995 MHz with a bandwidth of 226 MHz. The frequency ranges of the two channels are 3882-3942 MHz and 4048-4108 MHz, respectively. It is fabricated using double-sided Y Ba2Cu3O7 (YBCO) thin films on a piece of 30× 10 mm 2 MgO substrate. The experimental results show that the insertion loss is less than 0.1 dB for both channel filters and the isolation between the two channels is higher than 40 dB. Good agreement has been achieved between simulations and measurements to illustrate the effectiveness of the proposed approach. Moreover, the triplexer is also designed and measured and the scalability is verified by simulation and experiments.

  8. Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification.

    PubMed

    Cannata, Fabio; Brunet, Erika; Perrouault, Loïc; Roig, Victoria; Ait-Si-Ali, Slimane; Asseline, Ulysse; Concordet, Jean-Paul; Giovannangeli, Carine

    2008-07-15

    The inefficiency of gene modification by homologous recombination can be overcome by the introduction of a double-strand break (DSB) in the target. Engineering the endonucleases needed, however, remains a challenging task that limits widespread application of nuclease-driven gene modification. We report here that conjugates of orthophenanthroline (OP), a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences, are synthetic nucleases efficient at stimulating targeted genome modification. We show that in cultured cells, OP-TFO conjugates induce targeted DSBs. An OP-TFO with a unique target was highly efficient, and mutations at the target site were found in approximately 10% of treated cells, including small deletions most likely introduced during DSB repair by nonhomologous end joining. Importantly, we found that when homologous donor DNA was cotransfected, targeted gene modification took place in >1.5% of treated cells. Because triplex-forming sequences are frequent in human and mouse genes, OP-TFO conjugates therefore constitute an important class of site-specific nucleases for targeted gene modification. Harnessing DNA-damaging molecules to predetermined genomic sites, as achieved here, should also provide inroads into mechanisms of DNA repair and cancer. PMID:18599454

  9. The Effect of Small Cosolutes that Mimic Molecular Crowding Conditions on the Stability of Triplexes Involving Duplex DNA

    PubMed Central

    Aviñó, Anna; Mazzini, Stefania; Gargallo, Raimundo; Eritja, Ramon

    2016-01-01

    Triplex stability is studied in crowding conditions using small cosolutes (ethanol, acetonitrile and dimethylsulfoxide) by ultraviolet (UV), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The results indicate that the triplex is formed preferentially when the triplex forming oligonucleotide (TFO) is RNA. In addition, DNA triplexes (D:D·D) are clearly less stable in cosolute solutions while the stability of the RNA triplexes (R:D·D) is only slightly decreased. The kinetic of triplex formation with RNA-TFO is slower than with DNA-TFO and the thermal stability of the triplex is increased with the salt concentration in EtOH-water solutions. Accordingly, RNA could be considered a potential molecule to form a stable triplex for regulatory purposes in molecular crowding conditions. PMID:26861295

  10. The Effect of Small Cosolutes that Mimic Molecular Crowding Conditions on the Stability of Triplexes Involving Duplex DNA.

    PubMed

    Aviñó, Anna; Mazzini, Stefania; Gargallo, Raimundo; Eritja, Ramon

    2016-01-01

    Triplex stability is studied in crowding conditions using small cosolutes (ethanol, acetonitrile and dimethylsulfoxide) by ultraviolet (UV), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The results indicate that the triplex is formed preferentially when the triplex forming oligonucleotide (TFO) is RNA. In addition, DNA triplexes (D:D·D) are clearly less stable in cosolute solutions while the stability of the RNA triplexes (R:D·D) is only slightly decreased. The kinetic of triplex formation with RNA-TFO is slower than with DNA-TFO and the thermal stability of the triplex is increased with the salt concentration in EtOH-water solutions. Accordingly, RNA could be considered a potential molecule to form a stable triplex for regulatory purposes in molecular crowding conditions. PMID:26861295

  11. Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasI/R and gyrB genes.

    PubMed

    Aghamollaei, Hosseine; Moghaddam, Mehrdad M; Kooshki, Hamid; Heiat, Mohammad; Mirnejad, Reza; Barzi, Nastaran S

    2015-01-01

    Pseudomonas aeruginosa is a nosocomial pathogen, which, due to its inherent and acquired resistance to a wide range of antibiotics, causes high mortality rates. Therefore, rapid detection of the bacterium with high specificity and sensitivity plays a critical role in the control of the pathogenic bacterium. The aim of this study was to evaluate the accuracy and specificity of a prompt detection of the bacterium based on a triplex polymerase chain reaction that amplifies the lasI, lasR and gyrB genes. For this purpose, 30 clinical isolates of P. aeruginosa and 30 wound biopsy samples were retrieved from clinical diagnostic laboratories. After the extraction of the chromosomal DNA, the desired genes were amplified using uniplex and triplex PCR with appropriate primers. The specificity of the primers was evaluated by a comparison of the PCR results for P. aeruginosa clinical samples and non-Pseudomonas species control samples. The sensitivity of the primers was determined using a serial dilution of the genomic DNA template (100 ng to 100 fg) and by a comparison of the PCR and bacterial culture results. The results showed that the triplex PCR assay was positive for all of the samples (100%), while the PCR identifications were negative for non-Pseudomonas species. Additionally, at 10(-4) and 10(-5) diluted genomic DNA from P. aeruginosa (10 pg and 1 pg), the triplex PCR test was positive for the Las and gyrB genes in all of the samples, respectively. Based on these results, the designed primers can be used for the rapid, specific and sensitive diagnosis of P. aeruginosa in a triplex PCR assay. PMID:25863575

  12. A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase*

    PubMed Central

    Guo, Manhong; Hundseth, Kristian; Ding, Hao; Vidhyasagar, Venkatasubramanian; Inoue, Akira; Nguyen, Chi-Hung; Zain, Rula; Lee, Jeremy S.; Wu, Yuliang

    2015-01-01

    Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome. PMID:25561740

  13. 30. SECOND FLOOR EAST SIDE APARTMENT WEST BEDROOM INTERIOR SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    30. SECOND FLOOR EAST SIDE APARTMENT WEST BEDROOM INTERIOR SHOWING PAIRED 6-LIGHT OVER 6-LIGHT DOUBLE-HUNG, WOOD-FRAME WINDOWS THROUGH NORTH WALL. ORIGINAL LOUVERED DOORS FRAME CLOSET AT PHOTO LEFT. VIEW TO NORTH. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  14. 37. SECOND FLOOR WEST SIDE APARTMENT EAST BEDROOM INTERIOR SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    37. SECOND FLOOR WEST SIDE APARTMENT EAST BEDROOM INTERIOR SHOWING PAIRED 6-LIGHT OVER 6-LIGHT DOUBLE-HUNG, WOOD-FRAME WINDOWS ON NORTH WALL. VIEW TO NORTHEAST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  15. 16. FIRST FLOOR APARTMENT KITCHEN INTERIOR SHOWING OPEN DOORWAY TO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    16. FIRST FLOOR APARTMENT KITCHEN INTERIOR SHOWING OPEN DOORWAY TO LIVING ROOM AND PAIRED 6-LIGHT OVER 6-LIGHT DOUBLE-HUNG, WOOD-FRAME WINDOWS OVER SINK. VIEW TO EAST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  16. 31. SECOND FLOOR WEST SIDE APARTMENT LIVING ROOM INTERIOR SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. SECOND FLOOR WEST SIDE APARTMENT LIVING ROOM INTERIOR SHOWING PAIRED 4-LIGHT OVER 4-LIGHT DOUBLE-HUNG, WOOD-FRAME WINDOWS FLANKING ENTRY DOOR WITH UNUSUAL 8-LIGHT WINDOW. OPEN DOORWAY TO PHOTO LEFT LEADS TO KITCHEN. VIEW TO WEST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  17. 38. SECOND FLOOR WEST SIDE APARTMENT WEST BEDROOM INTERIOR SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    38. SECOND FLOOR WEST SIDE APARTMENT WEST BEDROOM INTERIOR SHOWING PAIRED 6-LIGHT OVER 6-LIGHT DOUBLE-HUNG, WOOD-FRAME WINDOWS ON WEST WALL AND OPEN DOORWAY TO LIVING ROOM. VIEW TO WEST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  18. 24. SECOND FLOOR EAST SIDE APARTMENT LIVING ROOM INTERIOR SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. SECOND FLOOR EAST SIDE APARTMENT LIVING ROOM INTERIOR SHOWING DOORWAY INTO KITCHEN AT PHOTO CENTER LEFT AND OPEN DOORWAY INTO BATHROOM AT PHOTO RIGHT. VIEW TO SOUTHWEST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  19. 32. SECOND FLOOR WEST SIDE APARTMENT LIVING ROOM INTERIOR SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    32. SECOND FLOOR WEST SIDE APARTMENT LIVING ROOM INTERIOR SHOWING DOORWAY INTO KITCHEN AT PHOTO CENTER RIGHT, AND OPEN DOORWAY IN BATHROOM AT PHOTO LEFT. VIEW TO SOUTHWEST. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  20. 25. SECOND FLOOR EAST SIDE APARTMENT KITCHEN INTERIOR SHOWING GROUP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    25. SECOND FLOOR EAST SIDE APARTMENT KITCHEN INTERIOR SHOWING GROUP OF THREE 6-LIGHT WOOD-FRAME CASEMENT WINDOWS OVER THE SINK, AND OPEN DOORWAY TO TOP OF EXTERIOR STAIR LANDING AND WALKWAY AT REAR OF HOUSE. WALKWAY IS VISIBLE THROUGH KITCHEN WINDOWS. VIEW TO SOUTH. - Lee Vining Creek Hydroelectric System, Triplex Cottage, Lee Vining Creek, Lee Vining, Mono County, CA

  1. Triggered Excited-State Intramolecular Proton Transfer Fluorescence for Selective Triplex DNA Recognition.

    PubMed

    Wang, Ying; Hu, Yuehua; Wu, Tao; Zhou, Xiaoshun; Shao, Yong

    2015-12-01

    The triplex DNA has received much interest due to its various applications in gene regulation, molecular switch, and sensor development. However, realizing a highly selective recognition using a fluorescence probe specific only for the triplex topology is still a great challenge. Herein, we found that relative to the structural analogues of natural robinetin, myricetin, quercetin, kaempferol, morin, rutin, baicalin, luteolin, naringenin, genistein, chrysin, galangin, isorhamnetin, and several synthetic flavonoids, fisetin (FIS) is the brightest emitter when targeting the triplex DNA in contrast to binding with ss-DNA, ds-DNA (with or without an abasic site), i-motif, and DNA/RNA G-quadruplexes. Only the triplex association triggers the FIS green fluorescence that is relaxed from the tautomer favorable for excited-state intramolecular proton transfer (ESIPT). FIS can stabilize the triplex structure and primarily interact with the two terminals of the triplex via a 2:1 binding mode. This work demonstrates the potential of FIS as a DNA structure-selective switch-on ESIPT probe when evolving the triplex-forming oligonucleotides and developing the novel triplex-based sensors and switches. PMID:26556582

  2. Potent triple helix stabilization by 5',3'-modified triplex-forming oligonucleotides.

    PubMed

    Ben Gaied, Nouha; Zhao, Zhengyun; Gerrard, Simon R; Fox, Keith R; Brown, Tom

    2009-07-20

    Anthraquinone and pyrene analogues attached to the 3' and/or 5' termini of triplex-forming oligonucleotides (TFOs) by various linkers increased the stability of parallel triple helices. The modifications are simple to synthesize and can be introduced during standard solid-phase oligonucleotide synthesis. Potent triplex stability was achieved by using doubly modified TFOs, which in the most favourable cases gave an increase in melting temperature of 30 degrees C over the unmodified counterparts and maintained their selectivity for the correct target duplex. Such TFOs can produce triplexes with melting temperatures of 40 degrees C at pH 7 even though they do not contain any triplex-stabilizing base analogues. These studies have implications for the design of triplex-forming oligonucleotides for use in biology and nanotechnology. PMID:19554592

  3. Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

    PubMed Central

    Buske, Fabian A.; Bauer, Denis C.; Mattick, John S.; Bailey, Timothy L.

    2012-01-01

    Double-stranded DNA is able to form triple-helical structures by accommodating a third nucleotide strand in its major groove. This sequence-specific process offers a potent mechanism for targeting genomic loci of interest that is of great value for biotechnological and gene-therapeutic applications. It is likely that nature has leveraged this addressing system for gene regulation, because computational studies have uncovered an abundance of putative triplex target sites in various genomes, with enrichment particularly in gene promoters. However, to draw a more complete picture of the in vivo role of triplexes, not only the putative targets but also the sequences acting as the third strand and their capability to pair with the predicted target sites need to be studied. Here we present Triplexator, the first computational framework that integrates all aspects of triplex formation, and showcase its potential by discussing research examples for which the different aspects of triplex formation are important. We find that chromatin-associated RNAs have a significantly higher fraction of sequence features able to form triplexes than expected at random, suggesting their involvement in gene regulation. We furthermore identify hundreds of human genes that contain sequence features in their promoter predicted to be able to form a triplex with a target within the same promoter, suggesting the involvement of triplexes in feedback-based gene regulation. With focus on biotechnological applications, we screen mammalian genomes for high-affinity triplex target sites that can be used to target genomic loci specifically and find that triplex formation offers a resolution of ∼1300 nt. PMID:22550012

  4. Triplex inducer-directed self-assembly of single-walled carbon nanotubes: a triplex DNA-based approach for controlled manipulation of nanostructures

    PubMed Central

    Zhao, Chao; Qu, Konggang; Xu, Can; Ren, Jinsong; Qu, Xiaogang

    2011-01-01

    As a promising strategy for artificially control of gene expression, reversible assembly of nanomaterials and DNA nanomachine, DNA triplex formation has received much attention. Carbon nanotubes as gene and drug delivery vector or as ‘building blocks’ in nano/microelectronic devices have been successfully explored. Therefore, studies on triplex DNA-based carbon nanotube hybrid materials are important for development of smart nanomaterials and for gene therapy. In this report, a small molecule directed single-walled carbon nanotubes (SWNTs) self-assembly assay has been developed by disproportionation of SWNTs–dT22·dA22 duplex into triplex dT22·dA22·dT22 and dA22 by a triplex formation inducer, coralyne. This has been studied by circular dichroism, light scattering (LS) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), electrophoretic mobility shift assay and supported by using DNA random sequence. In contrast, SWNTs do not aggregate under the same experimental conditions when the small molecules used can not induce dT22·dA22·dT22 triplex formation. Therefore, this novel small molecule-directed SWNTs self-assembly assay has also been used for screening of triplex inducers in our studies. PMID:21227925

  5. Design optimization of integrated BiDi triplexer optical filter based on planar lightwave circuit

    NASA Astrophysics Data System (ADS)

    Xu, Chenglin; Hong, Xiaobin; Huang, Wei-Ping

    2006-05-01

    Design optimization of a novel integrated bi-directional (BiDi) triplexer filter based on planar lightwave circuit (PLC) for fiber-to-the premise (FTTP) applications is described. A multi-mode interference (MMI) device is used to filter the up-stream 1310nm signal from the down-stream 1490nm and 1555nm signals. An array waveguide grating (AWG) device performs the dense WDM function by further separating the two down-stream signals. The MMI and AWG are built on the same substrate with monolithic integration. The design is validated by simulation, which shows excellent performance in terms of filter spectral characteristics (e.g., bandwidth, cross-talk, etc.) as well as insertion loss.

  6. Design optimization of integrated BiDi triplexer optical filter based on planar lightwave circuit.

    PubMed

    Xu, Chenglin; Hong, Xiaobin; Huang, Wei-Ping

    2006-05-29

    Design optimization of a novel integrated bi-directional (BiDi) triplexer filter based on planar lightwave circuit (PLC) for fiber-to-the premise (FTTP) applications is described. A multi-mode interference (MMI) device is used to filter the up-stream 1310nm signal from the down-stream 1490nm and 1555nm signals. An array waveguide grating (AWG) device performs the dense WDM function by further separating the two down-stream signals. The MMI and AWG are built on the same substrate with monolithic integration. The design is validated by simulation, which shows excellent performance in terms of filter spectral characteristics (e.g., bandwidth, cross-talk, etc.) as well as insertion loss. PMID:19516623

  7. Fluorescent 2-Aminopyridine Nucleobases for Triplex-Forming Peptide Nucleic Acids.

    PubMed

    Cheruiyot, Samwel K; Rozners, Eriks

    2016-08-17

    Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex-forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2-aminopyridine (M) nucleobase to a G-C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3-(1-phenylethynyl)-M and phenylpyrrolo-M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple-helix formation with cognate double-stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3-(1-phenylethynyl)-M may be used as a fluorescent nucleobase to study PNA-RNA triple-helix formation. PMID:27223320

  8. Synthesis and triplex formation of oligonucleotides containing 8-thioxodeoxyadenosine and 5-methyl-2-thiodeoxycytosine.

    PubMed

    Ohkubo, Akihiro; Miyata, Kenichi; Tsunoda, Hirosuke; Seio, Kohji; Sekine, Mitsuo

    2009-01-01

    For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides (TFO) containing 8-thioxodeoxyadenine (s(8)A) residues in place of the protonated cytosines (Cs) required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)A exhibited much stronger hybridization ability than C under neutral conditions when four s(8)A bases were arranged in a consecutive sequence. Moreover, we also synthesized TFOs containing 5-methyl-2-thiocytosines and examined their ability of triplex formation. PMID:19749240

  9. Transposon Tn7 Preferentially Inserts into GAA•TTC Triplet Repeats under Conditions Conducive to Y•R•Y Triplex Formation

    PubMed Central

    Mancuso, Miriam; Sammarco, Mimi C.; Grabczyk, Ed

    2010-01-01

    Background Expansion of an unstable GAA•TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Structure formation by the repeat has been implicated in both frataxin repression and GAA•TTC instability. The GAA•TTC sequence is capable of adopting multiple non-B DNA structures including Y•R•Y and R•R•Y triplexes. Lower pH promotes the formation of Y•R•Y triplexes by GAA•TTC. Here we used the bacterial transposon Tn7 as an in vitro tool to probe whether GAA•TTC repeats can attract a well-characterized recombinase. Methodology/Principal Findings Tn7 showed a pH-dependent preference for insertion into uninterrupted regions of a Friedreich ataxia patient-derived repeat, inserting 48, 39 and 14 percent of the time at pH 7, pH 8 and pH 9, respectively. Moreover, Tn7 also showed orientation and region specific insertion within the repeat at pH 7 and pH 8, but not at pH 9. In contrast, transposon Tn5 showed no strong preference for or against the repeat during in vitro transposition at any pH tested. Y•R•Y triplex formation was reduced in predictable ways by transposon interruption of the GAA•TTC repeat. However, transposon interruptions in the GAA•TTC repeats did not increase the in vitro transcription efficiency of the templates. Conclusions/Significance We have demonstrated that transposon Tn7 will recognize structures that form spontaneously in GAA•TTC repeats and insert in a specific orientation within the repeat. The conditions used for in vitro transposition span the physiologically relevant range suggesting that long GAA•TTC repeats can form triplex structures in vivo, attracting enzymes involved in DNA repair, recombination and chromatin modification. PMID:20559546

  10. Synthesis of C-5, C-2' and C-4'-neomycin-conjugated triplex forming oligonucleotides and their affinity to DNA-duplexes.

    PubMed

    Tähtinen, Ville; Granqvist, Lotta; Virta, Pasi

    2015-08-01

    Neomycin-conjugated homopyrimidine oligo 2'-deoxyribonucleotides have been synthesized on a solid phase and their potential as triplex forming oligonucleotides (TFOs) with DNA-duplexes has been studied. For the synthesis of the conjugates, C-5, C-2' and C-4'-tethered alkyne-modified nucleoside derivatives were used as an integral part of the standard automated oligonucleotide chain elongation. An azide-derived neomycin was then conjugated to the incorporated terminal alkynes by Cu(I)-catalyzed 1,3-dipolar cycloaddition (the click chemistry). Concentrated ammonia released the desired conjugates in acceptable purity and yields. The site of conjugation was expectedly important for the Hoogsteen-face recognition: C-5-conjugation showed a notable positive effect, whereas the influence of the C-2' and C-4'-modification remained marginal. In addition to conventional characterization methods (UV- and CD-spectroscopy), (19)F NMR spectroscopy was applied for the monitoring of triplex/duplex/single strand-conversions. PMID:26118338

  11. Tethered naphthalene diimide-based intercalators for DNA triplex stabilization

    PubMed Central

    Gianolio, Diego A.; Segismundo, Joanna M.; McLaughlin, Larry W.

    2000-01-01

    The synthesis and triplex stabilizing properties of oligodeoxyribonucleotides functionalized at the 5′- and/or 3′-termini with a naphthalene diimide-based (NDI) intercalator is described. The NDI intercalator was prepared in a single step from the corresponding dianhydride and was attached to the 5′-terminus of an oligodeoxyribonucleotide following a reverse coupling procedure. The DMT protecting group was removed and the sequence phosphitylated to generate the phosphoramidite derivative on the 5′-terminus of the support-bound oligodeoxyribonucleotide. The NDI intercalator with a free hydroxyl was then added in the presence of tetrazole. Attachment of the NDI to the 3′-terminus relied upon a tethered amino group that could be functionalized first with the naphthalene dianhydride, which was subsequently converted to the diimide. Using both procedures, an oligonucleotide conjugate was prepared having the NDI intercalator at both the 5′- and 3′-termini. Thermal denaturation studies were used to determine the remarkable gain in stability for triplexes formed when the NDI-conjugated oligonucleotide was present as the third strand in the complex. PMID:10773082

  12. DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity.

    PubMed

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao; He, Yuxian; Liu, Keliang

    2015-08-01

    DNA triplexes with hydrophobic modifications were designed and evaluated for their activity as inhibitors of the cell fusion of human immunodeficiency virus type 1 (HIV-1). Triplex inhibitors displayed low micromolar activities in the cell-cell fusion assay and nanomolar activities in the anti-HIV-1 pseudovirus test. Helix structure and the presence of sufficient numbers of hydrophobic regions were essential for the antifusion activity. Results from native polyacrylamide gel electrophoresis and a fluorescent resonance energy transfer-based inhibitory assay indicated that these triplexes may interact with the primary pocket at the glycoprotein 41 (gp41) N-heptad repeat, thereby inhibiting formation of the HIV-1 gp41 6-helical bundle. Triplex-based complexes may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery. PMID:26192705

  13. Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA.

    PubMed

    Sau, Sujay P; Kumar, Pawan; Anderson, Brooke A; Østergaard, Michael E; Deobald, Lee; Paszczynski, Andrzej; Sharma, Pawan K; Hrdlicka, Patrick J

    2009-11-28

    Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases. PMID:19885469

  14. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

    PubMed Central

    Mukherjee, Anirban; Vasquez, Karen M.

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  15. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  16. Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

    PubMed Central

    Liu, Jing; Xu, Ren-Huan; Jin, You-Xin; Wang, De-Bao

    2001-01-01

    Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed. PMID:11160902

  17. Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides

    PubMed Central

    Zhou, Yuan; Kierzek, Elzbieta; Loo, Zi Ping; Antonio, Meraldo; Yau, Yin Hoe; Chuah, York Wieo; Geifman-Shochat, Susana; Kierzek, Ryszard; Chen, Gang

    2013-01-01

    Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2′-O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine–purine–pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2′-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA. PMID:23658228

  18. Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides.

    PubMed

    Zhou, Yuan; Kierzek, Elzbieta; Loo, Zi Ping; Antonio, Meraldo; Yau, Yin Hoe; Chuah, York Wieo; Geifman-Shochat, Susana; Kierzek, Ryszard; Chen, Gang

    2013-07-01

    Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2'-O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine-purine-pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2'-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA. PMID:23658228

  19. Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence.

    PubMed

    Shchyolkina, Anna K; Kaluzhny, Dmitry N; Borisova, Olga F; Arndt-Jovin, Donna J; Jovin, Thomas M; Zhurkin, Victor B

    2016-06-01

    Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na(+) than for Li(+). Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance. PMID:26308235

  20. Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy.

    PubMed

    Hu, Hongyan; Huang, Xiangyi; Ren, Jicun

    2016-05-01

    Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA-based nanomaterials because of its direct recognition of natural double-stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single-strand DNA (ssDNA) fluorescent probes and fluorescent probe-double-strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (Ka ) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20-mer perfectly matched ssDNA probe and three single-base mismatched ssDNA probes with 146-mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single-base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26377428

  1. Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence

    PubMed Central

    Shchyolkina, Anna K.; Kaluzhny, Dmitry N.; Borisova, Olga F.; Arndt-Jovin, Donna J.; Jovin, Thomas M.; Zhurkin, Victor B.

    2016-01-01

    Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na+ than for Li+. Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance. PMID:26308235

  2. A Triplex Ribozyme Expression System Based on a Single Hairpin Ribozyme

    PubMed Central

    Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A.

    2008-01-01

    Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with trans-cleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies. PMID:18707243

  3. A triplex ribozyme expression system based on a single hairpin ribozyme.

    PubMed

    Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

    2008-09-01

    Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies. PMID:18707243

  4. Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks

    PubMed Central

    Lange, Sabine S.; Reddy, Madhava C.; Vasquez, Karen M.

    2009-01-01

    Psoralen is a chemotherapeutic agent that acts by producing DNA interstrand crosslinks (ICLs), which are especially cytotoxic and mutagenic because their complex chemical nature makes them difficult to repair. Proteins from multiple repair pathways, including nucleotide excision repair (NER), are involved in their removal in mammalian cells, but the exact nature of their repair is poorly understood. We have shown previously that HMGB1, a protein involved in chromatin structure, transcriptional regulation, and inflammation, can bind cooperatively to triplex-directed psoralen ICLs with RPA, and that mammalian cells lacking HMGB1 are hyper-sensitive to psoralen ICLs. However, whether this effect is mediated by a role for HMGB1 in DNA damage recognition is still unknown. Given HMGB1’s ability to bind to damaged DNA and its interaction with the RPA protein, we hypothesized that HMGB1 works together with the NER damage recognition proteins to aid in the removal of ICLs. We show here that HMGB1 is capable of binding to triplex-directed psoralen ICLs with the dedicated NER damage recognition complex XPC-RAD23B, as well as RPA, and that they form a high molecular weight complex on these lesions. In addition, we demonstrate that HMGB1 interacts with XPC-RAD23B and XPA in the absence of DNA. These findings directly demonstrate interactions between HMGB1 and the NER damage recognition proteins, and suggest that HMGB1 may affect ICL repair by enhancing the interactions between NER damage recognition factors. PMID:19446504

  5. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

    PubMed Central

    Kolganova, N. A.; Shchyolkina, A. K.; Chudinov, A. V.; Zasedatelev, A. S.; Florentiev, V. L.; Timofeev, E. N.

    2012-01-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  6. Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

    PubMed

    Holder, Isabelle T; Wagner, Stefanie; Xiong, Peiwen; Sinn, Malte; Frickey, Tancred; Meyer, Axel; Hartig, Jörg S

    2015-12-01

    Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon. PMID:26450966

  7. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases.

    PubMed

    Rogers, Faye A; Lloyd, Janice A; Tiwari, Meetu Kaushik

    2014-01-01

    Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis. PMID:25483840

  8. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides.

    PubMed

    Kolganova, N A; Shchyolkina, A K; Chudinov, A V; Zasedatelev, A S; Florentiev, V L; Timofeev, E N

    2012-09-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  9. Mechanisms of triplex DNA-mediated inhibition of transcription initiation in cells.

    PubMed

    Jain, Aklank; Magistri, Marco; Napoli, Sara; Carbone, Giuseppina M; Catapano, Carlo V

    2010-03-01

    Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF(II)130 and TAF(II)250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells. PMID:20045441

  10. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases

    PubMed Central

    Rogers, Faye A; Lloyd, Janice A; Tiwari, Meetu Kaushik

    2014-01-01

    Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis. PMID:25483840

  11. Intrastrand triplex DNA repeats in bacteria: a source of genomic instability

    PubMed Central

    Holder, Isabelle T.; Wagner, Stefanie; Xiong, Peiwen; Sinn, Malte; Frickey, Tancred; Meyer, Axel; Hartig, Jörg S.

    2015-01-01

    Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon. PMID:26450966

  12. Theoretical studies on conformation comparison of braid-like and triplex DNA

    NASA Astrophysics Data System (ADS)

    Yang, Linjing; Bai, Chunli; Liu, Ciquan; Shi, Xiufan; Lee, Imshik

    1999-03-01

    Based on the experimental data of scanning tunneling microscopy (STM), models of three-stranded braid-like DNAs composed by three kinds of base triplets AAA, TAT and GCA were constructed. We investigated the braid-like DNAs and their comparative triplex DNAs using a molecular mechanics method. The three strands of braid-like DNAs are proven equivalent, while those of triplex DNAs are not. The conformational energies for braid-like DNAs were found to be higher than that for triplex DNAs. Each period in one strand of braid-like DNA has 18 nucleotides, half of which are right-handed, while the other half are left-handed. Additional discussions concerning sugar puckering modes and the H-bonds are also included.

  13. New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction.

    PubMed

    Mizuta, Masahiro; Banba, Jun-ichi; Kanamori, Takashi; Tawarada, Ryuya; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2008-07-30

    New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies. PMID:18611007

  14. Triplex-directed recognition of a DNA nanostructure assembled by crossover strand exchange.

    PubMed

    Rusling, David A; Nandhakumar, Iris S; Brown, Tom; Fox, Keith R

    2012-04-24

    DNA has been widely exploited for the self-assembly of nanosized objects and arrays that offer the potential to act as scaffolds for the spatial positioning of molecular components with nanometer precision. Methods that allow the targeting of components to specific locations within these structures are therefore highly sought after. Here we report that the triplex approach to DNA recognition, which relies on the specific binding of an oligonucleotide within the major groove of double-helical DNA, can be exploited to recognize specific loci within a DNA double-crossover tile and array, a nanostructure assembled by crossover strand exchange. The oligonucleotide can be targeted to both crossover and non-crossover strands and, surprisingly, across the region spanning the crossover junction itself. Moreover, by attaching biotin to the end of the oligonucleotide, we show that streptavidin molecules can be recruited to precise locations within a DX array, with an average spacing of 31.9 (±1.3) nm. This is a promising approach that could be exploited to introduce other components compatible with oligonucleotide synthesis into the wide variety of DNA nanostructures assembled by crossover strand exchange, such as those generated by DNA origami. PMID:22443318

  15. Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

    PubMed Central

    Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

    2013-01-01

    Fluorescent probes for the detection of a double-stranded DNA were prepared by labeling a triplex-forming DNA oligonucleotide with a thiazole orange (TO) dimer unit. They belong to ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes which we have previously reported. The excitonic interaction between the two TO molecules was expected to effectively suppress the background fluorescence of the probes. The applicability of the ECHO probes for the detection of double-stranded DNA was confirmed by examining the thermal stability and photophysical and kinetic properties of the DNA triplexes formed by the ECHO probes. PMID:23445822

  16. Triplex formation by a psoralen-conjugated oligodeoxyribonucleotide containing the base analog 8-oxo-adenine.

    PubMed Central

    Miller, P S; Bi, G; Kipp, S A; Fok, V; DeLong, R K

    1996-01-01

    Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents. PMID:8604317

  17. Long-Term Observation of Triplex Surgery for Cataract after Phakic 6H Implantation for Super High Myopia

    PubMed Central

    Liu, Xin; Wang, Xiaoying; Lu, Yi; Zheng, Tianyu; Zhou, Xingtao

    2016-01-01

    Purpose. To analyze the safety, effectiveness, and stability of triplex surgery for phakic 6H anterior chamber phakic intraocular lens explantation and phacoemulsification with in-the-bag IOL implantation for super high myopia in long-term observations. Methods. This retrospective case series evaluated 16 eyes of 10 patients who underwent triplex surgery. Best corrected visual acuity (BCVA), endothelial cell density (ECD), and associated adverse events were evaluated. Results. The mean follow-up time after the triplex surgery was 46 ± 14 months. The mean logMAR BCVA was significantly improved after triplex surgery (P = 0.047). One eye developed endophthalmitis five days postoperatively and underwent pars plana vitrectomy (PPV). Five eyes with preoperative severe endothelial cell loss developed corneal decompensation and underwent keratoplasty at a mean time of 9.4 ± 2.6 months after the triplex surgery. One eye had graft failure and underwent a second keratoplasty. The eye developed rhegmatogenous retinal detachment and underwent PPV with silicone oil 18 months later. ECD before the triplex surgery was not significantly different compared with that at last follow-up (P = 0.495) apart from these five eyes. Three eyes (18.8%) developed posterior capsule opacification. Conclusions. Triplex surgery was safe and effective for phakic 6H related complicated cataracts. Early extraction before severe ECD loss is recommended. PMID:27190642

  18. Triplex staples: DNA double-strand cross-linking at internal and terminal sites using psoralen-containing triplex-forming oligonucleotides.

    PubMed

    Li, Hong; Broughton-Head, Victoria J; Peng, Guomei; Powers, Vicki E C; Ovens, Matthew J; Fox, Keith R; Brown, Tom

    2006-01-01

    A method has been developed to attach 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen to the 5 position of thymine bases during solid-phase oligonucleotide synthesis. UV irradiation of triplex-forming oligonucleotides (TFOs) containing internally attached psoralens produces photoadducts at TpA steps within target duplexes, thus relaxing the constraints on selection of psoralen target sequences. Photoreaction of TFOs containing two psoralens, located at the 5'- and 3'-ends, has been used to create double-strand cross-links (triplex staples) at both termini of the TFO. Such complexes have no free single-stranded ends. TFOs containing 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, 3-methyl-2-aminopyridine, and 5-(3-aminoprop-2-ynyl)deoxyuridine formed photoadducts with target duplexes under near-physiological conditions. PMID:17105237

  19. Improved synthesis of daunomycin conjugates with triplex-forming oligonucleotides. The polypurine tract of HIV-1 as a target.

    PubMed

    Capobianco, Massimo L; De Champdoré, Marcella; Arcamone, Federico; Garbesi, Anna; Guianvarc'h, Dominique; B Arimondo, Paola

    2005-05-01

    Triple helix-forming oligonucleotides (TFOs) are promising agents for the control of gene expression, as they can selectively bind to a chosen oligopyrimidine.oligopurine region of a gene of interest thus interfering with its expression. The stability of the triplex formed by the TFO and the duplex is often too poor for successful applications of TFOs in vivo and the conjugation of a DNA intercalating moiety to the TFO is a common way to enhance the TFO affinity for its target. In a previous work, we investigated the properties of daunomycin conjugated TFO (dauno-TFO) and found that this class of compounds showed a higher degree of affinity than native oligonucleotides for an oligopyrimidine.oligopurine duplex target and that the presence of the amino sugar increases such stability. Here, we report a significantly improved synthetic procedure for the preparation of the conjugates, based on the protection of the daunosamine moiety by N-trifluoroacetylation. This protecting group is removed as a final step from the conjugation product by mild basic hydrolysis to give the desired dauno-TFO. Compared to the previous synthetic procedure, the improvement is important. The synthesis is now more reproducible and no side products are formed. Moreover, the thus protected daunomycin derivative is very stable, up to at least one year. Two dauno-TFOs, differing by the length of the oligonucleotide moiety, were prepared to target the polypurine tract (PPT) of HIV-1. Triplex formation by these compounds with model duplexes was studied by UV spectroscopy, thermal gradient gel electrophoresis (TGGE) and gel electrophoretic mobility shift. The experimental results demonstrate that dauno-TFOs bind to the PPT of HIV-1 more strongly than the unconjugated TFOs. PMID:15809156

  20. Direct visualization of triplex DNA molecular dynamics by fluorescence resonance energy transfer and atomic force microscopy measurements

    NASA Astrophysics Data System (ADS)

    Chang, Chia-Ching; Lin, Po-Yen; Chen, Yen-Fu; Chang, Chia-Seng; Kan, Lou-Sing

    2007-11-01

    We have detected the dynamics of 17-mer DNA triplex dissociation mechanism at the molecular level. Fluorescence resonance energy transfer (FRET) was used as an indicator of intermolecular interaction in nanometer range, whereas atomic force microscopy (AFM) was employed to address single molecule with sub-angstrom precision. The maximum rupture force of DNA triplex was found at pH 4.65, consistent with macroscopic observations. These results indicated that the FRET together with an AFM detection system could be used to reveal the DNA triplex interaction in nanometer scale unambiguously.

  1. Remarkable stabilization of antiparallel DNA triplexes by strong stacking effects of consecutively modified nucleobases containing thiocarbonyl groups.

    PubMed

    Yamada, Kenji; Hattori, Yusaku; Inde, Takeshi; Kanamori, Takashi; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2013-02-01

    The consecutive arrangement of 2'-deoxy-6-thioguanosines (s(6)Gs) and 4-thiothymidines (s(4)Ts) in antiparallel triplex-forming oligonucleotides (TFOs) considerably stabilized the resulting antiparallel triplexes with high base recognition ability by the strong stacking effects of thiocarbonyl groups. This result was remarkable because chemical modifications of the sugar moieties and nucleobases of antiparallel TFOs generally destabilize triplex structures. Moreover, in comparison with unmodified TFOs, it was found that TFOs containing s(6)Gs and s(4)Ts could selectively bind to the complementary DNA duplex but not to mismatched DNA duplexes or single-stranded RNA. PMID:23287737

  2. Inhibition of transcription by platinated triplex-forming oligonucleotides.

    PubMed

    Graham, Mindy K; Miller, Paul S

    2012-12-01

    Platinated triplex-forming oligonucleotides (TFOs) consisting of 2'-methoxythymidine and 2'-methoxy-5-methylcytidine and an N-7 platinated deoxyguanosine ((Pt)G) at the 5'-((Pt)G-TFO), 3'-(TFO-G(Pt)), or 3'- and 5'-((Pt)G-TFO-G(Pt)) ends of the TFO form mono-((Pt)G-TFO and TFO-G(Pt)) and interstrand ((Pt)G-TFO-G(Pt)) cross-links with target DNA as a result of reaction of the (Pt)G with guanines adjacent to the homopurine TFO binding site in the target. The extent of cross-linking is greatest when the (Pt)G is located on the 3' end of the TFO and the target guanine is on the same strand as the TFO binding site. Multiple, contiguous deoxyguanosines in the TFO binding site or a cytosine adjacent to the G(Pt) of the TFO significantly reduce cross-linking. DNA reporter plasmids in which platinated TFOs were cross-linked at a site in the transcribed region between a CMV promoter and a luciferase reporter gene were transfected into Chinese hamster ovary cells, and luciferase expression was compared with that for the corresponding non-cross-linked plasmid. Luciferase expression was inhibited 95 % when TFO-G(Pt) was bound and cross-linked to the transcribed strand, demonstrating that the cross-linked TFO was able to block transcription elongation. Further inhibition (99 %) was observed in nucleotide excision repair (NER) deficient cells, suggesting that NER may repair this lesion. The 3'-G(Pt) group of TFO-G(Pt) protects the TFO from degradation by exonucleases found in mammalian serum. Taken together, these results suggest that platinated TFOs of the type TFO-G(Pt) may find applications as agents for suppressing DNA transcription and consequently inhibiting gene expression in mammalian cells. PMID:22965663

  3. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity.

    PubMed

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M

    2015-12-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA-RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA-RNA and RNA-RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA-RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  4. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity

    PubMed Central

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M.

    2015-01-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA–RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA–RNA and RNA–RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA–RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  5. Development of artificial nucleic acid that recognizes a CG base pair in triplex DNA formation.

    PubMed

    Hari, Yoshiyuki

    2013-01-01

    An oligonucleotide that can form a triplex with double-stranded DNA is called a triplex-forming oligonucleotide (TFO). TFOs have gained considerable attention because of their potential as gene targeting tools. However, triplex DNA formation involves inherent problems for practical use. The most important problem is that natural nucleotides in TFO do not have sufficient affinity and base pair-selectivity to pyrimidine-purine base pair, like a CG or TA base pair, within dsDNA. This suggests that dsDNA region including a CG or TA base pair cannot be targeted. Therefore, artificial nucleotides, especially with non-natural nucleobases, capable of direct recognition of a CG or TA base pair via hydrogen bond formation have been developed; however, nucleotides with better selectivity and stronger affinity are necessary for implementing this dsDNA-targeting technology using TFOs. Under such a background, we considered that facile and efficient synthesis of various nucleobase derivatives in TFOs would be useful for finding an ideal nucleobase for recognition of a CG or TA base pair because detailed and rational exploration of nucleobase structures is facilitated. Recently, to develop a nucleobase recognizing a CG base pair, we have used post-elongation modification, i.e., modification after oligonucleotide synthesis, for the facile synthesis of nucleobase derivatives. This review mainly summarizes our recent findings on the development of artificial nucleobases and nucleotides for recognition of a CG base pair in triplexes formed between dsDNA and TFOs. PMID:24189561

  6. Recognition of triplex forming oligodeoxynucleotides incorporating abasic sites by 5-arylcytosine residues in duplex DNAs.

    PubMed

    Mizuta, Masahiro; Banba, Jun-Ichi; Kanamori, Takashi; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2007-01-01

    In this paper, we reported our attempt to use a 5arylcytosine (dC(ar)) and the abasic site () as an artificial base pair for DNA triplex. The idea was confirmed by the molecular modeling studied in which the aromatic group of (ph) which protrudes in the major groove was buried into the cleft formed by the residue in the TFO. We synthesized three kinds of dC(ar) and the oligonucleotides incorporating them. Our UV-melting experiments revealed that the DNA triplex containing the dC(ph).phi was more stable than that containing dC.phi pair. Moreover, the dC.phi pair was more stable than any other dC.Y pairs such as dC(ph).G, dC(ph).C, dC(ph).T and dC(ph).A. These results indicated the possibility that the appropriate pair of dC(Ar) and could be the new sequence code of DNA triplex. We also carried out the Tm analyses of other TFOs incorporating dC(Ar) and , and clarified the stability of these triplexes. PMID:18029568

  7. Development and Physical Characteristics of Novel Zero-Porosity Vascular Graft “Triplex®”

    PubMed Central

    Takamoto, Shinichi

    2013-01-01

    We developed a novel large-diameter graft “Triplex®” that uses a non-biodegradable material as a coating material. This time, in order to demonstrate the physical properties of Triplex® grafts, we conducted physical tests in accordance with the international guidelines, using the collagen coated vascular grafts (Hemashield, Boston Scientific, Natick, Massachusetts, USA) as the controls. The grafts were tested with regard to strength (burst strength, circumferential tensile strength, longitudinal tensile strength), suture retention strength, integral water permeability, water leakage (needle puncture, after using clamp), and change in luminal diameter following pacing stress according to ISO7198 and FDA guidance. As indicated by the results, we experimentally demonstrated that uniquely designed vascular graft Triplex® led to less blood leakage from the vascular graft and less leakage from the needle puncture, although it has fundamental physical properties comparable to those of the vascular grafts using biodegradable material that has been utilized conventionally in clinical settings. Triplex ®is expected to play its role as a clinically beneficial next-generation vascular graft. PMID:23641287

  8. DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene

    PubMed Central

    Carbone, Giuseppina M.; McGuffie, Eileen; Napoli, Sara; Flanagan, Courtney E.; Dembech, Chiara; Negri, Umberto; Arcamone, Federico; Capobianco, Massimo L.; Catapano, Carlo V.

    2004-01-01

    Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells. The 11mer daunomycin-conjugated GT (dauno-GT11) TFO targeted a sequence upstream of the P2 promoter, a site known to be critical for transcription of the c-myc gene. Band-shift assays showed that the dauno-GT11 formed triplex DNA with enhanced stability compared to the unmodified TFO. Band shift and footprinting experiments demonstrated that binding of dauno-GT11 was highly sequence-specific with exclusive binding to the 11 bp target site in the c-myc promoter. The daunomycin-conjugated TFO inhibited transcription in vitro and reduced c-myc promoter activity in prostate and breast cancer cells. The daunomycin-conjugated TFO was taken up by cells with a distinctive intracellular distribution compared to free daunomycin. However, cationic lipid-mediated delivery was required for enhanced cellular uptake, nuclear localization and biological activity of the TFO in cells. Dauno-GT11 reduced transcription of the endogenous c-myc gene in cells, but did not affect expression of non-target genes, such as ets-1 and ets-2, which contained very similar target sequences in their promoters. Daunomycin-conjugated control oligonucleotides unable to form triplex DNA with the target sequence did not have any effect in these assays, indicating that daunomycin was not directly responsible for the activity of daunomycin-conjugated TFO. Thus, attachment of daunomycin resulted in increased triplex stability and biological activity of the 11mer GT-rich TFO without

  9. Triplex molecular beacons for sensitive recognition of melamine based on abasic-site-containing DNA and fluorescent silver nanoclusters.

    PubMed

    Wang, Ya; Sun, Qianqian; Zhu, Linling; Zhang, Junying; Wang, Fengyang; Lu, Linlin; Yu, Haijun; Xu, Zhiai; Zhang, Wen

    2015-05-01

    A melamine aptamer derived from an abasic-site-containing triplex molecular beacon (tMB) was designed and developed for sensitive recognition of melamine by integrating tMBs and fluorescent silver nanoclusters (Ag NCs). PMID:25865656

  10. Aptamer-modified PLGA nanoparticle delivery of triplex forming oligonucleotide for targeted prostate cancer therapy.

    PubMed

    Jiao, J; Zou, Q; Zou, M H; Guo, R M; Zhu, S; Zhang, Y

    2016-01-01

    Presented study aimed to prepare A10 aptamer-modified poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles loaded with triplex forming oligonucleotides(TFO) for targeted prostate cancer therapy. We first synthesized a PLGA-PEG-Apt copolymer. The PLGA-PEG-Apt nanoparticles (NP-Apt) were loaded with TFO using double emulsion solvent evaporation method. Carboxy-fluorescein labeled TFO-NP-Apt, TFO-NP and TFO were prepared for cellular uptake experiments. Cell counting kit-8 (CCK-8) test was used to determine the ability of TFO-NP-Apt to inhibit LNCaP cell proliferation. RT-PCR and Western blot was conducted to analyze AR gene expressing. Then, a mouse model of prostate cancer was used to evaluate the anti-cancer effect of TFO-NP-Apt in vivo. We confirmed that the PLGA-PEG-Apt conjugation was successful. The TFO encapsulation efficiency and drug loading percentage were 46.1± 3.6% and 40.8±5.3%, respectively. TFO-NP-Apt showed a more efficient cellular uptake than TFO-NP or TFO in LNCaP cells. TFO-NP-Apt was significantly more cytotoxic than TFO-NP and TFO in the CCK-8 test (p<0.001). TFO-NP-Apt silenced the AR gene better than unconjugated Apt, naked TFO, NP or saline. TFO-NP-Apt were more effective than TFO-NP, naked TFO, NP and saline at inhibiting prostate cancer growth in vivo (p<0.05). Aptamer-modified TFO-loaded PLGA nanoparticles may prove useful in targeted therapy for advanced prostate cancer. PMID:27268920

  11. Stabilization of a Bimolecular Triplex by 3'-S-Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation.

    PubMed

    Evans, Kathryn; Bhamra, Inder; Wheelhouse, Richard T; Arnold, John R P; Cosstick, Richard; Fisher, Julie

    2015-05-01

    Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single-stranded nucleic acids are also relevant in third-strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson-Crick (W-C) base-paired DNA duplexes and a Hoogsteen base-paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA-like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3'-S-phosphorothiolate (3'-SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. (1) H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3'-SP modifications is more compact. The 3'-SP modification was previously shown to stabilise G-quadruplex and i-motif structures and therefore is now proposed as a generic solution to stabilising multi-stranded DNA structures. PMID:25802084

  12. Thermal stability of G-rich anti-parallel DNA triplexes upon insertion of LNA and α-L-LNA.

    PubMed

    Kosbar, Tamer R; Sofan, Mamdouh A; Abou-Zeid, Laila; Pedersen, Erik B

    2015-05-14

    G-rich anti-parallel DNA triplexes were modified with LNA or α-L-LNA in their Watson-Crick and TFO strands. The triplexes were formed by targeting a pyrimidine strand to a putative hairpin formed by Hoogsteen base pairing in order to use the UV melting method to evaluate the stability of the triplexes. Their thermal stability was reduced when the TFO strand was modified with LNA or α-L-LNA. The same trend was observed when the TFO strand and the purine Watson-Crick strand both were modified with LNA. When all triad components were modified with α-L-LNA and LNA in the middle of the triplex, the thermal melting was increased. When the pyrimidine sequence was modified with a single insertion of LNA or α-L-LNA the ΔTm increased. Moreover, increasing the number of α-L-LNA in the pyrimidine target sequence to six insertions, leads to a high increase in the thermal stability. The conformational S-type structure of α-L-LNA in anti-parallel triplexes is preferable for triplex stability. PMID:25833006

  13. Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides.

    PubMed

    Duca, Maria; Guianvarc'h, Dominique; Oussedik, Kahina; Halby, Ludovic; Garbesi, Anna; Dauzonne, Daniel; Monneret, Claude; Osheroff, Neil; Giovannangeli, Carine; Arimondo, Paola B

    2006-01-01

    Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning. PMID:16598074

  14. Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides

    PubMed Central

    Duca, Maria; Guianvarc'h, Dominique; Oussedik, Kahina; Halby, Ludovic; Garbesi, Anna; Dauzonne, Daniel; Monneret, Claude; Osheroff, Neil; Giovannangeli, Carine; Arimondo, Paola B.

    2006-01-01

    Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents ‘poison’ the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13–14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug–TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning. PMID:16598074

  15. "The Show"

    ERIC Educational Resources Information Center

    Gehring, John

    2004-01-01

    For the past 16 years, the blue-collar city of Huntington, West Virginia, has rolled out the red carpet to welcome young wrestlers and their families as old friends. They have come to town chasing the same dream for a spot in what many of them call "The Show". For three days, under the lights of an arena packed with 5,000 fans, the state's best…

  16. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India

    PubMed Central

    2016-01-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  17. A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.

    PubMed

    Burrell, Matthew R; Burton, Nicolas P; Maxwell, Anthony

    2010-01-01

    We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase. PMID:19997889

  18. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India.

    PubMed

    Rajput, Manoj Kumar

    2016-02-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  19. Transcriptional regulation mechanism mediated by miRNA-DNA•DNA triplex structure stabilized by Argonaute.

    PubMed

    Toscano-Garibay, Julia D; Aquino-Jarquin, Guillermo

    2014-11-01

    Transcription regulation depends on interactions between repressor or activator proteins with promoter sequences, while post-transcriptional regulation typically relies on microRNA (miRNA) interaction with sequences in 5' and 3'-Untranslated regions (UTRs) of messenger RNA (mRNA). However, several pieces of evidence suggest that miRNA:Argonaute (AGO) complexes may also suppress transcription through RNA interference (RNAi) components and epigenetic mechanisms. However, recent observations suggest that miRNA-induced transcriptional silencing could be exerted by an unknown mechanism independent of chromatin modifiers. The RNA-DNA•DNA triplex structure has emerged as an important RNA tertiary motif in which successive non-canonical base pairs form between a DNA-DNA duplex and a third strand. Frequently, promoters have Purine (PU)-rich tracts, and some Triplex-forming oligonucleotides (TFOs) targeting these regulatory regions have been shown to inhibit transcription selectively. Here, we summarize observations suggesting that miRNAs exert regulation over promoter regions through miRNA-DNA•DNA triplex structure formation stabilized by AGO proteins which represents a plausible model of RNA-mediated Transcriptional gene silencing (TGS). PMID:25086339

  20. Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA.

    PubMed

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W; Palleschi, Giuseppe; Ricci, Francesco

    2010-11-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  1. Structural determinants of photoreactivity of triplex forming oligonucleotides conjugated to psoralens.

    PubMed

    Krishnan, Rajagopal; Oh, Dennis H

    2010-01-01

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

  2. Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

    PubMed Central

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W.; Palleschi, Giuseppe; Ricci, Francesco

    2011-01-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe’s redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  3. Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

    PubMed Central

    Krishnan, Rajagopal; Oh, Dennis H.

    2010-01-01

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

  4. Unusual Thermal Stability of RNA/[RP-PS]-DNA/RNA Triplexes Containing a Homopurine DNA Strand

    PubMed Central

    Guga, Piotr; Boczkowska, Małgorzata; Janicka, Magdalena; Maciaszek, Anna; Kuberski, Sławomir; Stec, Wojciech J.

    2007-01-01

    Homopurine deoxyribonucleoside phosphorothioates, as short as hexanucleotides and possessing all internucleotide linkages of RP configuration, form a triple helix with two RNA or 2′-OMe-RNA strands, with Watson-Crick and Hoogsteen complementarity. Melting temperature and fluorescence quenching experiments strongly suggest that the Hoogsteen RNA strand is parallel to the homopurine [RP-PS]-oligomer. Remarkably, these triplexes are thermally more stable than complexes formed by unmodified homopurine DNA molecules of the same sequence. The triplexes formed by phosphorothioate DNA dodecamers containing 4–6 dG residues are thermally stable at pH 7.4, although their stability increases significantly at pH 5.3. FTIR measurements suggest participation of the C2-carbonyl group of the pyrimidines in the stabilization of the triplex structure. Formation of triple-helix complexes with exogenously delivered PS-oligos may become useful for the reduction of RNA accessibility in vivo and, hence, selective suppression/inhibition of the translation process. PMID:17218459

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

    PubMed

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

    2016-01-01

    The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes. PMID:26603177

  6. Modeling dissolved organic carbon in temperate forest soils: TRIPLEX-DOC model development and validation

    NASA Astrophysics Data System (ADS)

    Wu, H.; Peng, C.; Moore, T. R.; Hua, D.; Li, C.; Zhu, Q.; Peichl, M.; Arain, M. A.; Guo, Z.

    2014-05-01

    Even though dissolved organic carbon (DOC) is the most active carbon (C) cycling in soil organic carbon (SOC) pools, it receives little attention from the global C budget. DOC fluxes are critical to aquatic ecosystem inputs and contribute to the C balance of terrestrial ecosystems, but few ecosystem models have attempted to integrate DOC dynamics into terrestrial C cycling. This study introduces a new process-based model, TRIPLEX-DOC, that is capable of estimating DOC dynamics in forest soils by incorporating both ecological drivers and biogeochemical processes. TRIPLEX-DOC was developed from Forest-DNDC, a biogeochemical model simulating C and nitrogen (N) dynamics, coupled with a new DOC process module that predicts metabolic transformations, sorption/desorption, and DOC leaching in forest soils. The model was validated against field observations of DOC concentrations and fluxes at white pine forest stands located in southern Ontario, Canada. The model was able to simulate seasonal dynamics of DOC concentrations and the magnitudes observed within different soil layers, as well as DOC leaching in the age sequence of these forests. Additionally, TRIPLEX-DOC estimated the effect of forest harvesting on DOC leaching, with a significant increase following harvesting, illustrating that land use change is of critical importance in regulating DOC leaching in temperate forests as an important source of C input to aquatic ecosystems.

  7. Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement

    PubMed Central

    Firman, Keith; Szczelkun, Mark D.

    2000-01-01

    The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triplex, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease. Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential. This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400 ± 32 bp/s at 20°C. Furthermore, the data can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can ‘reset’ in either direction whenever the DNA is released. PMID:10790375

  8. In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplex-forming oligonucleotide.

    PubMed Central

    Ing, N H; Beekman, J M; Kessler, D J; Murphy, M; Jayaraman, K; Zendegui, J G; Hogan, M E; O'Malley, B W; Tsai, M J

    1993-01-01

    Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone receptor-dependent transcription in vitro. To approach in vivo conditions, triplex-forming oligonucleotides were tested in cell transfection studies. The derivation of the oligonucleotides with cholesterol enhanced their cellular uptake and nuclear concentration by at least four-fold. The cholesterol-derivatized triplex-forming oligonucleotide specifically inhibited transcription of the PRE-containing reporter gene in cells when applied to the medium at micromolar concentrations. This is the first demonstration of steroid-responsive gene inhibition by triplex formation and joins the growing body of evidence indicating that oligonucleotides have therapeutic potential. Images PMID:8332487

  9. An isocytidine derivative with a 2-amino-6-methylpyridine unit for selective recognition of the CG interrupting site in an antiparallel triplex DNA.

    PubMed

    Okamura, Hidenori; Taniguchi, Yosuke; Sasaki, Shigeki

    2014-11-01

    Sequence-specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non-natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N(2)-arylmethyl isodC derivatives and incorporated them into triplex-forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2-amino-6-methylpyridine moiety (AP-isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs. PMID:25186222

  10. Interstrand cross-link formation in duplex and triplex DNA by modified pyrimidines.

    PubMed

    Peng, Xiaohua; Hong, In Seok; Li, Hong; Seidman, Michael M; Greenberg, Marc M

    2008-08-01

    DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2'-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and

  11. Targeting the human androgen receptor gene with platinated triplex-forming oligonucleotides.

    PubMed

    Graham, Mindy K; Brown, Terry R; Miller, Paul S

    2015-04-01

    Platinum-derivatized homopyrimidine triplex-forming oligonucleotides (Pt-TFOs) consisting of 2'-O-methyl-5-methyluridine, 2'-O-methyl-5-methylcytidine, and a single 3'-N7-trans-chlorodiammine platinum(II)-2'-deoxyguanosine were designed to cross-link to the transcribed strand at four different sequences in the human androgen receptor (AR) gene. Fluorescence microscopy showed that a fluorescein-tagged Pt-TFO localizes in both the cytoplasm and nucleus when it is transfected into LAPC-4 cells, a human prostate cancer cell line, using Lipofectamine 2000. A capture assay employing streptavidin-coated magnetic beads followed by polymerase chain reaction (PCR) amplification was used to demonstrate that 5'-biotin-conjugated Pt-TFOs cross-link in vitro to their four designated AR gene targets in genomic DNA extracted from LAPC-4 cells. Similarly, the capture assay was used to examine cross-linking between the 5'-biotin-conjugated Pt-TFOs and the AR gene in LAPC-4 cells in culture. Three of the four Pt-TFOs cross-linked to their designated target, suggesting that different regions of the AR gene are not uniformly accessible to Pt-TFO cross-linking. LAPC-4 cells were transfected with fluorescein-tagged Pt-TFO or a control oligonucleotide that does not bind or cross-link to AR DNA. The levels of AR mRNA in highly fluorescent cells isolated by fluorescence-activated cell sorting were determined by RT-qPCR, and the levels of AR protein were monitored by immunofluorescence microscopy. Decreases in mRNA and protein levels of 40 and 30%, respectively, were observed for fluorescein-tagged Pt-TFO versus control treated cells. Although the levels of knockdown of AR mRNA and protein were modest, the results suggest that Pt-TFOs hold potential as agents for controlling gene expression by cross-linking to DNA and disrupting transcription. PMID:25768916

  12. Kinetic study of the binding of triplex-forming oligonucleotides containing partial cationic modifications to double-stranded DNA.

    PubMed

    Hari, Yoshiyuki; Ijitsu, Shin; Akabane-Nakata, Masaaki; Yoshida, Takuya; Obika, Satoshi

    2014-07-15

    Several triplex-forming oligonucleotides (TFOs) partially modified with 2'-O-(2-aminoethyl)- or 2'-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5'-region of the TFOs significantly increased the kon values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3'-region. The kon value of a TFO with three adjacent cationic modifications at the 5'-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation. PMID:24865415

  13. Anti-parallel triplexes: Synthesis of 8-aza-7-deazaadenine nucleosides with a 3-aminopropynyl side-chain and its corresponding LNA analog.

    PubMed

    Kosbar, Tamer R; Sofan, Mamdouh A; Waly, Mohamed A; Pedersen, Erik B

    2015-05-15

    The phosphoramidites of DNA monomers of 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine (Y) and 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine LNA (Z) are synthesized, and the thermal stability at pH 7.2 and 8.2 of anti-parallel triplexes modified with these two monomers is determined. When, the anti-parallel TFO strand was modified with Y with one or two insertions at the end of the TFO strand, the thermal stability was increased 1.2°C and 3°C at pH 7.2, respectively, whereas one insertion in the middle of the TFO strand decreased the thermal stability 1.4°C compared to the wild type oligonucleotide. In order to be sure that the 3-aminopropyn-1-yl chain was contributing to the stability of the triplex, the nucleobase X without the aminopropynyl group was inserted in the same positions. In all cases the thermal stability was lower than the corresponding oligonucleotides carrying the 3-aminopropyn-1-yl chain, especially at the end of the TFO strand. On the other hand, the thermal stability of the anti-parallel triplex was dramatically decreased when the TFO strand was modified with the LNA monomer analog Z in the middle of the TFO strand (ΔTm=-9.1°C). Also the thermal stability decreased about 6.1°C when the TFO strand was modified with Z and the Watson-Crick strand with adenine-LNA (A(L)). The molecular modeling results showed that, in case of nucleobases Y and Z a hydrogen bond (1.69 and 1.72Ǻ, respectively) was formed between the protonated 3-aminopropyn-1-yl chain and one of the phosphate groups in Watson-Crick strand. Also, it was shown that the nucleobase Y made a good stacking and binding with the other nucleobases in the TFO and Watson-Crick duplex, respectively. In contrast, the nucleobase Z with LNA moiety was forced to twist out of plane of Watson-Crick base pair which is weakening the stacking interactions with the TFO nucleobases and the binding with the duplex part. PMID:25868748

  14. A pyrimidine motif DNA triplex with a third N3prime;rarr;P5$prime; phosphoramidate d-C,T strand studied by FTIR and UV spectroscopy

    NASA Astrophysics Data System (ADS)

    Mondragón-Sánchez, J. A.; Liquier, J.; Gryaznov, S. M.; Taillandier, E.

    2003-12-01

    Formation of a pyrimidine motif triple stranded structure containing a N3'→P5' phosphoramidate 5'-d(TTC-TCC-TTT-CTT)-3' third strand targeting the 5'-d(AAG-AGG-AAA-GAA)-3' sequence has been followed by Fourier transform infrared (FTIR) spectroscopy and UV spectroscopy. The use of a N3'→P5' phosphoramidate d-C,T third strand is aimed at increasing triplex stability at neutral pH. FTIR spectroscopy measurements at neutral pH show a biphasic melting profile ( Tm at 25 and 54 °C). The triple helix is stabilized by the formation of T *A-T base triplets, in spite of the presence of four unprotonated cytosines in the 12mer third d-C,T phosphoramidate strand and therefore of the absence of C +*G rad C base triplets. All N3'→P5' phosphoramidate nucleoside sugars in this triple helix adopt an S-type (C2' endo) conformation. No triple helix has been detected at neutral pH when a natural isosequential phosphodiester third strand was used. By decreasing the pH, the FTIR spectra show the formation of C +*G rad C base triplets in addition to the already formed T *A rad T base triplets. The melting of this stabilized triple helix is observed at a temperature higher than that of the initial Watson-Crick duplex. The existence of N-type sugars is then detected. When the concentration is decreased, at neutral pH, UV spectroscopy measurements show that the intermolecular triple helix formed by three short 12mer strands is no longer stable. In dilute solution at acidic pH the triplex is more stable than the initial Watson-Crick duplex.

  15. DNA Triplexes-Guided Assembly of G-Quadruplexes for Constructing Label-free Fluorescent Logic Gates.

    PubMed

    Xu, Lijun; Hong, Shanni; Shen, Xiaoqiang; Zhou, Lu; Wang, Jine; Zhang, Jianye; Pei, Renjun

    2016-07-01

    Assembly of G-quadruplexes guided by DNA triplexes in a controlled manner is achieved for the first time. The folding of triplex sequences in acidic conditions brings two separated guanine-rich sequences together and subsequently a G-quadruplex structure is formed in the presence of K(+) . Based on this novel platform, label-free fluorescent logic gates, such as AND, INHIBIT, and NOR, are constructed with ions as input and the fluorescence of a G-quadruplex-specific fluorescent probe NMM as output. PMID:27224871

  16. A triplex real-time PCR for differential detection of classical, variant and Bartha-K61 vaccine strains of pseudorabies virus.

    PubMed

    Meng, Xing-Yu; Luo, Yuzi; Liu, Yan; Shao, Lina; Sun, Yuan; Li, Yongfeng; Li, Su; Ji, Shengwei; Qiu, Hua-Ji

    2016-09-01

    Pseudorabies (PR), also known as Aujeszky's disease, is an economically important infectious disease of pigs and other animals caused by pseudorabies virus (PRV). Since late 2011, increasing numbers of PR outbreaks have been reported on many Bartha-K61-vaccinated pig farms in China, and emerging PRV variants that differ from classical PRV strains genetically and antigenically have been confirmed to be responsible for the outbreaks. Accordingly, there is a need to differentiate diverse PRV strains co-circulating in the field. Here, we developed and evaluated a triplex real-time PCR for differential detection of wild-type PRV (classical and variant strains) and gE/gI gene-deleted vaccine strains based on three differently labeled TaqMan probes. The detection limits of the assay were 0.5 TCID50 for classical strains, 0.2 TCID50 for variant strains and 0.05 TCID50 for vaccine strains. The sensitivity was also determined to be 50, 50 and 5 copies for the TJ, SC and Bartha-K61 strain, respectively. The assay did not show cross-reactivity with several common porcine viruses. Reproducibility tests showed that the inter- and intra-assay coefficients of variation were less than 3 %. When testing a total of 234 clinical swine samples, the agreement between the triplex real-time PCR and virus isolation was 100 % (234/234) for classical strains, 99.5 % (233/234) for variant strains, and 100 % (234/234) for the Bartha-K61 vaccine strain. The results demonstrate that this method is sensitive and specific and will be useful for rapid detection and differentiation of diverse PRV strains. PMID:27316441

  17. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome. PMID:25408401

  18. Design of ultra-compact triplexer with function-expansion based topology optimization.

    PubMed

    Zhang, Zejun; Tsuji, Yasuhide; Yasui, Takashi; Hirayama, Koichi

    2015-02-23

    In this paper, in order to optimize wavelength selective photonic devices using the function-expansion-based topology optimization method, several expansion functions are considered and the influence on the optimized structure based on each expansion function was investigated. Although the Fourier series is conventionally used in the function-expansion-based method, the optimized structure sometimes has a complicated refractive index distribution. Therefore, we employed a sampling function and a pyramid function to obtain a simpler structure through the optimal design. A triplexer was designed by using our method, and the comparison between the optimized structures based on the three expansion functions was also discussed in detail. PMID:25836433

  19. Binding properties of ruthenium(II) complexes [Ru(bpy)2(ppn)](2+) and [Ru(phen)2(ppn)](2+) with triplex RNA: As molecular "light switches" and stabilizers for poly(U)·poly(A)*poly(U) triplex.

    PubMed

    Li, Jia; Sun, Yanmei; Zhu, Zhiyuan; Zhao, Hong; Tan, Lifeng

    2016-08-01

    Stable RNA triplexes play key roles in many biological processes, while triplexes are thermodynamically less stable than the corresponding duplexes due to the Hoogsteen base pairing. To understand the factors affecting the stabilization of RNA triplexes by octahedral ruthenium(II) complexes, the binding of [Ru(bpy)2(ppn)](2+) (1, bpy=2,2'-bipyridine, ppn=2,4-diaminopyrimido[5,6-b]dipyrido[2,3-f:2',3'-h]quinoxaline) and [Ru(phen)2(ppn)](2+) (2, phen=1,10-phenanthroline) to poly(U)·poly(A)*poly(U) (· denotes the Watson-Crick base pairing and * denotes the Hoogsteen base pairing) has been investigated. The main results obtained here suggest that complexes 1 and 2 can serve as molecular "light switches" and stabilizers for poly(U)·poly(A)*poly(U), while the effectiveness of complex 2 are more marked, suggesting that the hydrophobicity of ancillary ligands has a significant effect on the two Ru(II) complexes binding to poly(U)·poly(A)*poly(U). This study further advances our knowledge on the binding of RNA triplexes with metal complexes, particularly with octahedral ruthenium polypyridyl complexes. PMID:27287059

  20. pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine.

    PubMed

    Seela, Frank; Shaikh, Khalil I

    2006-11-01

    Triplex-forming oligonucleotides (TFOs) containing 2'-deoxyisoguanosine (2), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2'-deoxyribonucleoside) were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19, 20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides containing 1 was carried out by Pd(0)[PPh3]4-PPh3 followed by 25% aq. NH3. Formation of the 31-mer single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-stranded 31-mer oligonucleotides the protonated dC in the dCH(+)-dG-dC base triad was replaced by 2'-deoxyisoguanosine (1), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine N7-(2'-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions. These triplexes contain "purine" nucleosides in the third pyrimidine rich strand of the oligonucleotide hairpin. PMID:17047881

  1. Triplex DNA: A new platform for polymerase chain reaction – based biosensor

    PubMed Central

    Li, Yubin; Miao, Xiangmin; Ling, Liansheng

    2015-01-01

    Non - specific PCR amplification and DNA contamination usually accompany with PCR process, to overcome these problems, here we establish a sensor for thrombin by sequence - specific recognition of the PCR product with molecular beacon through triplex formation. Probe A and probe B were designed for the sensor, upon addition of thrombin, two probes hybridized to each other and the probe B was extended in the presence of Klenow Fragment polymerase and dNTPs. The PCR amplification occurred with further addition of Taq DNA Polymerase and two primers, the PCR product was recognized by molecular beacon through triplex formation. The fluorescence intensity increased with the logarithm of the concentration of thrombin over the range from 1.0 × 10−12 M to 1.0 × 10−7 M, with a detection limit of 261 fM. Moreover, the effect of DNA contamination and non - specific amplification could be ignored completely in the proposed strategy. PMID:26268575

  2. High-resolution atomic force microscopy of duplex and triplex DNA molecules

    NASA Astrophysics Data System (ADS)

    Klinov, Dmitry; Dwir, Benjamin; Kapon, Eli; Borovok, Natalia; Molotsky, Tatiana; Kotlyar, Alexander

    2007-06-01

    Double-stranded poly(dG)-poly(dC) and triple-stranded poly(dG)-poly(dG)-poly(dC) DNA were deposited on the modified surface of highly oriented pyrolitic graphite (HOPG) and visualized using atomic force microscopy with high-resolution (radius of ~1 nm) tips. The high resolution attained by this technique enabled us to detect single-stranded regions in double-stranded poly(dG)-poly(dC) and double-stranded and single-stranded regions in poly(dG)-poly(dG)-poly(dC) triplexes, as well as to resolve the helical pitch of the triplex molecules. We could also follow the reaction of G-strand extension in poly(dG)-poly(dC) by the Klenow exo- fragment of DNA polymerase I. This approach to molecular visualization could serve as a useful tool for the investigation of irregular structures in canonical DNA and other biopolymers, as well as studies of the molecular mechanisms of DNA replication and transcription.

  3. Triplex DNA: A new platform for polymerase chain reaction-based biosensor.

    PubMed

    Li, Yubin; Miao, Xiangmin; Ling, Liansheng

    2015-01-01

    Non-specific PCR amplification and DNA contamination usually accompany with PCR process, to overcome these problems, here we establish a sensor for thrombin by sequence-specific recognition of the PCR product with molecular beacon through triplex formation. Probe A and probe B were designed for the sensor, upon addition of thrombin, two probes hybridized to each other and the probe B was extended in the presence of Klenow Fragment polymerase and dNTPs. The PCR amplification occurred with further addition of Taq DNA Polymerase and two primers, the PCR product was recognized by molecular beacon through triplex formation. The fluorescence intensity increased with the logarithm of the concentration of thrombin over the range from 1.0 × 10(-12) M to 1.0 × 10(-7) M, with a detection limit of 261 fM. Moreover, the effect of DNA contamination and non - specific amplification could be ignored completely in the proposed strategy. PMID:26268575

  4. DNA-triplex conformation from normal mode and hydrogen bond stability calculations.

    NASA Astrophysics Data System (ADS)

    Chen, Y. Z.; Prohofsky, E. W.; Powell, J. W.; White, A. P.

    1996-03-01

    Triple-stranded DNAs are of potential applications in genome mapping and in the treatment of genetic disorders with little side-effect. Despite significant interests, structural information of DNA triplexes is limited and sometimes conflicting. For instance, two structural models with different conformation have been proposed for a DNA-triplex Poly(dA)\\cdot2Poly(dT). We propose that the sensitivity of normal modes and hydrogen-bond stability on conformation can be used to determine the structure of biomolecules difficult to access by other methods. The structural model representative of the true conformation should have normal modes in agreement with observations, and have most stable hydrogen bonds which melt at observed temperatures. We carried out calculations on the two models of Poly(dA)\\cdot2Poly(dT) and found that one model is consistent with observations at high humidity and thus most likely a good approximation to the true conformation in that environment. Our method has potential application in structural prediction for other biomolecules.

  5. Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids--TINA.

    PubMed

    Schneider, Uffe V; Mikkelsen, Nikolaj D; Jøhnk, Nina; Okkels, Limei M; Westh, Henrik; Lisby, Gorm

    2010-07-01

    Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (T(m)) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that Delta T(m) of base mismatches on PT is remarkably high (between 7.4 and 15.2 degrees C) compared to antiparallel duplexes (between 3.8 and 9.4 degrees C). The specificity of PT by Delta T(m) increases when shorter TFOs and higher pH are chosen. To increase Delta Tms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3' (preferable) or 5' of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays. PMID:20338879

  6. Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids—TINA

    PubMed Central

    Schneider, Uffe V.; Mikkelsen, Nikolaj D.; Jøhnk, Nina; Okkels, Limei M.; Westh, Henrik; Lisby, Gorm

    2010-01-01

    Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (Tm) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that ΔTm of base mismatches on PT is remarkably high (between 7.4 and 15.2°C) compared to antiparallel duplexes (between 3.8 and 9.4°C). The specificity of PT by ΔTm increases when shorter TFOs and higher pH are chosen. To increase ΔTms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3′ (preferable) or 5′ of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays. PMID:20338879

  7. A web-based search engine for triplex-forming oligonucleotide target sequences.

    PubMed

    Gaddis, Sara S; Wu, Qi; Thames, Howard D; DiGiovanni, John; Walborg, Earl F; MacLeod, Michael C; Vasquez, Karen M

    2006-01-01

    Triplex technology offers a useful approach for site-specific modification of gene structure and function both in vitro and in vivo. Triplex-forming oligonucleotides (TFOs) bind to their target sites in duplex DNA, thereby forming triple-helical DNA structures via Hoogsteen hydrogen bonding. TFO binding has been demonstrated to site-specifically inhibit gene expression, enhance homologous recombination, induce mutation, inhibit protein binding, and direct DNA damage, thus providing a tool for gene-specific manipulation of DNA. We have developed a flexible web-based search engine to find and annotate TFO target sequences within the human and mouse genomes. Descriptive information about each site, including sequence context and gene region (intron, exon, or promoter), is provided. The engine assists the user in finding highly specific TFO target sequences by eliminating or flagging known repeat sequences and flagging overlapping genes. A convenient way to check for the uniqueness of a potential TFO binding site is provided via NCBI BLAST. The search engine may be accessed at spi.mdanderson.org/tfo. PMID:16764543

  8. Modulation of psoralen DNA crosslinking kinetics associated with a triplex-forming oligonucleotide.

    PubMed

    Oh, Dennis H; Suzara, Vincent; Krishnan, Rajagopal

    2008-01-01

    A triplex-forming oligonucleotide (TFO), HPRT3, conjugated to a psoralen derivative, was designed to target a psoralen reaction site within the HPRT gene. HPRT3 bound with high affinity to a synthetic duplex target sequence. At a uniform UVA radiation dose, the ratio of psoralen monoadducts (MA) to interstrand crosslinks decreased and inverted with increasing TFO concentration. As the TFO concentration increased from 10 nm to 10 microm, the efficiency of psoralen MA formation remained relatively constant but the efficiency of interstrand crosslink formation increased several-fold. Neither shortening the TFO to reduce its dissociation constant nor altering the DNA sequences flanking the TFO binding site altered the concentration dependence of MA and crosslink yields. The psoralen photokinetics associated with 10 nm HPRT3 converted to those associated with 10 microm HPRT3 with the addition of other unrelated TFOs at 10 microm that do not specifically interact with the HPRT3 target sequence. Glycerol at concentrations of 0.5% (vol/vol) or higher also mimicked high TFO concentrations in enhancing crosslink formation. These results demonstrate that while psoralen may be targeted to react at a particular sequence by TFOs, photoreactivity associated with triplex formation is also modulated by sequence-independent factors that may affect the local macromolecular environment. PMID:18435621

  9. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    PubMed Central

    Birkedal, Henrik

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies. PMID:21686249

  10. Local delivery of gene-modifying triplex-forming molecules to epidermis

    PubMed Central

    Rogers, Faye A.; Hu, Rong-Hua; Milstone, Leonard M.

    2012-01-01

    Epidermal keratinocytes are particularly suitable candidates for in situ gene correction. Intraperitoneal administration of a triplex-forming oligonucleotide (TFO) was shown previously to introduce DNA base changes in a reporter gene in skin, without identifying which cells had been targeted. We extend those previous experiments using two triplex-forming molecules (TFMs), a peptide nucleic acid (PNA-Antp) and a TFO (AG30), and two lines of transgenic mice that have the chromosomally integrated λsupFG1 shuttle-reporter transgene. Successful in vivo genomic modification occurs in epidermis and dermis in CD1 transgenic mice following either intraperitoneal or intradermal delivery of the PNA-Antennapedia conjugate. FITC-PNA-Antp accumulates in nuclei of keratinocytes and, after intradermal delivery of the PNA-Antp, chromosomally modified, keratin 5 positive basal keratinocytes persist for at least 10 days. In hairless (SKH1) mice with the λsupFG1 transgene, intradermal delivery of the TFO, AG30, introduces gene modifications in both tail and back skin and those chromosomal modifications persist in basal keratinocytes for 10 days. Hairless mice should facilitate comparison of various targeting agents and methods of delivery. Gene targeting by repeated local administration of oligonucleotides may prove clinically useful for judiciously selected disease-causing genes in the epidermis. PMID:23014335

  11. Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic Vibrio cholerae.

    PubMed

    Chua, Ang Lim; Elina, Husni Tan; Lim, Boon Huat; Yean, Chan Yean; Ravichandran, Manickam; Lalitha, Pattabhiraman

    2011-04-01

    Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free. PMID:21183596

  12. Probing the recognition surface of a DNA triplex: Binding studies with intercalator-neomycin conjugates

    PubMed Central

    Xue, Liang; Xi, Hongjuan; Kumar, Sunil; Gray, David; Davis, Erik; Hamilton, Paris; Skirba, Michael; Arya, Dev P.

    2012-01-01

    Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA)•2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalenedimide-neomycin (3) and anthraquinone-neomycin (4) were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2), in our investigations. A combination of experiments including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC) revealed that all four conjugates (1–4) stabilized poly(dA)•2poly(dT) much greater than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (Tm3→2) at which poly(dA)•2poly(dT) dissociated into poly(dA)•poly(dT) and poly(dT) increased dramatically (> 12 °C) in the presence of intercalator-neomycin (1–4) even at a very low concentration (2 µM). In contrast to intercalator-neomycin conjugates, the increment of Tm3→2 of poly(dA)•2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin (1–4) to poly(dA)•2poly(dT) was also confirmed by competition dialysis and fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compound 1–4 had slightly larger binding site size (~7–7.5) with poly(dA)•2poly(dT) as compared to neomycin (~6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA)•2poly(dT) were derived from an integrated van’t Hoff equation using the Tm3→2 values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA)•2poly(dT) increased in the order neomycin < 1 < 3 < 4 < 2. Amongst them, the binding constant [(2.7 ± 0.3) × 108 M−1] of 2 with poly(dA)•2poly(dT) was the highest, almost 1000 fold more

  13. Synthesis and properties of triplex-forming oligonucleotides containing 2'-O-(2-methoxyethyl)-5-(3-aminoprop-1-ynyl)-uridine.

    PubMed

    Lou, Chenguang; Xiao, Qiang; Brennan, Lavinia; Light, Mark E; Vergara-Irigaray, Nuria; Atkinson, Elizabeth M; Holden-Dye, Lindy M; Fox, Keith R; Brown, Tom

    2010-09-01

    2'-O-(2-Methoxyethyl)-5-(3-aminoprop-1-ynyl)-uridine phosphoramidite (MEPU) has been synthesized from d-ribose and 5-iodouracil and incorporated into triplex-forming oligonucleotides (TFOs) by automated solid-phase oligonucleotide synthesis. The TFOs gave very high triplex stability with their target duplexes as measured by ultraviolet/fluorescence melting and DNase I footprinting. The incorporation of MEPU into TFOs renders them resistant to degradation by serum nucleases. PMID:20674370

  14. Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA.

    PubMed

    Taniguchi, Yosuke; Uchida, Yuko; Takaki, Tomoko; Aoki, Eriko; Sasaki, Shigeki

    2009-10-01

    We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid steric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was illustrated by the fact that the TFO containing WNA-betaAP in the 3'G-WNA-G-5' sequence formed a stable triplex with selectivity to the CG interrupting site where the previous WNA-betaC did not induce the triplex formation. PMID:19736014

  15. Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking.

    PubMed

    Abdallah, Hatem O; Ohayon, Yoel P; Chandrasekaran, Arun Richard; Sha, Ruojie; Fox, Keith R; Brown, Tom; Rusling, David A; Mao, Chengde; Seeman, Nadrian C

    2016-06-28

    The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5',8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability. PMID:27265774

  16. Correction of F508del CFTR in airway epithelium using nanoparticles delivering triplex-forming PNAs

    PubMed Central

    McNeer, Nicole Ali; Anandalingam, Kavitha; Fields, Rachel J.; Caputo, Christina; Kopic, Sascha; Gupta, Anisha; Quijano, Elias; Polikoff, Lee; Kong, Yong; Bahal, Raman; Geibel, John P; Glazer, Peter M.; Saltzman, W. Mark; Egan, Marie E.

    2015-01-01

    Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here, we use triplex-forming PNA molecules and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep sequencing reveals negligible off-target effects in partially homologous sites. Intranasal application of nanoparticles in CF mice produces changes in nasal epithelium potential differences consistent with corrected CFTR, with gene correction also detected in lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects. PMID:25914116

  17. Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

    NASA Technical Reports Server (NTRS)

    Su, L.; Chen, L.; Egli, M.; Berger, J. M.; Rich, A.

    1999-01-01

    Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

  18. High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.

    PubMed

    Wu, Qi; Gaddis, Sara S; MacLeod, Michael C; Walborg, Earl F; Thames, Howard D; DiGiovanni, John; Vasquez, Karen M

    2007-01-01

    Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted. PMID:17013831

  19. Stress-optic modulator in TriPleX platform using a ezoelectric lead zirconate titanate (PZT) thin film.

    PubMed

    Hosseini, Naser; Dekker, Ronald; Hoekman, Marcel; Dekkers, Matthijn; Bos, Jan; Leinse, Arne; Heideman, Rene

    2015-06-01

    We will demonstrate a stress-optic phase modulator in the passive SiN-based TriPleX platform using a layer of piezoelectric material. Regarding the stress-optic effect, the piezoelectric layer deposited on top of an optical waveguide is employed to control the phase of propagating light in the structure by applying an electrical field across the layer. In this work, it is demonstrated that the stress-optic effect lowers the power consumption by a factor of one million for quasi-DC operation and increases the modulation speed by three orders of magnitude, compared to currently used thermo-optic modulation in the TriPleX platform. PMID:26072771

  20. Cross-linking to an interrupted polypurine sequence with a platinum-modified triplex-forming oligonucleotide.

    PubMed

    Campbell, Meghan A; Miller, Paul S

    2009-08-01

    Triplex-forming oligonucleotides (TFOs) can bind specifically to polypurine sequences in double-stranded DNA. A single interruption of this polypurine tract can greatly destabilize triplex formation. The stability of triplexes can be significantly enhanced by covalently linking the TFO to its DNA target with reactive functional groups conjugated to the TFO. Covalently cross-linked TFOs are effective inhibitors of transcription of the target DNA sequence. We have designed a TFO with a platinum-modified base that can interact with and cross-link to a cytosine interruption in the polypurine tract of a target DNA duplex. The TFO contains an N(4)-(aminoalkyl)cytosine derivatized with cis-diamminediaquaplatinum(II) or trans-diamminediaquaplatinum(II). When bound to its target, the tethered platinum of the TFO can reach across the major groove and form an adduct with the guanine N7 of the interrupting C.G base pair. The optimal tether length is five methylene groups, and cross-linking is most efficient when the tether is modified with trans-diamminediaquaplatinum(II). Cross-linking requires that the TFO is bound to its designated DNA target. Addition of cyanide to the cross-linked TFO product reversed the cross-link, behavior that is consistent with the presence of a platinum-guanine adduct. The kinetics of the cross-linking reaction were studied and the half-life of the cross-linking reaction was approximately 3 h. Our results demonstrate that platinum-conjugated TFOs can be designed to cross-link with DNA targets that contain a single pyrimidine interruption. Modifications of this type may prove useful for expanding the DNA sequences that can be targeted by TFOs and increasing the stability of the resulting triplexes. PMID:19350290

  1. Effects of halogenated WNA derivatives on sequence dependency for expansion of recognition sequences in non-natural-type triplexes.

    PubMed

    Taniguchi, Yosuke; Nakamura, Ayako; Senko, Yusuke; Nagatsugi, Fumi; Sasaki, Shigeki

    2006-03-01

    Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their target duplexes are limited to homopurine/homopyrimidine sequences because of interruption of the pyrimidines bases in the purine region. This problem has not been fully solved despite a wide variety of studies. Recently, we have developed a bicyclic system as a novel scaffold for nucleoside analogues (WNA, W-shaped nucleoside analogues) and determined two useful compounds, WNA-betaT (2) and WNA-betaC (5), for highly stable and selective triplex formation at a TA and a CG interrupting site, respectively. However, subsequent investigations have shown that the triplex formation using WNA is dependent on the neighboring bases of the TFOs. In this study, we have synthesized new WNA derivatives having halogenated recognition bases or benzene rings and evaluated the effects of the modifications on the triplex stability as well as selectivity. It has been found that the WNA-betaT analogues holding 5-halogenated pyrimidine bases (WNA-beta(Br)U (3) and WNA-beta(F)U (4)) exhibit high CG-selectivity. On the other hand, the WNA-betaT derivatives having the bromo-substituted benzene ring (mBr-WNA-betaT (10) and oBr-WNA-betaT (11)) have shown high selectivity to a TA interrupting site with high stability in the sequences to which the original WNA-betaT do not bind. Thus, sequence-dependency has been overcome by the sequence-dependent use of WNA-betaT, mBr-WNA-betaT, and oBr-WNA-betaT. PMID:16497000

  2. Selective inhibition of transcription of the Ets2 gene in prostate cancer cells by a triplex-forming oligonucleotide

    PubMed Central

    Carbone, Giuseppina M.; McGuffie, Eileen M.; Collier, Angela; Catapano, Carlo V.

    2003-01-01

    The transcription factor Ets2 has a role in cancer development and represents an attractive therapeutic target. In this study, we designed a triplex-forming oligonucleotide (TFO) directed to a homopurine:homopyrimidine sequence in the Ets2 promoter. Transcription factors of the Sp family bound to this sequence and mutation of the Sp1 site reduced Ets2 promoter activity. The Ets2-TFO had high binding affinity for the target sequence and inhibited binding of Sp1/Sp3 to the overlapping site. This effect occurred with a high degree of sequence specificity. Mismatched oligonucleotides did not inhibit Sp1/Sp3 binding and mutations in the target sequence that abolished triplex formation prevented inhibition of Sp1/Sp3 binding by the TFO. The Ets2-TFO inhibited Ets2 promoter activity and expression of the endogenous gene in prostate cancer cells at nanomolar concentrations. The TFO did not affect reporter constructs with mutations in the TFO binding site and promoters of non-targeted genes. Expression of non-targeted genes was also not affected in TFO-treated cells. Collectively, these data demonstrated that the anti-transcriptional activity of the Ets2-TFO was sequence- and target-specific, and ruled out alternative, non-triplex mediated mechanisms of action. This anti-transcriptional approach may be useful to examine the effects of selective downregulation of Ets2 expression and may have therapeutic applications. PMID:12560478

  3. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome

    PubMed Central

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A.

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser. PMID:25324314

  4. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome.

    PubMed

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser. PMID:25324314

  5. Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

    PubMed Central

    Chin, Joanna Y; Reza, Faisal; Glazer, Peter M

    2013-01-01

    Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes. PMID:23337982

  6. Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.

    PubMed Central

    Collier, D A; Mergny, J L; Thuong, N T; Helene, C

    1991-01-01

    Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs intercalation to a single site in 3kb of DNA at the 5' triplex-duplex junction. Footprinting with diethylpyrocarbonate and dimethylsulphate revealed strong protection from modification of adenine residues within the triple-helix in concordance with their Hoogsteen pairing with the third strand, and a strong hypersensitivity to diethylpyrocarbonate at the first adenine of the duplex. This result indicates that intercalation at this site induces a conformational change at the 5' triplex-duplex junction. Furthermore, the same diethlypyrocarbonate hypersensitivity was observed with an unmodified triple-strand forming oligonucleotide and a range of intercalating molecules present in solution. Thus the 5' triplex-duplex junction is a strong binding site for some intercalating molecules and the junction undergoes a conformational change which is sensitive to diethylpyrocarbonate upon insertion of the planar aromatic chromophore. This conformational change can be used to direct a single-strand cut in duplex DNA to a defined site. Images PMID:1870975

  7. Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA.

    PubMed

    Sato, Takaya; Sato, Yusuke; Nishizawa, Seiichi

    2016-08-01

    We have developed a new fluorescent sensing probe for double-stranded RNA (dsRNA) by integrating thiazole orange (TO) as a base surrogate into triplex-forming PNA. Our probe forms the thermally stable triplex with the target dsRNA at acidic pH; and the triplex formation is accompanied by the remarkable light-up response of the TO unit. The binding of our probe to the target dsRNA proceeds very rapidly, allowing real-time monitoring of the triplex formation. Importantly, we found the TO base surrogate in our probe functions as a universal base for the base pair opposite the TO unit in the triplex formation. Furthermore, the TO unit is significantly more responsive for the fully matched dsRNA sequence compared to the mismatch-containing sequences, which enables the analysis of the target dsRNA sequence at the single-base pair resolution. The binding and sensing functions of our probe are described for the development of fluorescent probes applicable to sensing biologically relevant dsRNA. PMID:27442229

  8. Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo

    PubMed Central

    McNeer, Nicole A.; Schleifman, Erica B.; Cuthbert, Amy; Brehm, Michael; Jackson, Andrew; Cheng, Christopher; Anandalingam, Kavitha; Kumar, Priti; Shultz, Leonard D.; Greiner, Dale L.

    2013-01-01

    In vivodelivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD-scid IL2rγnull mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA, and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen, and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an EGFP- β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo. PMID:23076379

  9. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    PubMed

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis. PMID:23971699

  10. Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

    PubMed Central

    Brunet, Erika; Corgnali, Maddalena; Perrouault, Loïc; Roig, Victoria; Asseline, Ulysse; Sørensen, Mads D.; Babu, B. Ravindra; Wengel, Jesper; Giovannangeli, Carine

    2005-01-01

    Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5′-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life ∼10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations ∼0.1 μM for acridine-conjugated TFO/LNA (or ∼2 μM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated. PMID:16049028

  11. A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide

    PubMed Central

    Ruigrok, Vincent J. B.; Westra, Edze R.; Brouns, Stan J. J.; Escudé, Christophe; Smidt, Hauke; van der Oost, John

    2013-01-01

    Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA–protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ∼18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA–protein interactions. PMID:23571753

  12. Triplex DNA logic gate based upon switching on/off their structure by Ag(+)/cysteine.

    PubMed

    Xiao, Zhiyou; Zhu, Houya; Xin, Aiping; Li, Yubin; Ling, Liansheng

    2015-11-01

    The formation of intramolecular triplex DNA can be regulated by Ag(+) and Cys (cysteine), which switch off/on the fluorescence of the oligonucleotides, 5'-TAMRA-TTC TCT TCC TCT TCC TTC TGA CGA CAG TTG ACT CTT CCT TCT CCT TCT CTT-BHQ-2-3' (Oligo 1) and 3'-GAA GGA AGA GGA AGA GAA-5' (Oligo 2). Based on this principle, sensors for Ag(+) and Cys are developed. The sensor for Ag(+) has a linear range of 2.5 nM-40 nM and a detection limit of 1.8 nM, whereas the sensor for Cys has a linear range of 10.0 nM-120.0 nM and a detection limit of 8.2 nM. Furthermore, the fluorescence is reversible with the alternate addition of Ag(+) and Cys. We constructed a DNA logic gate using Ag(+) and Cys as the input, and the fluorescence intensity as the output. The DNA logic gate is simple; moreover, it has a fast response and good reversibility. PMID:26359516

  13. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.

    PubMed

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2009-10-01

    Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells. PMID:19726585

  14. Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms.

    PubMed

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2008-08-01

    Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells. PMID:18628293

  15. Targeting DNA with triplex-forming oligonucleotides to modify gene sequence.

    PubMed

    Simon, Philippe; Cannata, Fabio; Concordet, Jean-Paul; Giovannangeli, Carine

    2008-08-01

    Molecules that interact with DNA in a sequence-specific manner are attractive tools for manipulating gene sequence and expression. For example, triplex-forming oligonucleotides (TFOs), which bind to oligopyrimidine.oligopurine sequences via Hoogsteen hydrogen bonds, have been used to inhibit gene expression at the DNA level as well as to induce targeted mutagenesis in model systems. Recent advances in using oligonucleotides and analogs to target DNA in a sequence-specific manner will be discussed. In particular, chemical modification of TFOs has been used to improve binding to chromosomal target sequences in living cells. Various oligonucleotide analogs have also been found to expand the range of sequences amenable to manipulation, including so-called "Zorro" locked nucleic acids (LNAs) and pseudo-complementary peptide nucleic acids (pcPNAs). Finally, we will examine the potential of TFOs for directing targeted gene sequence modification and propose that synthetic nucleases, based on conjugation of sequence-specific DNA ligands to DNA damaging molecules, are a promising alternative to protein-based endonucleases for targeted gene sequence modification. PMID:18460344

  16. Triplex-stimulated intermolecular recombination at a single-copy genomic target.

    PubMed

    Knauert, Melissa P; Kalish, Jennifer M; Hegan, Denise C; Glazer, Peter M

    2006-09-01

    Gene targeting via homologous recombination offers a potential strategy for therapeutic correction of mutations in disease-related human genes. However, there is a need to improve the efficiency of site-specific recombination by transfected donor DNAs. Oligonucleotide-mediated triple helix formation has been shown to constitute a DNA lesion sufficient to provoke DNA repair and thereby stimulate recombination. However, the ability of triplex-forming oligonucleotides (TFOs) to induce recombination between a target locus and a donor DNA has so far been demonstrated only with multicopy episomal targets in mammalian cells. Using cell lines containing the firefly luciferase reporter gene, we have now established the ability of TFOs to induce gene correction by exogenous donor DNAs at a single-copy chromosomal locus. We find that cotransfection of TFOs and short, single-stranded DNA donor molecules into mammalian cells yields gene correction in a dose-dependent manner at frequencies up to 0.1%, which is five- to ninefold above background. We demonstrate both oligonucleotide-specific and target site-specific effects. We also find that recombination can be induced by both parallel and antiparallel triple helix formation. These results provide further support for the development of TFOs as reagents to stimulate site-specific correction of defective human genes. PMID:16731047

  17. Targeted generation of DNA strand breaks using pyrene-conjugated triplex-forming oligonucleotides.

    PubMed

    Benfield, Aaron P; Macleod, Michael C; Liu, Yaobin; Wu, Qi; Wensel, Theodore G; Vasquez, Karen M

    2008-06-10

    Gene targeting by triplex-forming oligonucleotides (TFOs) has proven useful for gene modulation in vivo. Photoreactive molecules have been conjugated to TFOs to direct sequence-specific damage in double-stranded DNA. However, the photoproducts are often repaired efficiently in cells. This limitation has led to the search for sequence-specific photoreactive reagents that can produce more genotoxic lesions. Here we demonstrate that photoactivated pyrene-conjugated TFOs (pyr-TFOs) induce DNA strand breaks near the pyrene moiety with remarkably high efficiency and also produce covalent pyrene-DNA adducts. Free radical scavenging experiments demonstrated a role for singlet oxygen activated by the singlet excited state of pyrene in the mechanism of pyr-TFO-induced DNA damage. In cultured mammalian cells, the effect of photoactivated pyr-TFO-directed DNA damage was to induce mutations, in the form of deletions, approximately 7-fold over background levels, at the targeted site. Thus, pyr-TFOs represent a potentially powerful new tool for directing DNA strand breaks to specific chromosomal locations for biotechnological and potential clinical applications. PMID:18473480

  18. Modelling methane emissions from natural wetlands by development and application of the TRIPLEX-GHG model

    USGS Publications Warehouse

    Zhu, Qing; Liu, Jinxun; Peng, C.; Chen, H.; Fang, X.; Jiang, H.; Yang, G.; Zhu, D.; Wang, W.; Zhou, X.

    2014-01-01

    A new process-based model TRIPLEX-GHG was developed based on the Integrated Biosphere Simulator (IBIS), coupled with a new methane (CH4) biogeochemistry module (incorporating CH4 production, oxidation, and transportation processes) and a water table module to investigate CH4 emission processes and dynamics that occur in natural wetlands. Sensitivity analysis indicates that the most sensitive parameters to evaluate CH4 emission processes from wetlands are r (defined as the CH4 to CO2 release ratio) and Q10 in the CH4 production process. These two parameters were subsequently calibrated to data obtained from 19 sites collected from approximately 35 studies across different wetlands globally. Being heterogeneously spatially distributed, r ranged from 0.1 to 0.7 with a mean value of 0.23, and the Q10 for CH4 production ranged from 1.6 to 4.5 with a mean value of 2.48. The model performed well when simulating magnitude and capturing temporal patterns in CH4 emissions from natural wetlands. Results suggest that the model is able to be applied to different wetlands under varying conditions and is also applicable for global-scale simulations.

  19. Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

    PubMed

    Hégarat, Nadia; Novopashina, Darya; Fokina, Alesya A; Boutorine, Alexandre S; Venyaminova, Alya G; Praseuth, Danièle; François, Jean-Christophe

    2014-03-01

    Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells. PMID:24423253

  20. A H+/Ag+ dual-target responsive label-free light-up probe based on a DNA triplex.

    PubMed

    Xu, Lijun; Guo, Yahui; Wang, Jine; Zhou, Lu; Zhang, Yuanyuan; Hong, Shanni; Wang, Zhili; Zhang, Jianye; Pei, Renjun

    2015-05-01

    We developed a dual-target responsive sensor for label-free light-up fluorescent detection of protons (H(+)) and silver ions (Ag(+)) using an "OR'' logic gate. Berberine, a cost-effective and non-toxic indicator, partially intercalates the formed triplex DNA in the presence of H(+) or Ag(+), generating enhanced fluorescence. The designed Ag(+) probe has high selectivity and desirable sensitivity, which is necessary for practical use. The robust "OR" logic gate is capable of a rapid and reversible response to the H(+) and/or Ag(+) inputs. PMID:25663002

  1. Design and construction of a VHGT-attached WDM-type triplex transceiver module using polymer PLC hybrid integration technology

    NASA Astrophysics Data System (ADS)

    Jerábek, Vitezslav; Hüttel, Ivan; Prajzler, Václav; Busek, K.; Seliger, P.

    2008-11-01

    We report about design and construction of the bidirectional transceiver TRx module for subscriber part of the passive optical network PON for a fiber to the home FTTH topology. The TRx module consists of a epoxy novolak resin polymer planar lightwave circuit (PLC) hybrid integration technology with volume holographic grating triplex filter VHGT, surface-illuminated photodetectors and spot-size converted Fabry-Pérot laser diode in SMD package. The hybrid PLC has composed from a two parts-polymer optical waveguide including VHGT filter section and a optoelectronic microwave section. The both parts are placed on the composite substrate.

  2. Transplatin-conjugated triplex-forming oligonucleotides form adducts with both strands of DNA.

    PubMed

    Campbell, Meghan A; Miller, Paul S

    2009-12-01

    Triplex-forming oligonucleotides (TFOs) can bind to polypurine x polypyrimidine tracts in DNA and, as a consequence, perturb the normal functioning of a targeted gene. The effectiveness of such antigene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here, we report that attachment of N-7-platinated guanine nucleosides to the 3'- and/or 5'-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3',5'-bis-platinated TFOs can result in the formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37 degrees C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast, high levels (approximately 86%) of adduct formation by platinated 2'-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2'-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinum-conjugated TFOs may be good candidates for use as antigene agents. PMID:19950917

  3. Evaluation of a triplex real-time PCR system to detect the plant-pathogenic molds Alternaria spp., Fusarium spp. and C. purpurea.

    PubMed

    Grube, Sabrina; Schönling, Jutta; Prange, Alexander

    2015-12-01

    This article describes the development of a triplex real-time PCR system for the simultaneous detection of three major plant-pathogenic mold genera (Alternaria spp., Fusarium spp. and the species Claviceps purpurea). The designed genus-specific primer-probe systems were validated for sensitivity, specificity and amplification in the presence of background DNA. PMID:26545945

  4. Gold nanoparticles-based colorimetric investigation of triplex formation under weak alkalic pH environment with the aid of Ag +

    NASA Astrophysics Data System (ADS)

    Xiong, Cen; Wu, Chengke; Zhang, Hong; Ling, Liansheng

    2011-09-01

    A novel colorimetric method for investigating triplex formation between oligonucleotide modified Au nanoparticles (AuNPs) under weak alkalic pH environment is developed based upon the specific recognition property of Ag + with CGC triads. Oligonucleotide 5'-SH-T 12-CTTCTTTCCTTTCTTC-3' (oligo-1) is modified on the surface of AuNPs. Upon addition of oligonucleotide 5'-GAAGAAAGGAAAGAAG-3' (oligo-2), triplex formation between oligo-1 modified AuNPs occurred at pH 8.0 with the aid of Ag +, triggers the aggregation of AuNPs, accompany with the solution color change from red to purple. The melting temperature demonstrates a 31 °C increase for the triplex DNA compose of 10 T•A∘T triads and 6 C•G∘C triads upon addition of Ag +, the disassociation constant ( Kd) between Ag + and C•G∘C triads is 3.6 μM. Moreover, triplex formation between AuNPs depending on Ag + can be used to recognize Ag + ion with the naked eye, as well as UV-vis absorption spectroscopy.

  5. Small molecule binding to a G-hairpin and a G-triplex: a new insight into anticancer drug design targeting G-rich regions.

    PubMed

    Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Teulade-Fichou, Marie-Paule; Mergny, Jean-Louis; Sugiyama, Hiroshi

    2015-06-01

    To gain new insights into G-quadruplex-drug interactions, we captured the solution-state structures of the complexes between a drug-like small molecule and a G-hairpin/G-triplex. Our results indicated that the ligand initially binds to the intermediates and induces stepwise folding into a quadruplex. PMID:25951794

  6. In vitro selection of oligonucleotides that bind double-stranded DNA in the presence of triplex-stabilizing agents

    PubMed Central

    Ayel, Elodie; Escudé, Christophe

    2010-01-01

    A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine–oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand–DNA complexes. PMID:20007154

  7. Stable DNA Nanomachine Based on Duplex-Triplex Transition for Ratiometric Imaging Instantaneous pH Changes in Living Cells.

    PubMed

    Yang, Mengqi; Zhang, Xiaoling; Liu, Haipeng; Kang, Huaizhi; Zhu, Zhi; Yang, Wen; Tan, Weihong

    2015-06-16

    DNA nanomachines are becoming useful tools for molecular recognition, imaging, and diagnostics and have drawn gradual attention. Unfortunately, the present application of most DNA nanomachines is limited in vitro, so expanding their application in organism has become a primary focus. Hence, a novel DNA nanomachine named t-switch, based on the DNA duplex-triplex transition, is developed for monitoring the intracellular pH gradient. Our strategy is based on the DNA triplex structure containing C(+)-G-C triplets and pH-dependent Förster resonance energy transfer (FRET). Our results indicate that the t-switch is an efficient reporter of pH from pH 5.3 to 6.0 with a fast response of a few seconds. Also the uptake of the t-switch is speedy. In order to protect the t-switch from enzymatic degradation, PEI is used for modification of our DNA nanomachine. At the same time, the dynamic range could be extended to pH 4.6-7.8. The successful application of this pH-depended DNA nanomachine and motoring spatiotemporal pH changes associated with endocytosis is strong evidence of the possibility of self-assembly DNA nanomachine for imaging, targeted therapies, and controllable drug delivery. PMID:26016566

  8. Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis, duplex and triplex studies.

    PubMed

    Kumar, Pawan; Sharma, Pawan K; Hansen, Jonas; Jedinak, Lukas; Reslow-Jacobsen, Charlotte; Hornum, Mick; Nielsen, Poul

    2016-02-15

    In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2'-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2'-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker. PMID:26778611

  9. Regulation of Transcription through Light-Activation and Light-Deactivation of Triplex-Forming Oligonucleotides in Mammalian Cells

    PubMed Central

    Govan, Jeane M.; Uprety, Rajendra; Hemphill, James; Lively, Mark O.

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the first temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO, and leading to the inhibition of gene transcription. Furthermore, the synthesis and site-specific incorporation of caged deoxycytidine nucleotides within TFO inhibitor sequences was developed, and allows for the light-deactivation of TFO function and thus photochemical activation of gene expression. After UV-induced removal of the caging groups, the TFO forms a DNA dumbbell structure, rendering it inactive, releasing it from the DNA, and activating transcription. These are the first examples of light-regulated TFOs and their application in the photochemical activation and deactivation of gene expression. In addition, hairpin loop structures were found to significantly increase the efficacy of phosphodiester DNA-based TFOs in tissue culture. PMID:22540192

  10. Regulation of transcription through light-activation and light-deactivation of triplex-forming oligonucleotides in mammalian cells.

    PubMed

    Govan, Jeane M; Uprety, Rajendra; Hemphill, James; Lively, Mark O; Deiters, Alexander

    2012-07-20

    Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO and leading to the inhibition of transcription. Furthermore, the synthesis and site-specific incorporation of caged deoxycytidine nucleotides within TFO inhibitor sequences was developed, allowing for the light-deactivation of TFO function and thus photochemical activation of gene expression. After UV-induced removal of the caging groups, the TFO forms a DNA dumbbell structure, rendering it inactive, releasing it from the DNA, and activating transcription. These are the first examples of light-regulated TFOs and their application in the photochemical activation and deactivation of gene expression. In addition, hairpin loop structures were found to significantly increase the efficacy of phosphodiester DNA-based TFOs in tissue culture. PMID:22540192

  11. Glass-NiP-CoFeP triplex-shell particles with hollow cores and tunable magnetic properties.

    PubMed

    An, Zhenguo; Zhang, Jingjie

    2013-02-01

    Low density (0.55-0.92g/mL, depending on the shell thickness and composition) glass-metal-metal triplex-shell hollow particles (TSHP) were prepared by a three-step route. First, micrometer-sized silicate glass particles with hollow cores, uniform shells, and high sphericity were prepared through spray drying and subsequent melting. NiP shell was uniformly assembled to the previously obtained glass hollow particles by silver seed induced chemical reduction of Ni(2+) by sodium hypophosphite, and glass-NiP double-shell hollow particles (DSHP) with compact and uniform shells were formed. The as-formed NiP particles further acted as the seeds for the directed formation and assembly of the CoFeP shell on the NiP shell to form the final glass-NiP-CoFeP triplex-shell hollow particles (TSHP). The influences of the component of the reaction system on the composition, structure, and magnetic properties of the hollow particles were studied. The multishell hollow particles thus obtained may have some promising applications in the fields of low-density magnetic materials, conduction, microwave absorbers, catalysis, etc. This work provides an additional strategy to fabricate multishell structured hollow particles with tailored shell composition and magnetic properties, which can be extended to the controlled preparation of multishell composite particles with the shells consisting of metal, oxides, or other compounds. PMID:23281871

  12. Growth inhibition and apoptosis induced by daunomycin-conjugated triplex-forming oligonucleotides targeting the c-myc gene in prostate cancer cells

    PubMed Central

    Napoli, Sara; Negri, Umberto; Arcamone, Federico; Capobianco, Massimo L.; Carbone, Giuseppina M.; Catapano, Carlo V.

    2006-01-01

    Covalent attachment of intercalating agents to triplex-forming oligonucleotides (TFOs) is a promising strategy to enhance triplex stability and biological activity. We have explored the possibility to use the anticancer drug daunomycin as triplex stabilizing agent. Daunomycin-conjugated TFOs (dauno-TFOs) bind with high affinity and maintain the sequence-specificity required for targeting individual genes in the human genome. Here, we examined the effects of two dauno-TFOs targeting the c-myc gene on gene expression, cell proliferation and survival. The dauno-TFOs were directed to sequences immediately upstream (dauno-GT11A) and downstream (dauno-GT11B) the major transcriptional start site in the c-myc gene. Both dauno-TFOs were able to down-regulate promoter activity and transcription of the endogenous gene. Myc-targeted dauno-TFOs inhibited growth and induced apoptosis of prostate cancer cells constitutively expressing the gene. Daunomycin-conjugated control oligonucleotides with similar sequences had only minimal effects, confirming that the activity of dauno-TFOs was sequence-specific and triplex-mediated. To test the selectivity of dauno-TFOs, we examined their effects on growth of normal human fibroblasts, which express low levels of c-myc. Despite their ability to inhibit c-myc transcription, both dauno-TFOs failed to inhibit growth of normal fibroblasts at concentrations that inhibited growth of prostate cancer cells. In contrast, daunomycin inhibited equally fibroblasts and prostate cancer cells. Thus, daunomycin per se did not contribute to the antiproliferative activity of dauno-TFOs, although it greatly enhanced their ability to form stable triplexes at the target sites and down-regulate c-myc. Our data indicate that dauno-TFOs are attractive gene-targeting agents for development of new cancer therapeutics. PMID:16449206

  13. FC-TRIPLEX Chagas/Leish IgG1: a multiplexed flow cytometry method for differential serological diagnosis of chagas disease and leishmaniasis.

    PubMed

    Teixeira-Carvalho, Andréa; Campos, Fernanda Magalhães Freire; Geiger, Stefan Michael; Rocha, Roberta Dias Rodrigues; de Araújo, Fernanda Fortes; Vitelli-Avelar, Danielle Marquete; Andrade, Mariléia Chaves; Araújo, Márcio Sobreira Silva; Lemos, Elenice Moreira; de Freitas Carneiro Proietti, Anna Bárbara; Sabino, Ester Cerdeira; Caldas, Rafaella Gaiotti; Freitas, Carolina Renata Camargos; Campi-Azevedo, Ana Carolina; Elói-Santos, Silvana Maria; Martins-Filho, Olindo Assis

    2015-01-01

    Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4 °C, and -20 °C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis. PMID:25875961

  14. FC-TRIPLEX Chagas/Leish IgG1: A Multiplexed Flow Cytometry Method for Differential Serological Diagnosis of Chagas Disease and Leishmaniasis

    PubMed Central

    Teixeira-Carvalho, Andréa; Campos, Fernanda Magalhães Freire; Geiger, Stefan Michael; Rocha, Roberta Dias Rodrigues; de Araújo, Fernanda Fortes; Vitelli-Avelar, Danielle Marquete; Andrade, Mariléia Chaves; Araújo, Márcio Sobreira Silva; Lemos, Elenice Moreira; de Freitas Carneiro Proietti, Anna Bárbara; Sabino, Ester Cerdeira; Caldas, Rafaella Gaiotti; Freitas, Carolina Renata Camargos; Campi-Azevedo, Ana Carolina; Elói-Santos, Silvana Maria; Martins-Filho, Olindo Assis

    2015-01-01

    Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4°C, and –20°C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis. PMID:25875961

  15. Television Quiz Show Simulation

    ERIC Educational Resources Information Center

    Hill, Jonnie Lynn

    2007-01-01

    This article explores the simulation of four television quiz shows for students in China studying English as a foreign language (EFL). It discusses the adaptation and implementation of television quiz shows and how the students reacted to them.

  16. A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

    PubMed Central

    Bliem, Rupert; Schauer, Sonja; Plicka, Helga; Obwaller, Adelheid; Sommer, Regina; Steinrigl, Adolf; Alam, Munirul; Reischer, Georg H.; Farnleitner, Andreas H.

    2015-01-01

    Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 102 to 2.3 × 104 cell equivalents liter−1, whereas GR-corrected abundances ranged from 4.7 × 103 to 1.6 × 106 cell equivalents liter−1. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. PMID:25724966

  17. A novel triplex quantitative PCR strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments.

    PubMed

    Bliem, Rupert; Schauer, Sonja; Plicka, Helga; Obwaller, Adelheid; Sommer, Regina; Steinrigl, Adolf; Alam, Munirul; Reischer, Georg H; Farnleitner, Andreas H; Kirschner, Alexander

    2015-05-01

    Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6×10(2) to 2.3×10(4) cell equivalents liter(-1), whereas GR-corrected abundances ranged from 4.7×10(3) to 1.6×10(6) cell equivalents liter(-1). GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. PMID:25724966

  18. Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

    NASA Astrophysics Data System (ADS)

    Vasquez, Karen M.; Christensen, Jesper; Li, Lei; Finch, Rick A.; Glazer, Peter M.

    2002-04-01

    Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

  19. T.C.G triplet in an antiparallel purine.purine.pyrimidine DNA triplex. Conformational studies by NMR.

    PubMed

    Dittrich, K; Gu, J; Tinder, R; Hogan, M; Gao, X

    1994-04-12

    The antiparallel purine.purine.pyrimidine DNA triplex, RRY6, which contains a T.C.G inverted triplet in the center of the sequence, was examined by proton and phosphorous two-dimensional NMR spectroscopy. The local conformation of the T.C.G triplet (T4.C11.G18) and the effect of this triplet on the global helical structure were analyzed in detail. The formation of the T.C.G triplet is confirmed by a set of cross-strand NOEs, including unusual cross-strand NOEs between the third strand and the pyrimidine strand as opposed to the purine strand of the duplex. NMR data suggest that the T.C.G triplet may be present in an equilibrium between a non-hydrogen-bonded form and a T(O4)-C(NH2) hydrogen-bonded form and that there is a distortion of the in-plane alignment of the three bases. The flanking G.G.C base triplets are well-defined on the 5'-side of T4, but somewhat interrupted on the 3'-side of T4. The effect of the third strand binding on the Watson-Crick duplex was probed by an NMR study of the free duplex RY6. NMR parameters are affected mostly around the T.C.G inversion site. The perturbations extend to at least two adjacent base triplets on either side. The binding of the third purine strand and the accommodation of a central T.C.G inversion in RRY6 does not require a readjustment in sugar pucker, which remains in the range of C2'-endo. 31P resonances of RRY6 distribute over a range of 2.2 ppm. The H-P coupling patterns of the third strand differ from those of the duplex. General spectral patterns defined by the marker protons of the RRY and YRY triplexes are compared. PMID:8155628

  20. The Wordpath Show.

    ERIC Educational Resources Information Center

    Anderton, Alice

    The Intertribal Wordpath Society is a nonprofit educational corporation formed to promote the teaching, status, awareness, and use of Oklahoma Indian languages. The Society produces "Wordpath," a weekly 30-minute public access television show about Oklahoma Indian languages and the people who are teaching and preserving them. The show aims to…

  1. A Holographic Road Show.

    ERIC Educational Resources Information Center

    Kirkpatrick, Larry D.; Rugheimer, Mac

    1979-01-01

    Describes the viewing sessions and the holograms of a holographic road show. The traveling exhibits, believed to stimulate interest in physics, include a wide variety of holograms and demonstrate several physical principles. (GA)

  2. Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.

    PubMed

    Semenyuk, A; Darian, E; Liu, J; Majumdar, A; Cuenoud, B; Miller, P S; Mackerell, A D; Seidman, M M

    2010-09-14

    The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence. PMID:20701359

  3. Targeting of an Interrupted Polypurine:Polypyrimidine Sequence in Mammalian Cells by a Triplex-Forming Oligonucleotide Containing a Novel Base Analogue†

    PubMed Central

    Semenyuk, A.; Darian, E.; Liu, J.; Majumdar, A.; Cuenoud, B.; Miller, P. S.; MacKerell, A. D.; Seidman, M. M.

    2010-01-01

    The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2′-OMeguanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence. PMID:20701359

  4. Orchestration of Structural, Stereoelectronic, and Hydrogen-Bonding Effects in Stabilizing Triplexes from Engineered Chimeric Collagen Peptides (Pro(X)-Pro(Y)-Gly)6 Incorporating 4(R/S)-Aminoproline.

    PubMed

    Umashankara, Muddegowda; Sonar, Mahesh V; Bansode, Nitin D; Ganesh, Krishna N

    2015-09-01

    Collagens are an important family of structural proteins found in the extracellular matrix with triple helix as the characteristic structural motif. The collagen triplex is made of three left-handed polyproline II (PPII) helices with each PPII strand consisting of repetitive units of the tripeptide motif X-Y-Gly, where the amino acids X and Y are most commonly proline (Pro) and 4R-hydroxyproline (Hyp), respectively. A C4-endo pucker at X-site and C4-exo pucker at Y-site have been proposed to be the key for formation of triplex, and the nature of pucker is dependent on both the electronegativity and stereochemistry of the substituent. The present manuscript describes a new class of collagen analogues-chimeric cationic collagens-wherein both X- and Y-sites in collagen triad are simultaneously substituted by a combination of 4(R/S)-(OH/NH2/NH3(+)/NHCHO)-prolyl units and triplex stabilities measured at different pHs and in EG:H2O. Based on the results a model has been proposed with the premise that any factors which specifically favor the ring puckers of C4-endo at X-site and C4-exo at Y-site stabilize the PPII conformation and hence the derived triplexes. The pH-dependent triplex stability uniquely observed with ionizable 4-amino substituent on proline enables one to define the critical combination of factors C4-(exo/endo), intraresidue H-bonding, stereoelectronic (R/S) and n → π* interactions in dictating the triplex strength. The ionizable NH2 substituent at C4 in R/S configuration is thus a versatile probe for delineating the triplex stabilizing factors and the results have potential for designing of collagen analogues with customized properties for material and biological applications. PMID:26274096

  5. Show What You Know

    ERIC Educational Resources Information Center

    Eccleston, Jeff

    2007-01-01

    Big things come in small packages. This saying came to the mind of the author after he created a simple math review activity for his fourth grade students. Though simple, it has proven to be extremely advantageous in reinforcing math concepts. He uses this activity, which he calls "Show What You Know," often. This activity provides the perfect…

  6. The Ozone Show.

    ERIC Educational Resources Information Center

    Mathieu, Aaron

    2000-01-01

    Uses a talk show activity for a final assessment tool for students to debate about the ozone hole. Students are assessed on five areas: (1) cooperative learning; (2) the written component; (3) content; (4) self-evaluation; and (5) peer evaluation. (SAH)

  7. Honored Teacher Shows Commitment.

    ERIC Educational Resources Information Center

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  8. Talk Show Science.

    ERIC Educational Resources Information Center

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  9. Stage a Water Show

    ERIC Educational Resources Information Center

    Frasier, Debra

    2008-01-01

    In the author's book titled "The Incredible Water Show," the characters from "Miss Alaineus: A Vocabulary Disaster" used an ocean of information to stage an inventive performance about the water cycle. In this article, the author relates how she turned the story into hands-on science teaching for real-life fifth-grade students. The author also…

  10. Showing What They Know

    ERIC Educational Resources Information Center

    Cech, Scott J.

    2008-01-01

    Having students show their skills in three dimensions, known as performance-based assessment, dates back at least to Socrates. Individual schools such as Barrington High School--located just outside of Providence--have been requiring students to actively demonstrate their knowledge for years. The Rhode Island's high school graduating class became…

  11. Taking in a Show.

    PubMed

    Boden, Timothy W

    2016-01-01

    Many medical practices have cut back on education and staff development expenses, especially those costs associated with conventions and conferences. But there are hard-to-value returns on your investment in these live events--beyond the obvious benefits of acquired knowledge and skills. Major vendors still exhibit their services and wares at many events, and the exhibit hall is a treasure-house of information and resources for the savvy physician or administrator. Make and stick to a purposeful plan to exploit the trade show. You can compare products, gain new insights and ideas, and even negotiate better deals with representatives anxious to realize returns on their exhibition investments. PMID:27249887

  12. Not a "reality" show.

    PubMed

    Wrong, Terence; Baumgart, Erica

    2013-01-01

    The authors of the preceding articles raise legitimate questions about patient and staff rights and the unintended consequences of allowing ABC News to film inside teaching hospitals. We explain why we regard their fears as baseless and not supported by what we heard from individuals portrayed in the filming, our decade-long experience making medical documentaries, and the full un-aired context of the scenes shown in the broadcast. The authors don't and can't know what conversations we had, what documents we reviewed, and what protections we put in place in each televised scene. Finally, we hope to correct several misleading examples cited by the authors as well as their offhand mischaracterization of our program as a "reality" show. PMID:23631336

  13. Public medical shows.

    PubMed

    Walusinski, Olivier

    2014-01-01

    In the second half of the 19th century, Jean-Martin Charcot (1825-1893) became famous for the quality of his teaching and his innovative neurological discoveries, bringing many French and foreign students to Paris. A hunger for recognition, together with progressive and anticlerical ideals, led Charcot to invite writers, journalists, and politicians to his lessons, during which he presented the results of his work on hysteria. These events became public performances, for which physicians and patients were transformed into actors. Major newspapers ran accounts of these consultations, more like theatrical shows in some respects. The resultant enthusiasm prompted other physicians in Paris and throughout France to try and imitate them. We will compare the form and substance of Charcot's lessons with those given by Jules-Bernard Luys (1828-1897), Victor Dumontpallier (1826-1899), Ambroise-Auguste Liébault (1823-1904), Hippolyte Bernheim (1840-1919), Joseph Grasset (1849-1918), and Albert Pitres (1848-1928). We will also note their impact on contemporary cinema and theatre. PMID:25273491

  14. The Great Cometary Show

    NASA Astrophysics Data System (ADS)

    2007-01-01

    its high spatial and spectral resolution, it was possible to zoom into the very heart of this very massive star. In this innermost region, the observations are dominated by the extremely dense stellar wind that totally obscures the underlying central star. The AMBER observations show that this dense stellar wind is not spherically symmetric, but exhibits a clearly elongated structure. Overall, the AMBER observations confirm that the extremely high mass loss of Eta Carinae's massive central star is non-spherical and much stronger along the poles than in the equatorial plane. This is in agreement with theoretical models that predict such an enhanced polar mass-loss in the case of rapidly rotating stars. ESO PR Photo 06c/07 ESO PR Photo 06c/07 RS Ophiuchi in Outburst Several papers from this special feature focus on the later stages in a star's life. One looks at the binary system Gamma 2 Velorum, which contains the closest example of a star known as a Wolf-Rayet. A single AMBER observation allowed the astronomers to separate the spectra of the two components, offering new insights in the modeling of Wolf-Rayet stars, but made it also possible to measure the separation between the two stars. This led to a new determination of the distance of the system, showing that previous estimates were incorrect. The observations also revealed information on the region where the winds from the two stars collide. The famous binary system RS Ophiuchi, an example of a recurrent nova, was observed just 5 days after it was discovered to be in outburst on 12 February 2006, an event that has been expected for 21 years. AMBER was able to detect the extension of the expanding nova emission. These observations show a complex geometry and kinematics, far from the simple interpretation of a spherical fireball in extension. AMBER has detected a high velocity jet probably perpendicular to the orbital plane of the binary system, and allowed a precise and careful study of the wind and the shockwave

  15. Direct identification of mycobacteria from liquid media using a triplex real-time PCR coupled with pyrosequencing method.

    PubMed

    Kim, Jeong-Uk; Cha, Choong-Hwan; Park, Seon-Hee

    2015-12-01

    Culture in enriched broth, as well as on a solid medium, is recommended for primary isolation of mycobacteria. With the introduction of liquid mycobacterial culture methods, a substantial workload regarding the identification of culture-recovered mycobacterial species, particularly Mycobacterium tuberculosis complex (MTC), has been imposed on our laboratory. We thus developed a triplex, real-time PCR coupled with pyrosequencing assay that can directly identify mycobacterial species from liquid media, which can reduce the workload. In this assay, real-time PCR simultaneously detects MTC and Mycobacterium xenopi, and amplifies the region of 16S rRNA gene containing hypervariable region A for pyrosequencing analysis; subsequent, pyrosequencing identifies many other nontuberculous mycobacteria. The assay was evaluated using 333 DNA samples directly prepared from liquid media, including 24 reference strains and 309 clinical isolates. Three hundred and twenty-eight (98.5%) of the 333 samples were correctly identified. The remaining five were determined as indeterminate. In conclusion, this coupled assay would be an alternative method for rapid identification of mycobacteria directly from liquid media in a clinical laboratory with a high workload in regions where tuberculosis is endemic. PMID:26471200

  16. Triplex Forming Oligonucleotides against Type α 1(I) Collagen attenuates Liver Fibrosis induced by Bile Duct ligation

    PubMed Central

    Panakanti, Ravikiran; Pratap, Akshay; Yang, Ningning; Jackson, John S.; Mahato, Ram I.

    2010-01-01

    Liver fibrosis is a consequence of chronic liver disorders which lead to the accumulation of extracellular matrix (ECM). Particularly, there is an increased accumulation of collagen in the fibrotic liver. We have therefore used a triplex forming oligonucleotide (TFO) against the type α 1 (I) collagen and evaluated, whether it can attenuate liver fibrosis induced by common bile duct ligation (CBDL) in rats. There was a significant decrease in hydroxyproline levels and Masson’s trichrome staining for collagen in TFO-treated CBDL groups compared to non-treated CBDL group. There was over expression of type α1(I) collagen, α-smooth muscle actin (α-SMA) and TGF-β1 expression in the CBDL group compared to TFO-treated CBDL group. Also, the serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were less in the TFO treated group compared to non-treated CBDL group. There was also less neutrophils accumulation in TFO treated CBDL group assayed by myeloperoxidase (MPO) assay. These results suggests that TFO can be used to downregulate type 1 collagen gene expression and can alleviate liver fibrosis induced by common bile duct ligation. PMID:20816672

  17. Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

    PubMed

    McNeer, Nicole Ali; Anandalingam, Kavitha; Fields, Rachel J; Caputo, Christina; Kopic, Sascha; Gupta, Anisha; Quijano, Elias; Polikoff, Lee; Kong, Yong; Bahal, Raman; Geibel, John P; Glazer, Peter M; Saltzman, W Mark; Egan, Marie E

    2015-01-01

    Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects. PMID:25914116

  18. Fabrication of a TFF-Attached WDM-Type Triplex Transceiver Module Using Silica PLC Hybrid Integration Technology

    NASA Astrophysics Data System (ADS)

    Han, Young-Tak; Park, Yoon-Jung; Park, Sang-Ho; Shin, Jang-Uk; Lee, Chul-Wook; Ko, Hyunsung; Baek, Yongsoon; Park, Chul-Hee; Kwon, Yoon-Koo; Hwang, Wol-Yon; Oh, Kwang-Ryong; Sung, Heekyung

    2006-12-01

    An optical triplex transceiver (TRx) module, which consists of thin-film filter (TFF)-attached wavelength-division multiplexer (WDM) and photodiode (PD) carriers, has been fabricated using a silica planar lightwave circuit (PLC) hybrid integration technology. Two types of TFFs were attached to a diced sidewall of a silica-terraced PLC platform to realize the TFF-attached WDM. The PD carriers with a 45° mirror, on which receiving surface-illuminated PDs were bonded, were assembled with the PLC platform to form receiver (Rx) parts. As the main performances of the packaged TRx module, a very clear transmitter (Tx) eye pattern and minimum Rx sensitivity of -25.7 dBm were obtained under a 1.25-Gb/s Tx Rx operation for digital applications. For an analog Rx application, a module responsivity of about 0.8 A/W was achieved, and a second-order intermodulation distortion value of less than -70 dBc at an optical modulation index of 40% was obtained under a two-tone test of 400 and 450 MHz.

  19. Determination of navigation FDI thresholds using a Markov model. [Failure Detection and Identification in triplex inertial platform systems for Shuttle entry

    NASA Technical Reports Server (NTRS)

    Walker, B. K.; Gai, E.

    1978-01-01

    A method for determining time-varying Failure Detection and Identification (FDI) thresholds for single sample decision functions is described in the context of a triplex system of inertial platforms. A cost function consisting of the probability of vehicle loss due to FDI decision errors is minimized. A discrete Markov model is constructed from which this cost can be determined as a function of the decision thresholds employed to detect and identify the first and second failures. Optimal thresholds are determined through the use of parameter optimization techniques. The application of this approach to threshold determination is illustrated for the Space Shuttle's inertial measurement instruments.

  20. A versatile label-free and signal-on electrochemical biosensing platform based on triplex-forming oligonucleotide probe.

    PubMed

    Wang, Xiuzhong; Jiang, Aiwen; Hou, Ting; Li, Feng

    2015-08-26

    Nucleic acid and protein assays are very important in modern life sciences, and the recently developed triplex-forming oligonucleotide probes provide a unique means for biological analysis of different kinds of analytes. Herein, we report a label-free and signal-on electrochemical sensor for the detection of specific targets, which is based on the triple-helix structure formation between the hairpin molecular beacon and the capture probe through the intermolecular DNA hybridization induced by Watson-Crick and Hoogsteen base pairings. Upon the introduction of a specific target, the triple-helical stem region is dissembled to liberate the hemin aptamer, and a G-quadruplex- hemin complex can be formed in the presence of K(+) and hemin on the electrode surface to give an electrochemical response, thus signaling the presence of the target. With the use of Human Immunodeficiency Virus type 1 (HIV-1) as a proof-of-principle analyte, we first demonstrated this approach by using a molecular beacon, which consists of a central section with the DNA sequence complementary to HIV-1, flanked by two arm segments. This newly designed protocol provides an ultrasensitive electrochemical detection of HIV-1 with a limit of detection down to 0.054 nM, and also exhibit good selectivity. Therefore, the as-proposed strategy holds a great potential for early diagnosis in gene-related diseases, and with further development, it could be used as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules. PMID:26347170

  1. Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs.

    PubMed

    Ohkubo, Akihiro; Yamada, Kenji; Ito, Yu; Yoshimura, Kiichi; Miyauchi, Koichiro; Kanamori, Takashi; Masaki, Yoshiaki; Seio, Kohji; Yuasa, Hideya; Sekine, Mitsuo

    2015-07-13

    A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2'-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (d(g)C) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2'-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2'-OMe (g)C), 2'-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2'-OMe (g)Cs), d(g)C and 4S-(2-guanidoethyl)-4-thiothymidine ((gs)T). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine ((DA)Nac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing (DA)Nac, d(g)C, 2'-OMe-2-thiothymidine (2'-OMe (s)T) and 2'-OMe-8-thioxoadenosine (2'-OMe (s)A) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions. PMID:26013815

  2. Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs

    PubMed Central

    Ohkubo, Akihiro; Yamada, Kenji; Ito, Yu; Yoshimura, Kiichi; Miyauchi, Koichiro; Kanamori, Takashi; Masaki, Yoshiaki; Seio, Kohji; Yuasa, Hideya; Sekine, Mitsuo

    2015-01-01

    A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2’-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (dgC) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2’-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2’-OMegC), 2’-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2’-OMegCs), dgC and 4S-(2-guanidoethyl)-4-thiothymidine (gsT). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine (DANac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing DANac, dgC, 2’-OMe-2-thiothymidine (2’-OMesT) and 2’-OMe-8-thioxoadenosine (2’-OMesA) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions. PMID:26013815

  3. Double-stranded DNA-templated cleavage of oligonucleotides containing a P3'->N5' linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity.

    PubMed

    Ito, Kosuke Ramon; Kodama, Tetsuya; Tomizu, Masaharu; Negoro, Yoshinori; Orita, Ayako; Osaki, Tomohisa; Hosoki, Noritsugu; Tanaka, Takaya; Imanishi, Takeshi; Obika, Satoshi

    2010-11-01

    We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5'-amino-2',4'-BNA were used as a DNA-detecting probe. This modification introduced a P3'→N5' linkage (P-N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P-N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P-N linkage should change the reactivity by altering the microenvironment. We synthesized 5'-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5'-amino-2',4'-BNA to investigate the effect of 5'-elongation. In addition, 2',4'-BNA/LNA or 2',5'-linked DNA were introduced at the 3'- and/or 5'-neighboring residues of 5'-amino-2',4'-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P-N linkage greatly affected their reaction properties. Notably, 2',5'-linked DNA at the 3' position flanking 5'-amino-2',4'-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool. PMID:20615902

  4. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    PubMed

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  5. Development of a novel multi-triplex qPCR method for the assessment of bacterial community structure in oral populations.

    PubMed

    Ciric, Lena; Pratten, Jonathan; Wilson, Michael; Spratt, David

    2010-12-01

    Gingivitis and dental caries are two of the most predominant diseases in humans. Both conditions are easily treated with the removal of the plaque biofilm by brushing or the use of oral hygiene products. In both cases, pathogenic taxa found within the plaque biofilm are the causal agents of the disease. Actinomyces naeslundii, Fusobacterium nucleatum and Prevotalla intermedia have all been implicated in the development of gingivitis, while Streptococcus mutans is the main organism associated with dental caries. Many studies have so far focused on the use of culture methods to detect and enumerate the pathogenic taxa within plaque samples. However, these methods are both labour intensive and biased towards culturable taxa. In the present study, a novel high-throughput multi-triplex quantitative PCR method was developed with the aim to investigate the community dynamics associated with oral communities. Three triplex assays were designed targeting taxa associated with gingivitis and dental caries as well as oral health. Saliva samples collected from healthy individuals were used in order to validate the newly developed method. PMID:23766283

  6. Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers

    SciTech Connect

    Crum, C.P.; Nuovo, G.; Friedman, D.; Silverstein, S.J.

    1988-01-01

    The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded /sup 35/S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggest that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata. Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.

  7. LocARNAscan: Incorporating thermodynamic stability in sequence and structure-based RNA homology search

    PubMed Central

    2013-01-01

    Background The search for distant homologs has become an import issue in genome annotation. A particular difficulty is posed by divergent homologs that have lost recognizable sequence similarity. This same problem also arises in the recognition of novel members of large classes of RNAs such as snoRNAs or microRNAs that consist of families unrelated by common descent. Current homology search tools for structured RNAs are either based entirely on sequence similarity (such as blast or hmmer) or combine sequence and secondary structure. The most prominent example of the latter class of tools is Infernal. Alternatives are descriptor-based methods. In most practical applications published to-date, however, the information contained in covariance models or manually prescribed search patterns is dominated by sequence information. Here we ask two related questions: (1) Is secondary structure alone informative for homology search and the detection of novel members of RNA classes? (2) To what extent is the thermodynamic propensity of the target sequence to fold into the correct secondary structure helpful for this task? Results Sequence-structure alignment can be used as an alternative search strategy. In this scenario, the query consists of a base pairing probability matrix, which can be derived either from a single sequence or from a multiple alignment representing a set of known representatives. Sequence information can be optionally added to the query. The target sequence is pre-processed to obtain local base pairing probabilities. As a search engine we devised a semi-global scanning variant of LocARNA’s algorithm for sequence-structure alignment. The LocARNAscan tool is optimized for speed and low memory consumption. In benchmarking experiments on artificial data we observe that the inclusion of thermodynamic stability is helpful, albeit only in a regime of extremely low sequence information in the query. We observe, furthermore, that the sensitivity is bounded in particular by the limited accuracy of the predicted local structures of the target sequence. Conclusions Although we demonstrate that a purely structure-based homology search is feasible in principle, it is unlikely to outperform tools such as Infernal in most application scenarios, where a substantial amount of sequence information is typically available. The LocARNAscan approach will profit, however, from high throughput methods to determine RNA secondary structure. In transcriptome-wide applications, such methods will provide accurate structure annotations on the target side. Availability Source code of the free software LocARNAscan 1.0 and supplementary data are available at http://www.bioinf.uni-leipzig.de/Software/LocARNAscan. PMID:23601347

  8. VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. SHOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. SHOWS 499-501, MUNOZ HOUSE (AZ-73-37) ON FAR RIGHT - Antonio Bustamente House, 485-489 South Meyer Avenue & 186 West Kennedy Street, Tucson, Pima County, AZ

  9. 15. Detail showing lower chord pinconnected to vertical member, showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. Detail showing lower chord pin-connected to vertical member, showing floor beam riveted to extension of vertical member below pin-connection, and showing brackets supporting cantilevered sidewalk. View to southwest. - Selby Avenue Bridge, Spanning Short Line Railways track at Selby Avenue between Hamline & Snelling Avenues, Saint Paul, Ramsey County, MN

  10. Evidence for a group II intron-like catalytic triplex in the spliceosome

    PubMed Central

    Piccirilli, Joseph A.; Staley, Jonathan P.

    2014-01-01

    To catalyze pre-mRNA splicing, U6 snRNA positions two metals that interact directly with the scissile phosphates. The U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. In domain V, the ligands are organized by base-triple interactions, which also juxtapose the 3′ splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, crosslinking, and biochemistry in yeast that analogous triples form in U6 and promote catalytic metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5′ splice site, the triples also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing. PMID:24747940

  11. Asymmetric triplex metallohelices with high and selective activity against cancer cells

    NASA Astrophysics Data System (ADS)

    Faulkner, Alan D.; Kaner, Rebecca A.; Abdallah, Qasem M. A.; Clarkson, Guy; Fox, David J.; Gurnani, Pratik; Howson, Suzanne E.; Phillips, Roger M.; Roper, David I.; Simpson, Daniel H.; Scott, Peter

    2014-09-01

    Small cationic amphiphilic α-helical peptides are emerging as agents for the treatment of cancer and infection, but they are costly and display unfavourable pharmacokinetics. Helical coordination complexes may offer a three-dimensional scaffold for the synthesis of mimetic architectures. However, the high symmetry and modest functionality of current systems offer little scope to tailor the structure to interact with specific biomolecular targets, or to create libraries for phenotypic screens. Here, we report the highly stereoselective asymmetric self-assembly of very stable, functionalized metallohelices. Their anti-parallel head-to-head-to-tail ‘triplex’ strand arrangement creates an amphipathic functional topology akin to that of the active sub-units of, for example, host-defence peptides and p53. The metallohelices display high, structure-dependent toxicity to the human colon carcinoma cell-line HCT116 p53++, causing dramatic changes in the cell cycle without DNA damage. They have lower toxicity to human breast adenocarcinoma cells (MDA-MB-468) and, most remarkably, they show no significant toxicity to the bacteria methicillin-resistant Staphylococcus aureus and Escherichia coli.

  12. Development of SERS substrate using phage-based magnetic template for triplex assay in sepsis diagnosis.

    PubMed

    Nguyen, Anh H; Shin, Yesol; Sim, Sang Jun

    2016-11-15

    Development of a new substrate for surface-enhanced Raman scattering (SERS) is one area of interest for the improvement of SERS performance. Herein, we introduce a new method for developing new mesoporous SERS substrates using M13 phages that display cysteine-rich peptides on the pVIII major units, which is an alternative for thiol donor using chemical modifications. Together with the SERS substrate development, and the use of the SERS technique for sepsis diagnostics is a new approach in clinical settings. The substrates were characterized and magnetized with magnetic immuno colloids made of gold-coated magnetic nanoparticles and specific antibodies. Conventionally, the SERS-tags are prepared by using gold nanoparticles and are modified with Raman dyes to immobilize specific antibodies to capture the biomarkers in the serum samples. However, in this method the SERS-tags are bound to the mesoporous substrate via antibody/antigen interactions to form clusters or layer-by-layer assemblies of SERS-tags for Raman signal enhancement. The SERS spectra showed distinct peaks for tags corresponding to three typical sepsis-specific biomarkers for diagnostics with the limit of detection values of 27 pM, 103 pM, and 78 pM for C-reactive protein (CRP), procalcitonin (PCT), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), respectively. With such an approach, SERS can be used for clinical purposes and can be improved by phage display modification rather than chemical alternatives. PMID:27209579

  13. 28. MAP SHOWING LOCATION OF ARVFS FACILITY AS BUILT. SHOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    28. MAP SHOWING LOCATION OF ARVFS FACILITY AS BUILT. SHOWS LINCOLN BOULEVARD, BIG LOST RIVER, AND NAVAL REACTORS FACILITY. F.C. TORKELSON DRAWING NUMBER 842-ARVFS-101-2. DATED OCTOBER 12, 1965. INEL INDEX CODE NUMBER: 075 0101 851 151969. - Idaho National Engineering Laboratory, Advanced Reentry Vehicle Fusing System, Scoville, Butte County, ID

  14. 8. Detail showing concrete abutment, showing substructure of bridge, specifically ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. Detail showing concrete abutment, showing substructure of bridge, specifically west side of arch and substructure. - Presumpscot Falls Bridge, Spanning Presumptscot River at Allen Avenue extension, 0.75 mile west of U.S. Interstate 95, Falmouth, Cumberland County, ME

  15. Anti-Stokes luminescence of Ln2O2S:Er(3+), Yb(3+) in triplexes under 0.93, 1.06, 1.53, and 1.59-micron excitation, part 1

    NASA Astrophysics Data System (ADS)

    Kurochkin, A. V.; Mailibaeva, L. M.; Manashirov, O. Ya.; Sattarov, D. K.; Smirnov, V. B.

    1992-10-01

    Studies are reported of visible anti-Stokes luminescence of one of most efficient IR image converters, the oxysulphide Ln2O2S (Ln = Y,Gd, La), mono- and codoped by trivalent rare-earth ions. Studies of these anti-stokes luminophores involving also visual observations are performed in triplexes under low-intensity (compared with previous studies) excitation, 10-300 m W/cm(sup 2); lambda = 0.93 and 1.06 mu m, produced by various infrared sources. Mechanisms of anti-Stokes luminescence are examined. Experimental results allow us to explain the effects of technological (powder structure of the polycrystalline anti-Stokes luminophore, details of triplex preparation) and of physico-chemical factors on the quantum yield of anti-Stokes luminescence of (Ln(0.86)Yb(0.08)Er(0.06))(sub 2)O(sub 2)S.

  16. One-step triplex-polymerase chain reaction assay for the authentication of yellowfin (Thunnus albacares), bigeye (Thunnus obesus), and skipjack (Katsuwonus pelamis) tuna DNA from fresh, frozen, and canned tuna samples.

    PubMed

    Michelini, Elisa; Cevenini, Luca; Mezzanotte, Laura; Simoni, Patrizia; Baraldini, Mario; De Laude, Luca; Roda, Aldo

    2007-09-19

    A one-step triplex-polymerase chain reaction (PCR)-based assay was developed to discriminate between three tuna species, Thunnus albacares, Thunnus obesus, and Katsuwonus pelamis, even in highly processed food samples such as canned or cooked tuna. Diagnostic nucleotides were identified by direct sequencing and alignment of part of the mitochondrial cytochrome b gene of 30 authenticated exemplars, which allowed us to evaluate intraspecific variation and the genetic distance between three tuna species. The assay relies on a one-step triplex-PCR reaction in which in a single tube species-specific amplification products are generated only in the presence of the correct template nucleic acid and the species of origin of the DNA is indicated by the distinctive size of the PCR product. The identification of tuna species can be performed with a good accuracy, low cost, and with potential automation for large-scale high-throughput screenings in small in-house laboratories. PMID:17711337

  17. Double-stranded DNA-templated cleavage of oligonucleotides containing a P3′→N5′ linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity

    PubMed Central

    Ito, Kosuke Ramon; Kodama, Tetsuya; Tomizu, Masaharu; Negoro, Yoshinori; Orita, Ayako; Osaki, Tomohisa; Hosoki, Noritsugu; Tanaka, Takaya; Imanishi, Takeshi; Obika, Satoshi

    2010-01-01

    We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5′-amino-2′,4′-BNA were used as a DNA-detecting probe. This modification introduced a P3′→N5′ linkage (P–N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P–N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P–N linkage should change the reactivity by altering the microenvironment. We synthesized 5′-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5′-amino-2′,4′-BNA to investigate the effect of 5′-elongation. In addition, 2′,4′-BNA/LNA or 2′,5′-linked DNA were introduced at the 3′- and/or 5′-neighboring residues of 5′-amino-2′,4′-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P–N linkage greatly affected their reaction properties. Notably, 2′,5′-linked DNA at the 3′ position flanking 5′-amino-2′,4′-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool. PMID:20615902

  18. Planning a Successful Tech Show

    ERIC Educational Resources Information Center

    Nikirk, Martin

    2011-01-01

    Tech shows are a great way to introduce prospective students, parents, and local business and industry to a technology and engineering or career and technical education program. In addition to showcasing instructional programs, a tech show allows students to demonstrate their professionalism and skills, practice public presentations, and interact…

  19. Hey Teacher, Your Personality's Showing!

    ERIC Educational Resources Information Center

    Paulsen, James R.

    1977-01-01

    A study of 30 fourth, fifth, and sixth grade teachers and 300 of their students showed that a teacher's age, sex, and years of experience did not relate to students' mathematics achievement, but that more effective teachers showed greater "freedom from defensive behavior" than did less effective teachers. (DT)

  20. What Do Blood Tests Show?

    MedlinePlus

    ... shows the ranges for blood glucose levels after 8 to 12 hours of fasting (not eating). It shows the normal range and the abnormal ranges that are a sign of prediabetes or diabetes. Plasma Glucose Results (mg/dL)* Diagnosis 70 to 99 ...

  1. Satellite Movie Shows Erika Dissipate

    NASA Video Gallery

    This animation of visible and infrared imagery from NOAA's GOES-West satellite from Aug. 27 to 29 shows Tropical Storm Erika move through the Eastern Caribbean Sea and dissipate near eastern Cuba. ...

  2. National Orange Show Photovoltaic Demonstration

    SciTech Connect

    Dan Jimenez Sheri Raborn, CPA; Tom Baker

    2008-03-31

    National Orange Show Photovoltaic Demonstration created a 400KW Photovoltaic self-generation plant at the National Orange Show Events Center (NOS). The NOS owns a 120-acre state fairground where it operates an events center and produces an annual citrus fair known as the Orange Show. The NOS governing board wanted to employ cost-saving programs for annual energy expenses. It is hoped the Photovoltaic program will result in overall savings for the NOS, help reduce the State's energy demands as relating to electrical power consumption, improve quality of life within the affected grid area as well as increase the energy efficiency of buildings at our venue. In addition, the potential to reduce operational expenses would have a tremendous effect on the ability of the NOS to service its community.

  3. Creating Slide Show Book Reports.

    ERIC Educational Resources Information Center

    Taylor, Harriet G.; Stuhlmann, Janice M.

    1995-01-01

    Describes the use of "Kid Pix 2" software by fourth grade students to develop slide-show book reports. Highlights include collaboration with education majors from Louisiana State University, changes in attitudes of the education major students and elementary students, and problems with navigation and disk space. (LRW)

  4. Producing Talent and Variety Shows.

    ERIC Educational Resources Information Center

    Szabo, Chuck

    1995-01-01

    Identifies key aspects of producing talent shows and outlines helpful hints for avoiding pitfalls and ensuring a smooth production. Presents suggestions concerning publicity, scheduling, and support personnel. Describes types of acts along with special needs and problems specific to each act. Includes a list of resources. (MJP)

  5. Magic Carpet Shows Its Colors

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The upper left image in this display is from the panoramic camera on the Mars Exploration Rover Spirit, showing the 'Magic Carpet' region near the rover at Gusev Crater, Mars, on Sol 7, the seventh martian day of its journey (Jan. 10, 2004). The lower image, also from the panoramic camera, is a monochrome (single filter) image of a rock in the 'Magic Carpet' area. Note that colored portions of the rock correlate with extracted spectra shown in the plot to the side. Four different types of materials are shown: the rock itself, the soil in front of the rock, some brighter soil on top of the rock, and some dust that has collected in small recesses on the rock face ('spots'). Each color on the spectra matches a line on the graph, showing how the panoramic camera's different colored filters are used to broadly assess the varying mineral compositions of martian rocks and soils.

  6. ENVITEC shows off air technologies

    SciTech Connect

    McIlvaine, R.W.

    1995-08-01

    The ENVITEC International Trade Fair for Environmental Protection and Waste Management Technologies, held in June in Duesseldorf, Germany, is the largest air pollution exhibition in the world and may be the largest environmental technology show overall. Visitors saw thousands of environmental solutions from 1,318 companies representing 29 countries and occupying roughly 43,000 square meters of exhibit space. Many innovations were displayed under the category, ``thermal treatment of air pollutants.`` New technologies include the following: regenerative thermal oxidizers; wet systems for removing pollutants; biological scrubbers;electrostatic precipitators; selective adsorption systems; activated-coke adsorbers; optimization of scrubber systems; and air pollution monitors.

  7. ShowMe3D

    Energy Science and Technology Software Center (ESTSC)

    2012-01-05

    ShowMe3D is a data visualization graphical user interface specifically designed for use with hyperspectral image obtained from the Hyperspectral Confocal Microscope. The program allows the user to select and display any single image from a three dimensional hyperspectral image stack. By moving a slider control, the user can easily move between images of the stack. The user can zoom into any region of the image. The user can select any pixel or region from themore » displayed image and display the fluorescence spectrum associated with that pixel or region. The user can define up to 3 spectral filters to apply to the hyperspectral image and view the image as it would appear from a filter-based confocal microscope. The user can also obtain statistics such as intensity average and variance from selected regions.« less

  8. ShowMe3D

    SciTech Connect

    Sinclair, Michael B

    2012-01-05

    ShowMe3D is a data visualization graphical user interface specifically designed for use with hyperspectral image obtained from the Hyperspectral Confocal Microscope. The program allows the user to select and display any single image from a three dimensional hyperspectral image stack. By moving a slider control, the user can easily move between images of the stack. The user can zoom into any region of the image. The user can select any pixel or region from the displayed image and display the fluorescence spectrum associated with that pixel or region. The user can define up to 3 spectral filters to apply to the hyperspectral image and view the image as it would appear from a filter-based confocal microscope. The user can also obtain statistics such as intensity average and variance from selected regions.

  9. Pea Plants Show Risk Sensitivity.

    PubMed

    Dener, Efrat; Kacelnik, Alex; Shemesh, Hagai

    2016-07-11

    Sensitivity to variability in resources has been documented in humans, primates, birds, and social insects, but the fit between empirical results and the predictions of risk sensitivity theory (RST), which aims to explain this sensitivity in adaptive terms, is weak [1]. RST predicts that agents should switch between risk proneness and risk aversion depending on state and circumstances, especially according to the richness of the least variable option [2]. Unrealistic assumptions about agents' information processing mechanisms and poor knowledge of the extent to which variability imposes specific selection in nature are strong candidates to explain the gap between theory and data. RST's rationale also applies to plants, where it has not hitherto been tested. Given the differences between animals' and plants' information processing mechanisms, such tests should help unravel the conflicts between theory and data. Measuring root growth allocation by split-root pea plants, we show that they favor variability when mean nutrient levels are low and the opposite when they are high, supporting the most widespread RST prediction. However, the combination of non-linear effects of nitrogen availability at local and systemic levels may explain some of these effects as a consequence of mechanisms not necessarily evolved to cope with variance [3, 4]. This resembles animal examples in which properties of perception and learning cause risk sensitivity even though they are not risk adaptations [5]. PMID:27374342

  10. Casimir experiments showing saturation effects

    SciTech Connect

    Sernelius, Bo E.

    2009-10-15

    We address several different Casimir experiments where theory and experiment disagree. First out is the classical Casimir force measurement between two metal half spaces; here both in the form of the torsion pendulum experiment by Lamoreaux and in the form of the Casimir pressure measurement between a gold sphere and a gold plate as performed by Decca et al.; theory predicts a large negative thermal correction, absent in the high precision experiments. The third experiment is the measurement of the Casimir force between a metal plate and a laser irradiated semiconductor membrane as performed by Chen et al.; the change in force with laser intensity is larger than predicted by theory. The fourth experiment is the measurement of the Casimir force between an atom and a wall in the form of the measurement by Obrecht et al. of the change in oscillation frequency of a {sup 87}Rb Bose-Einstein condensate trapped to a fused silica wall; the change is smaller than predicted by theory. We show that saturation effects can explain the discrepancies between theory and experiment observed in all these cases.

  11. Mimas Showing False Colors #1

    NASA Technical Reports Server (NTRS)

    2005-01-01

    False color images of Saturn's moon, Mimas, reveal variation in either the composition or texture across its surface.

    During its approach to Mimas on Aug. 2, 2005, the Cassini spacecraft narrow-angle camera obtained multi-spectral views of the moon from a range of 228,000 kilometers (142,500 miles).

    The image at the left is a narrow angle clear-filter image, which was separately processed to enhance the contrast in brightness and sharpness of visible features. The image at the right is a color composite of narrow-angle ultraviolet, green, infrared and clear filter images, which have been specially processed to accentuate subtle changes in the spectral properties of Mimas' surface materials. To create this view, three color images (ultraviolet, green and infrared) were combined into a single black and white picture that isolates and maps regional color differences. This 'color map' was then superimposed over the clear-filter image at the left.

    The combination of color map and brightness image shows how the color differences across the Mimas surface materials are tied to geological features. Shades of blue and violet in the image at the right are used to identify surface materials that are bluer in color and have a weaker infrared brightness than average Mimas materials, which are represented by green.

    Herschel crater, a 140-kilometer-wide (88-mile) impact feature with a prominent central peak, is visible in the upper right of each image. The unusual bluer materials are seen to broadly surround Herschel crater. However, the bluer material is not uniformly distributed in and around the crater. Instead, it appears to be concentrated on the outside of the crater and more to the west than to the north or south. The origin of the color differences is not yet understood. It may represent ejecta material that was excavated from inside Mimas when the Herschel impact occurred. The bluer color of these materials may be caused by subtle differences in

  12. LncRNA Khps1 Regulates Expression of the Proto-oncogene SPHK1 via Triplex-Mediated Changes in Chromatin Structure.

    PubMed

    Postepska-Igielska, Anna; Giwojna, Alena; Gasri-Plotnitsky, Lital; Schmitt, Nina; Dold, Annabelle; Ginsberg, Doron; Grummt, Ingrid

    2015-11-19

    Although thousands of long noncoding RNAs (lncRNAs) have been discovered, very little is known about their mode of action. Here we functionally characterize an E2F1-regulated lncRNA named Khps1, which is transcribed in antisense orientation to the proto-oncogene SPHK1. Khps1 activates SPHK1 expression by recruiting the histone acetyltransferase p300/CBP to the SPHK1 promoter, which leads to local changes of the chromatin structure that ensures E2F1 binding and enhances transcription. Mechanistically, this is achieved by direct association of Khps1 with a homopurine stretch upstream of the transcription start site of SPHK1, which forms a DNA-RNA triplex that anchors the lncRNA and associated effector proteins to the gene promoter. The results reveal an lncRNA- and E2F1-driven regulatory loop in which E2F1-dependent induction of antisense RNA leads to changes in chromatin structure, facilitating E2F1-dependent expression of SPHK1 and restriction of E2F1-induced apoptosis. PMID:26590717

  13. Triplex real-time PCR--an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells.

    PubMed

    Rygiel, Karolina A; Grady, John P; Taylor, Robert W; Tuppen, Helen A L; Turnbull, Doug M

    2015-01-01

    Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (OL) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings. PMID:25989140

  14. Triplex-forming oligonucleotides targeting c-MYC potentiate the anti-tumor activity of gemcitabine in a mouse model of human cancer.

    PubMed

    Boulware, Stephen B; Christensen, Laura A; Thames, Howard; Coghlan, Lezlee; Vasquez, Karen M; Finch, Rick A

    2014-09-01

    Antimetabolite chemotherapy remains an essential cancer treatment modality, but often produces only marginal benefit due to the lack of tumor specificity, the development of drug resistance, and the refractoriness of slowly proliferating cells in solid tumors. Here, we report a novel strategy to circumvent the proliferation-dependence of traditional antimetabolite-based therapies. Triplex-forming oligonucleotides (TFOs) were used to target site-specific DNA damage to the human c-MYC oncogene, thereby inducing replication-independent, unscheduled DNA repair synthesis (UDS) preferentially in the TFO-targeted region. The TFO-directed UDS facilitated incorporation of the antimetabolite, gemcitabine (GEM), into the damaged oncogene, thereby potentiating the anti-tumor activity of GEM. Mice bearing COLO 320DM human colon cancer xenografts (containing amplified c-MYC) were treated with a TFO targeted to c-MYC in combination with GEM. Tumor growth inhibition produced by the combination was significantly greater than with either TFO or GEM alone. Specific TFO binding to the genomic c-MYC gene was demonstrated, and TFO-induced DNA damage was confirmed by NBS1 accumulation, supporting a mechanism of enhanced efficacy of GEM via TFO-targeted DNA damage-induced UDS. Thus, coupling antimetabolite chemotherapeutics with a strategy that facilitates selective targeting of cells containing amplification of cancer-relevant genes can improve their activity against solid tumors, while possibly minimizing host toxicity. PMID:23681918

  15. Triplex-forming oligonucleotides targeting c-MYC potentiate the anti-tumor activity of gemcitabine in a mouse model of human cancer

    PubMed Central

    Boulware, Stephen B.; Christensen, Laura A.; Thames, Howard; Coghlan, Lezlee; Vasquez, Karen M.; Finch, Rick A.

    2014-01-01

    Antimetabolite chemotherapy remains an essential cancer treatment modality, but often produces only marginal benefit due to the lack of tumor specificity, the development of drug resistance, and the refractoriness of slowly-proliferating cells in solid tumors. Here, we report a novel strategy to circumvent the proliferation-dependence of traditional antimetabolite-based therapies. Triplex-forming oligonucleotides (TFOs) were used to target site-specific DNA damage to the human c-MYC oncogene, thereby inducing replication-independent, unscheduled DNA repair synthesis (UDS) preferentially in the TFO-targeted region. The TFO-directed UDS facilitated incorporation of the antimetabolite, gemcitabine (GEM), into the damaged oncogene, thereby potentiating the anti-tumor activity of GEM. Mice bearing COLO 320DM human colon cancer xenografts (containing amplified c-MYC) were treated with a TFO targeted to c-MYC in combination with GEM. Tumor growth inhibition produced by the combination was significantly greater than with either TFO or GEM alone. Specific TFO binding to the genomic c-MYC gene was demonstrated, and TFO-induced DNA damage was confirmed by NBS1 accumulation, supporting a mechanism of enhanced efficacy of GEM via TFO-targeted DNA damage-induced UDS. Thus, coupling antimetabolite chemotherapeutics with a strategy that facilitates selective targeting of cells containing amplification of cancer-relevant genes can improve their activity against solid tumors, while possibly minimizing host toxicity. PMID:23681918

  16. Development of novel triplex single-step real-time PCR assay for detection of Hepatitis Virus B and C simultaneously.

    PubMed

    Prakash, Shantanu; Jain, Amita; Jain, Bhawana

    2016-05-01

    Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of them provide quality check of sample. In the present study we developed a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) with sample quality check, by using β-actin as housekeeping gene. The primers and probes were self-designed and assay was standardized. Assay was also destined to quantitate copy numbers of HBV and HCV. This novel assay was sensitive, specific, and reproducible for detection of HBV and HCV in serum/plasma. The assay also detected all genotypes of HBV and HCV. The detection limit was 60 IU/mL for HBV and 20 IU/mL for HCV. This assay is the first assay developed on single-step platform for nucleic acid detection of HBV and HCV with an extra edge over all other assays by providing inbuilt check for quality of sample. PMID:26914508

  17. Effect of lower bainite/martensite/retained austenite triplex microstructure on the mechanical properties of a low-carbon steel with quenching and partitioning process

    NASA Astrophysics Data System (ADS)

    Li, Wan-song; Gao, Hong-ye; Li, Zhong-yi; Nakashima, Hideharu; Hata, Satoshi; Tian, Wen-huai

    2016-03-01

    We present a study concerning Fe-0.176C-1.31Si-1.58Mn-0.26Al-0.3Cr (wt%) steel subjected to a quenching and partitioning (Q&P) process. The results of scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and tensile tests demonstrate that the microstructures primarily consist of lath martensite, retained austenite, lower bainite (LB), and a small amount of tempered martensite; moreover, few twin austenite grains were observed. In the microstructure, three types of retained austenite with different sizes and morphologies were observed: blocky retained austenite (~300 nm in width), film-like retained austenite (80-120 nm in width), and ultra- fine film-like retained austenite (30-40 nm in width). Because of the effect of the retained austenite/martensite/LB triplex microstructure, the specimens prepared using different quenching temperatures exhibit high ultimate tensile strength and yield strength. Furthermore, the strength effect of LB can partially counteract the decreasing strength effect of martensite. The formation of LB substantially reduces the amount of retained austenite. Analyses of the retained austenite and the amount of blocky retained austenite indicated that the carbon content is critical to the total elongation of Q&P steel.

  18. A study of the ultrasound-targeted microbubble destruction based triplex-forming oligodexinucleotide delivery system to inhibit tissue factor expression

    PubMed Central

    LIANG, WEIHUA; ZHANG, WEIWEI; ZHAO, SHIFU; LI, QIANNING; YANG, YIMING; LIANG, HUA; CENG, RONGCHUAN

    2015-01-01

    The efficiency of cellular uptake of triplex-forming oligodexinucleotides (TFO), and the inhibition of tissue factor (TF) is low. The aim of the present study was to improve the absorption of TFO, and increase the inhibition of TF induced by shear stress both in vitro and in vivo, by using an ultrasound-targeted microbubble destruction (UTMD)-based delivery system. TFO-conjugated lipid ultrasonic microbubbles (TFO-M) were first constructed and characterised. The absorption of TFO was observed by a fluorescence-based method, and the inhibition of TF by immunofluorescence and quantitative polymerase chain reaction. ECV304 human umbilical vein endothelial cells were subjected to fluid shear stress for 6 h after treatment with TFO conjugated lipid ultrasonic microbubbles without sonication (TFO-M group); TFO alone; TFO conjugated lipid ultrasonic microbubbles, plus immediate sonication (TFO+U group and TFO-M+U group); or mock treated with 0.9% NaCl only (SSRE group). The in vivo experiments were established in a similar manner to the in vitro experiments, except that TFO or TFO-M was injected into rats through the tail vein. Six hours after the preparation of a carotid stenosis model, the rats were humanely sacrificed. The transfection efficiency of TFO in the TFO-M+U group was higher as compared with the TFO-M and TFO+U group (P<0.01). The protein and mRNA expression of TF in the TFO-M+U group was significantly decreased both in vitro and in vivo (P<0.01), as compared with the TFO-M, TFO+U and SSRE groups. The UTMD-based TFO delivery system promoted the absorption of TFO and the inhibition of TF, and was therefore considered to be favorable for preventing thrombosis induced by shear stress. PMID:25355395

  19. Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle.

    PubMed Central

    Brennwald, P; Liao, X; Holm, K; Porter, G; Wise, J A

    1988-01-01

    We have cloned the gene encoding a novel small cytoplasmic RNA from the fission yeast Schizosaccharomyces pombe. Four lines of evidence support the idea that this RNA is a homolog of the 7SL RNA component of mammalian signal recognition particle (SRP), which targets presecretory proteins to the endoplasmic reticulum membrane. First, it shares limited but significant primary sequence homology with previously identified 7SL RNAs and can be folded into a similar secondary structure. Second, it possesses the 5' triphosphate characteristic of unprocessed RNA polymerase III transcripts, and moreover, it is the only fission yeast RNA in this size range with such a terminus. Third, its behavior in cell fractionation experiments suggests that it is part of a small ribonucleoprotein which forms salt-labile contacts with larger structures. Fourth, the particle containing S. pombe 7SL RNA resembles mammalian SRP in both size (11S) and affinity for DEAE-Sepharose. Disruption of the single-copy gene, designated slr1+, reveals that the RNA is indispensable for growth in fission yeast. This result is not surprising, since secretion is an essential cellular process. Images PMID:2837648

  20. Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle

    SciTech Connect

    Brennwald, P.; Liao, X.; Holm, K.; Porter, G.; Wise, J.A.

    1988-04-01

    The authors have cloned the gene encoding a novel small cytoplasmic RNA from the fission yeast Schizosaccharomyces pombe. Four lines of evidence support the idea that this RNA is a homolog of the 7SL RNA component of mammalian signal recognition particle (SRP), which targets presecretory proteins to the endoplasmic reticulum membrane. First, it shares limited but significant primary sequence homology with previously identified 7SL RNAs and can be folded into a similar secondary structure. Second, it possesses the 5' triphosphate characteristic of unprocessed RNA polymerase III transcripts, and moreover, it is the only fission yeast RNA in this size range with such a terminus. Third, its behavior in cell fractionation experiments suggests that it is part of a small ribonucleoprotein which forms salt-labile contacts with larger structures. Fourth, the particle containing S.pombe 7SLRNA resembles mammalian SRP in both size (11S) and affinity for DEAE-Sepharose. Disruption of the single-copy gene, designated slrl/sup +/, reveals that the RNA is indispensable for growth in fission yeast. This result is not surprising, since secretion is an essential cellular process.

  1. Conformational transitions of duplex and triplex nucleic acid helices: thermodynamic analysis of effects of salt concentration on stability using preferential interaction coefficients.

    PubMed Central

    Bond, J. P.; Anderson, C. F.; Record, M. T.

    1994-01-01

    For order-disorder transitions of double- and triple-stranded nucleic acid helices, the midpoint temperatures Tm depend strongly on a +/-, the mean ionic activity of uniunivalent salt. Experimental determinations of dTm/d ln a +/- and of the enthalpy change (delta H(o)) accompanying the transition in excess salt permit evaluation of delta gamma, the stoichiometrically weighted combination of preferential interaction coefficients, each of which reflects thermodynamic effects of interactions of salt ions with a reactant or product of the conformational transition (formula; see text) Here delta H(o) is defined per mole of nucleotide by analogy to delta gamma. Application of Eq. 1 to experimental values of delta H(o) and Tm yields values of delta gamma for the denaturation of B-DNA over the range of NaCl concentrations 0.01-0.20 M (Privalov et al. (1969), Biopolymers 8,559) and for each of four order-disorder transitions of poly rA.(poly rU)n, n = 1, 2 over the range of NaCl concentrations 0.01-1.0 M (Krakauer and Sturtevant (1968), Biopolymers 6, 491). For denaturation of duplexes and triplexes, delta gamma is negative and not significantly dependent on a +/-, but delta gamma is positive and dependent on a +/- for the disproportionation transition of poly rA.poly rU duplexes. Quantitative interpretations of these trends and magnitudes of delta gamma in terms of coulombic and excluded volume effects are obtained by fitting separately each of the two sets of thermodynamic data using Eq. 1 with delta gamma PB evaluated from the cylindrically symmetric Poisson-Boltzmann (PB) equation for a standard model of salt-polyelectrolyte solutions. The only structural parameters required by this model are: b, the mean axial distance between the projections of adjacent polyion charges onto the cylindrical axis; and a, the mean distance of closest approach between a salt ion center and the cylindrical axis. Fixing bMS and aMS for the multi-stranded (ordered) conformations, we

  2. Experimental Drug for Rheumatoid Arthritis Shows Promise

    MedlinePlus

    ... news/fullstory_158076.html Experimental Drug for Rheumatoid Arthritis Shows Promise Baricitinib helped patients who failed other ... HealthDay News) -- An experimental drug to treat rheumatoid arthritis showed promise in a new six-month trial. ...

  3. Experimental Genital Herpes Drug Shows Promise

    MedlinePlus

    ... gov/medlineplus/news/fullstory_159462.html Experimental Genital Herpes Drug Shows Promise Drug lowered viral activity, recurrence ... News) -- An experimental immune-boosting treatment for genital herpes shows promise, researchers report. The drug, called GEN- ...

  4. Alzheimer's Gene May Show Effects in Childhood

    MedlinePlus

    ... https://medlineplus.gov/news/fullstory_159854.html Alzheimer's Gene May Show Effects in Childhood Brain scans reveal ... 2016 WEDNESDAY, July 13, 2016 (HealthDay News) -- A gene related to Alzheimer's disease may start to show ...

  5. Triplex in-situ hybridization

    DOEpatents

    Fresco, Jacques R.; Johnson, Marion D.

    2002-01-01

    Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

  6. No-Show Analysis. Final Report.

    ERIC Educational Resources Information Center

    Kalsbeek, William D.; And Others

    The National Assessment of Educational Progress; Second Science Assessment No-Show Study assessed the magnitude and causation of nonresponse biases. A No-Show is defined as an individual who was selected as a sample respondent but failed to be present for regular assessment of the 17-year-old group. The procedure whereby a sample of eligible…

  7. Effects of Talk Show Viewing on Adolescents.

    ERIC Educational Resources Information Center

    Davis, Stacy; Mares, Marie-Louise

    1998-01-01

    Investigates the effects of talk-show viewing on high-school students' social-reality beliefs. Supports the hypothesis that viewers overestimate the frequency of deviant behaviors; does not find support for the hypothesis that viewers become desensitized to the suffering of others; and finds that talk-show viewing was positively related, among…

  8. Acculturation, Cultivation, and Daytime TV Talk Shows.

    ERIC Educational Resources Information Center

    Woo, Hyung-Jin; Dominick, Joseph R.

    2003-01-01

    Explores the cultivation phenomenon among international college students in the United States by examining the connection between levels of acculturation, daytime TV talk show viewing, and beliefs about social reality. Finds that students who scored low on acculturation and watched a great deal of daytime talk shows had a more negative perception…

  9. The Physics of Equestrian Show Jumping

    ERIC Educational Resources Information Center

    Stinner, Art

    2014-01-01

    This article discusses the kinematics and dynamics of equestrian show jumping. For some time I have attended a series of show jumping events at Spruce Meadows, an international equestrian center near Calgary, Alberta, often referred to as the "Wimbledon of equestrian jumping." I have always had a desire to write an article such as this…

  10. The Language of Show Biz: A Dictionary.

    ERIC Educational Resources Information Center

    Sergel, Sherman Louis, Ed.

    This dictionary of the language of show biz provides the layman with definitions and essays on terms and expressions often used in show business. The overall pattern of selection was intended to be more rather than less inclusive, though radio, television, and film terms were deliberately omitted. Lengthy explanations are sometimes used to express…

  11. Comparison of Weather Shows in Eastern Europe

    NASA Astrophysics Data System (ADS)

    Najman, M.

    2009-09-01

    Comparison of Weather Shows in Eastern Europe Television weather shows in Eastern Europe have in most cases in the high graphical standard. There is though a wast difference in duration and information content in the weather shows. There are few signs and regularities by which we can see the character of the weather show. The main differences are mainly caused by the income structure of the TV station. Either it is a fully privately funded TV relying on the TV commercials income. Or it is a public service TV station funded mainly by the national budget or fixed fee structure/tax. There are wast differences in duration and even a graphical presentation of the weather. Next important aspect is a supplier of the weather information and /or the processor. Shortly we can say, that when the TV show is produced by the national met office, the TV show consists of more scientific terms, synoptic maps, satellite imagery, etc. If the supplier is the private meteorological company, the weather show is more user-friendly, laical with less scientific terms. We are experiencing a massive shift in public weather knowledge and demand for information. In the past, weather shows consisted only of maps with weather icons. In todaýs world, even the laic weather shows consist partly of numerical weather model outputs - they are of course designed to be understandable and graphically attractive. Outputs of the numerical weather models used to be only a part of daily life of a professional meteorologist, today they are common part of life of regular people. Video samples are a part of this presentation.

  12. Spacecraft Image Mashup Shows Galactic Collision

    NASA Video Gallery

    This new composite image from the Chandra X-ray Observatory, the Hubble Space Telescope, and the Spitzer Space Telescope shows two colliding galaxies more than a 100 million years after they first ...

  13. Portable Zika Test Shows Promise in Monkeys

    MedlinePlus

    ... nih.gov/medlineplus/news/fullstory_158704.html Portable Zika Test Shows Promise in Monkeys Easy-to-use ... News) -- A fast, inexpensive test that detects the Zika virus in monkeys might be useful for doctors ...

  14. TRMM Satellite Shows Heavy Rainfall in Cristina

    NASA Video Gallery

    NASA's TRMM satellite rainfall data was overlaid on an enhanced visible/infrared image from NOAA's GOES-East satellite showing cloud and rainfall extent. Green areas indicate rainfall at over 20 mm...

  15. GOES Satellite Data Shows Tornado Development

    NASA Video Gallery

    This animation of NOAA's GOES-East satellite data shows the development and movement of the weather system that spawned tornadoes affecting the southern and eastern U.S. states on April 27-29, 2014...

  16. Lightweight magnesium-lithium alloys show promise

    NASA Technical Reports Server (NTRS)

    Adams, W. T.; Cataldo, C. E.

    1964-01-01

    Evaluation tests show that magnesium-lithium alloys are lighter and more ductile than other magnesium alloys. They are being used for packaging, housings, containers, where light weight is more important than strength.

  17. Portable Zika Test Shows Promise in Monkeys

    MedlinePlus

    ... https://medlineplus.gov/news/fullstory_158704.html Portable Zika Test Shows Promise in Monkeys Easy-to-use ... News) -- A fast, inexpensive test that detects the Zika virus in monkeys might be useful for doctors ...

  18. Malaria Vaccine Shows Promise in Small Study

    MedlinePlus

    ... nih.gov/medlineplus/news/fullstory_158765.html Malaria Vaccine Shows Promise in Small Study It protected more ... May 10, 2016 (HealthDay News) -- An experimental malaria vaccine protects a majority of adults against the mosquito- ...

  19. Malaria Vaccine Shows Promise in Small Study

    MedlinePlus

    ... https://medlineplus.gov/news/fullstory_158765.html Malaria Vaccine Shows Promise in Small Study It protected more ... May 10, 2016 (HealthDay News) -- An experimental malaria vaccine protects a majority of adults against the mosquito- ...

  20. 47 CFR 90.505 - Showing required.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... MOBILE RADIO SERVICES Developmental Operation § 90.505 Showing required. (a) Except as provided in... radio art, or is investigating new unexplored concepts in radio transmission and communications; (4)...

  1. Educational Outreach: The Space Science Road Show

    NASA Astrophysics Data System (ADS)

    Cox, N. L. J.

    2002-01-01

    The poster presented will give an overview of a study towards a "Space Road Show". The topic of this show is space science. The target group is adolescents, aged 12 to 15, at Dutch high schools. The show and its accompanying experiments would be supported with suitable educational material. Science teachers at schools can decide for themselves if they want to use this material in advance, afterwards or not at all. The aims of this outreach effort are: to motivate students for space science and engineering, to help them understand the importance of (space) research, to give them a positive feeling about the possibilities offered by space and in the process give them useful knowledge on space basics. The show revolves around three main themes: applications, science and society. First the students will get some historical background on the importance of space/astronomy to civilization. Secondly they will learn more about novel uses of space. On the one hand they will learn of "Views on Earth" involving technologies like Remote Sensing (or Spying), Communication, Broadcasting, GPS and Telemedicine. On the other hand they will experience "Views on Space" illustrated by past, present and future space research missions, like the space exploration missions (Cassini/Huygens, Mars Express and Rosetta) and the astronomy missions (Soho and XMM). Meanwhile, the students will learn more about the technology of launchers and satellites needed to accomplish these space missions. Throughout the show and especially towards the end attention will be paid to the third theme "Why go to space"? Other reasons for people to get into space will be explored. An important question in this is the commercial (manned) exploration of space. Thus, the questions of benefit of space to society are integrated in the entire show. It raises some fundamental questions about the effects of space travel on our environment, poverty and other moral issues. The show attempts to connect scientific with

  2. Liquid Crystal Research Shows Deformation By Drying

    NASA Technical Reports Server (NTRS)

    2003-01-01

    These images, from David Weitz's liquid crystal research, show ordered uniform sized droplets (upper left) before they are dried from their solution. After the droplets are dried (upper right), they are viewed with crossed polarizers that show the deformation caused by drying, a process that orients the bipolar structure of the liquid crystal within the droplets. When an electric field is applied to the dried droplets (lower left), and then increased (lower right), the liquid crystal within the droplets switches its alignment, thereby reducing the amount of light that can be scattered by the droplets when a beam is shone through them.

  3. Children's Art Show: An Educational Family Experience

    ERIC Educational Resources Information Center

    Bakerlis, Julienne

    2007-01-01

    In a time of seemingly rampant budget cuts in the arts in school systems throughout the country, a children's art show reaps many rewards. It can strengthen family-school relationships and community ties and stimulate questions and comments about the benefits of art and its significance in the development of young children. In this photo essay of…

  4. Show Them You Really Want the Job

    ERIC Educational Resources Information Center

    Perlmutter, David D.

    2012-01-01

    Showing that one really "wants" the job entails more than just really wanting the job. An interview is part Broadway casting call, part intellectual dating game, part personality test, and part, well, job interview. When there are 300 applicants for a position, many of them will "fit" the required (and even the preferred) skills listed in the job…

  5. Laser entertainment and light shows in education

    NASA Astrophysics Data System (ADS)

    Sabaratnam, Andrew T.; Symons, Charles

    2002-05-01

    Laser shows and beam effects have been a source of entertainment since its first public performance May 9, 1969, at Mills College in Oakland, California. Since 1997, the Photonics Center, NgeeAnn Polytechnic, Singapore, has been using laser shows as a teaching tool. Students are able to exhibit their creative skills and learn at the same time how lasers are used in the entertainment industry. Students will acquire a number of skills including handling three- phase power supply, operation of cooling system, and laser alignment. Students also acquire an appreciation of the arts, learning about shapes and contours as they develop graphics for the shows. After holography, laser show animation provides a combination of the arts and technology. This paper aims to briefly describe how a krypton-argon laser, galvanometer scanners, a polychromatic acousto-optic modulator and related electronics are put together to develop a laser projector. The paper also describes how students are trained to make their own laser animation and beam effects with music, and at the same time have an appreciation of the operation of a Class IV laser and the handling of optical components.

  6. Showing Enantiomorphous Crystals of Tartaric Acid

    ERIC Educational Resources Information Center

    Andrade-Gamboa, Julio

    2007-01-01

    Most of the articles and textbooks that show drawings of enantiomorphous crystals use an inadequate view to appreciate the fact that they are non-superimposable mirror images of one another. If a graphical presentation of crystal chirality is not evident, the main attribute of crystal enantiomorphism can not be recognized by students. The classic…

  7. Tilapia show immunization response against Ich

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study compares the immune response of Nile tilapia and red tilapia against parasite Ichthyophthirius multifiliis (Ich) using a cohabitation challenge model. Both Nile and red tilapia showed strong immune response post immunization with live Ich theronts by IP injection or immersion. Blood serum...

  8. A Talk Show from the Past.

    ERIC Educational Resources Information Center

    Gallagher, Arlene F.

    1991-01-01

    Describes a two-day activity in which elementary students examine voting rights, the right to assemble, and women's suffrage. Explains the game, "Assemble, Reassemble," and a student-produced talk show with five students playing the roles of leaders of the women's suffrage movement. Profiles Elizabeth Cady Stanton, Lucretia Mott, Susan B. Anthony,…

  9. Idaho State University Physics Road Show

    NASA Astrophysics Data System (ADS)

    Shropshire, Steve

    2009-05-01

    The ISU Physics Road Show services over 40 schools and 12,000 students each year. Exciting and informative demonstration shows are conducted during assemblies at elementary, middle, and junior high schools. Discussion will focus on efforts taken to maximize the educational impact to students and teachers. These efforts include supplemental information and materials provided to teachers, teacher workshops, and careful catering of subject material to state and national education standards. A few sample demonstrations will be performed, including the boiling green water sucker, a magnet strongly repelled from a cooled copper disc, an artificial geyser that shoots water 6 meters, and a few liquid nitrogen tricks. This program is supported in part by a grant from the Idaho Community Foundation.

  10. The Physics of Equestrian Show Jumping

    NASA Astrophysics Data System (ADS)

    Stinner, Art

    2014-04-01

    This article discusses the kinematics and dynamics of equestrian show jumping. For some time I have attended a series of show jumping events at Spruce Meadows, an international equestrian center near Calgary, Alberta, often referred to as the "Wimbledon of equestrian jumping." I have always had a desire to write an article such as this one, but when I searched the Internet for information and looked at YouTube presentations, I could only find simplistic references to Newton's laws and the conservation of mechanical energy principle. Nowhere could I find detailed calculations. On the other hand, there were several biomechanical articles with empirical reports of the results of kinetic and dynamic investigations of show jumping using high-speed digital cameras and force plates. They summarize their results in tables that give information about the motion of a horse jumping over high fences (1.40 m) and the magnitudes of the forces encountered when landing. However, they do not describe the physics of these results.

  11. Asteroid Ida - 6 Views Showing Rotation

    NASA Technical Reports Server (NTRS)

    1994-01-01

    This composite image shows the asteroid 243 Ida as seen from the Galileo spacecraft during its approach on August 28, 1993. The six views were shuttered through the camera's green filter and show Ida's rotation over a period of about 3 hours 18 minutes. The asteroid makes a complete rotation every 4 hours 38 minutes; therefore, this set of images spans about 3/4 of Ida's rotation period and shows most of Ida's surface. By combining the information in these views with that from the highest resolution images returned from the spacecraft in September 1993, the size and shape of this irregular body can now be determined accurately The asteroid appears to be about 58 kilometers (36 miles) long and about 23 kilometers wide, with a very irregular shape and volume of some 16,000 cubic kilometers. The images are arranged in chronological order from a time 3 hours 51 minutes before closest approach (upper left), through upper right, middle left, middle right lower left and lower right (33 minutes before closest approach). The six images show Ida at the same scale throughout. Ida's rotation axis is roughly vertical in these images, and the rotation causes the right-hand end of Ida to move toward the viewer as time progresses. The first image was taken from a range of about 171,000 km (106,000 miles) and provides an image resolution of about 1,700 meters per pixel (the highest resolution achieved for Ida is about 25 meters per pixel). The second, taken 70 minutes later, is from 119,000 kilometers, followed by 102,000 kilometers, 85,000 kilometers, 50,000 kilometers, and 25,000 kilometers. The features on Ida are less sharp in the earlier views because of the greater distances. Prominent in the middle three views is a deep depression across the short axis of the Asteroid. This feature tends to support the idea that Ida may have originally been formed from two or more separate large objects that collided softly and stuck together. Also visible in the lower left view is an

  12. Anti-Stokes luminescence o Ln2O2S: Er(3+), Yb(3+) in triplexes excited in the 0.9. 1.53 and 1.59-micron ranges, part 2

    NASA Astrophysics Data System (ADS)

    Kurochkin, A. V.; Mailibaeva, L. M.; Manashirov, O. Ya.; Sattarov, D. K.; Smirnov, V. B.

    1992-10-01

    Studies are reported of IR anti-Stokes luminescence of three Ln2O2S:Er(sup 3 +), Yb(sup 3 +) luminophores incorporated in triplexes under excitation by different IR sources in the 0.93, 1.53, and 1.59 mu m ranges. Spectra of three different anti-Stokes luminophores (Ln = Y, Fd and La) are examined and the fine structure of each individual anti-Stokes band is analyzed. The appearance of these bands is explained on the basis of energy structures of doping Er(sup 3 +) and Yb(sup 3 +) ions in the studied oxysulfides. The anti-Stokes luminescence spectra excited by IR sources with lambda(sub max) = 0.93 and 1.53 mu m are compared. For the same oxysulfides doped by Er(sup 3 +) only or by both Er(sup 3 +) and Yb(sup 3 +), differences in the fine structures of the 0.99-micron band are explained, this band being the most promising for conversion of IR radiation in the 1.4-1.7 -micron range. The results obtained indicate the possibility to develop, using the anti-Stokes luminophores studied, a uniform polychromatic IR image converter and to suggest ways for its further improvement.

  13. Color Voyager 2 Image Showing Crescent Uranus

    NASA Technical Reports Server (NTRS)

    1990-01-01

    This image shows a crescent Uranus, a view that Earthlings never witnessed until Voyager 2 flew near and then beyond Uranus on January 24, 1986. This planet's natural blue-green color is due to the absorption of redder wavelengths in the atmosphere by traces of methane gas. Uranus' diameter is 32,500 miles, a little over four times that of Earth. The hazy blue-green atmosphere probably extends to a depth of around 5,400 miles, where it rests above what is believed to be an icy or liquid mixture (an 'ocean') of water, ammonia, methane, and other volatiles, which in turn surrounds a rocky core perhaps a little smaller than Earth.

  14. Star Shows It Has The Right Stuff

    NASA Astrophysics Data System (ADS)

    2004-01-01

    Astronomers have used an observation by NASA's Chandra X-ray Observatory to make the best case yet that a star can be engulfed by its companion star and survive. This discovery will help astronomers better understand how closely coupled stars, and perhaps even stars and planets, evolve when one of the stars expands enormously in its red giant phase. The binary star system known as V471 Tauri comprises a white dwarf star (the primary) in a close orbit -- one thirtieth of the distance between Mercury and the Sun -- with a normal Sun-like star (the secondary). Chandra's data showed that the hot upper atmosphere of the secondary star has a deficit of carbon atoms relative to nitrogen atoms. "This deficit of carbon atoms is the first clear observational evidence that the normal star was engulfed by its companion in the past," according to Jeremy Drake of the Smithsonian Astrophysical Observatory in Cambridge, MA, who coauthored an article on V471 in The Astrophysical Journal Letters with Marek Sarna of the N. Copernicus Astronomical Center in Poland. The white dwarf star was once a star several times as massive as the Sun. Nuclear fusion reactions in the core of such a star convert carbon into nitrogen over a period of about a billion years. When the fuel in the core of the star is exhausted, the core collapses, triggering more energetic nuclear reactions that cause the star to expand and transform into a red giant before eventually collapsing to become a white dwarf. The carbon-poor material in the core of the red giant is mixed with outer part of the star, so its atmosphere shows a deficit of carbon, as compared with Sun-like stars. The X-ray spectra of a red giant star (top panel) and a Sun-like star (bottom panel) show the large difference in the peaks due to carbon atoms in the two stars. Theoretical calculations indicate that a red giant in a binary system can completely envelop its companion star and dramatically affect its evolution. During this common envelope

  15. Surveys show support for green 'activities'.

    PubMed

    Baillie, Jonathan

    2012-03-01

    Two independently conducted surveys on sustainability - one into the 'views and values' of NHS 'leaders', and the other questioning the public about the importance of the 'green agenda' in the NHS, and their opinions on how the service might most effectively reduce its carbon footprint, form the basis of Sustainability in the NHS: Health Check 2012, a new NHS Sustainable Development Unit (NHS SDU) publication. As HEJ editor Jonathan Baillie reports, the new document also presents updated data on the 'size' of the carbon footprint of the NHS in England, showing that, although good work by a number of Trusts in the past two years has seen healthcare-generated carbon emissions start to 'level off', the biggest contributors have been the current health service spending review, and the increased national availability of renewable energy. PMID:22515017

  16. Survey shows successes, failures of horizontal wells

    SciTech Connect

    Deskins, W.G.; McDonald, W.J.; Reid, T.B.

    1995-06-19

    Industry`s experience now shows that horizontal well technology must be applied thoughtfully and be site-specific to attain technical and economic success. This article, based on a comprehensive study done by Maurer Engineering for the US Department of Energy (DOE), addresses the success of horizontal wells in less-publicized formations, that is, other than the Austin chalk. Early excitement within the industry about the new technology reached a fever pitch at times, leaving some with the impression that horizontal drilling is a panacea for all drilling environments. This work gauges the overall success of horizontal technology in US and Canadian oil and gas fields, defines the applications where horizontal technology is most appropriate, and assesses its impact on oil recovery and reserves.

  17. 832 Karin Shows No Rotational Spectral Variations

    NASA Astrophysics Data System (ADS)

    Chapman, Clark R.; Enke, B.; Merline, W. J.; Nesvorny, D.; Tamblyn, P.; Young, E. F.

    2006-09-01

    Sasaki et al. (2004, 2005) claimed that 832 Karin, the brightest member of the very young (5.75 Myr) Karin cluster of the Koronis family, shows dramatically different colors as a function of rotational phase. It was interpreted that Karin is a fragment of the recently broken-up asteroid, showing the reddish space-weathered exterior surface of the precursor asteroid as well as an interior face, which has not had time to become space-weathered. On five nights during UT 7-14 January 2006, we observed Karin with the SpeX instrument, 0.8-2.5 microns, on the IRTF. We sampled its spectrum well throughout its rotation. We analyzed the data in 50 deg. intervals of rotational longitude; some longitudes were sampled during two different nights. We find that Karin exhibits minimal spectral variations with rotation, certainly nothing of the magnitude reported by Sasaki et al. Since our data resemble Sasaki et al.'s "blue" and "green" sets, we suggest that their "red" set is spurious. Indeed, it is difficult to understand how the reported color change could have occurred during such a modest interval ( 4%) of rotational longitude. (Note that we have not determined Karin's pole position nor the phase of the Sasaki et al. data within our own coverage, so the refutation of dramatic color change is not absolutely secure.) Karin and its family members are not quite as red as typical S-types, yet have shallow absorption bands. Perhaps the space-weathering process affecting these young asteroids has had time to reduce spectral contrast, but has not operated long enough to redden them -- an intermediate case of space weathering, which has gone to completion for older main-belt asteroids of these sizes. Supported by the NASA Planetary Astronomy Program. T. Sasaki et al. 2004. ApJ 615, L161-L164; T. Sasaki et al. 2005. LPSC XXXVI, 1590.pdf.

  18. NASA GIBS Use in Live Planetarium Shows

    NASA Astrophysics Data System (ADS)

    Emmart, C. B.

    2015-12-01

    The American Museum of Natural History's Hayden Planetarium was rebuilt in year 2000 as an immersive theater for scientific data visualization to show the universe in context to our planet. Specific astrophysical movie productions provide the main daily programming, but interactive control software, developed at AMNH allows immersive presentation within a data aggregation of astronomical catalogs called the Digital Universe 3D Atlas. Since 2006, WMS globe browsing capabilities have been built into a software development collaboration with Sweden's Linkoping University (LiU). The resulting Uniview software, now a product of the company SCISS, is operated by about fifty planetariums around that world with ability to network amongst the sites for global presentations. Public presentation of NASA GIBS has allowed authoritative narratives to be presented within the range of data available in context to other sources such as Science on a Sphere, NASA Earth Observatory and Google Earth KML resources. Specifically, the NOAA supported World Views Network conducted a series of presentations across the US that focused on local ecological issues that could then be expanded in the course of presentation to national and global scales of examination. NASA support of for GIBS resources in an easy access multi scale streaming format like WMS has tremendously enabled particularly facile presentations of global monitoring like never before. Global networking of theaters for distributed presentations broadens out the potential for impact of this medium. Archiving and refinement of these presentations has already begun to inform new types of documentary productions that examine pertinent, global interdependency topics.

  19. The earliest published electrocardiogram showing ventricular preexcitation.

    PubMed

    Von Knorre, Georg H

    2005-03-01

    When in 1930, Wolff, Parkinson, and White published what is today known as the WPW, or preexcitation syndrome, they, and subsequently others, found few comparable cases in the preceding literature. Among these the report of Cohn and Fraser, published in 1913, was the earliest. However, another even earlier documentation in a 1909 article by Hoffmann escaped notice till now. The ECG of a patient with paroxysmal tachycardia reveals a short PR interval and a delta-wave-induced widening of the QRS complex, even though the reproduced tachycardia was not preexcitation related. The interpretation of this poorly reproduced ECG can be confirmed by another and more detailed description of the patient in an electrocardiography textbook published in 1914 by the same author. Thus, the earliest publication of an ECG showing ventricular preexcitation now can be dated back to 1909. Moreover, the Hoffmann monograph contains two additional examples of the WPW syndrome not noticed until now. All three cases published by Hoffmann had their first ECG recordings in 1912 or earlier. PMID:15733183

  20. Temperature Data Shows Warming in 2001

    NASA Technical Reports Server (NTRS)

    2002-01-01

    TThe figure above depicts how much air temperatures near the Earth's surface changed relative to the global mean temperature from 1951 to 1980. NASA researchers used maps of urban areas derived from city lights data to account for the 'heat island' effect of cities. The red and orange colors show that temperatures are warmer in most regions of the world when compared to the 1951 to 1980 'normal' temperatures. Warming around the world has been widespread, but it is not present everywhere. The largest warming is in Northern Canada, Alaska and Siberia, as indicated by the deeper red colors. The lower 48 United States have become warmer recently, but only enough to make the temperatures comparable to what they were in the 1930s. The scale on the bottom of these temperature anomaly images represent degrees in Celsius. The negative numbers represent cooling and the positive numbers depict warming. Overall, the air temperature near the Earth's surface has warmed by 1oF (0.6oC) globally, on average, over the last century. For more information and additional images, read Satellites Shed Light on a Warmer World. Image courtesy Goddard Institute for Space Studies (GISS).

  1. Women showing off: notes on female exhibitionism.

    PubMed

    Balsam, Rosemary H

    2008-03-01

    The limitations of the phallocentric cast of earlier psychoanalytic formulations of "female exhibitionism" linger into the present. In part this connects to certain historical expectations for women's social behavior, and to the vicissitudes of Freud's insufficient knowledge of women in his libidinal psychosexual phasing used as a basis for analytic understanding. The contemporary fade of libido theory contributes to the neglect of such topics as they relate to the biological body. Yet ease and conflict regarding conscious and unconscious female body image representations related to that stepchild of theory-pregnancy and childbirth in particular-play a major role in female body display. Recognition of such body fantasies and female body meanings from early childhood into maturity tends to be marginalized within all of the psychoanalytic theories current today. The focus here on female exhibitionism suggests a normative spectrum for pleasurably active sex seeking and pleasurable procreative desire and fantasy that is present in a female's use of her body and which (of course, but secondarily) can become caught up in conflict. Two cases accenting analyses of female "showing off" behavior are included. PMID:18430704

  2. Fading Supernova Creates Spectacular Light Show

    NASA Technical Reports Server (NTRS)

    2003-01-01

    This image of SN 1987A, taken November 28, 2003 by the Advanced Camera for Surveys aboard NASA's Hubble Space Telescope (HST), shows many bright spots along a ring of gas, like pearls on a necklace. These cosmic pearls are being produced as superior shock waves unleashed during an explosion slam into the ring at more than a million miles per hour. The collision is heating the gas ring, causing its irnermost regions to glow. Astronomers detected the first of these hot spots in 1996, but now they see dozens of them all around the ring. With temperatures surging from a few thousand degrees to a million degrees, the flares are increasing in number. In the next few years, the entire ring will be ablaze as it absorbs the full force of the crash and is expected to become bright enough to illuminate the star's surroundings. Astronomers will then be able to obtain information on how the star ejected material before the explosion. The elongated and expanding object in the center of the ring is debris form the supernova blast which is being heated by radioactive elements, principally titanium 44, that were created in the explosion. This explosion was first observed by astronomers seventeen years ago in 1987, although the explosion took place about 160,000 years ago.

  3. Bacteriophages show promise as antimicrobial agents.

    PubMed

    Alisky, J; Iczkowski, K; Rapoport, A; Troitsky, N

    1998-01-01

    The emergence of antibiotic-resistant bacteria has prompted interest in alternatives to conventional drugs. One possible option is to use bacteriophages (phage) as antimicrobial agents. We have conducted a literature review of all Medline citations from 1966-1996 that dealt with the therapeutic use of phage. There were 27 papers from Poland, the Soviet Union, Britain and the U.S.A. The Polish and Soviets administered phage orally, topically or systemically to treat a wide variety of antibiotic-resistant pathogens in both adults and children. Infections included suppurative wound infections, gastroenteritis, sepsis, osteomyelitis, dermatitis, empyemas and pneumonia; pathogens included Staphylococcus, Streptococcus, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella and Salmonella spp. Overall, the Polish and Soviets reported success rates of 80-95% for phage therapy, with rare, reversible gastrointestinal or allergic side effects. However, efficacy of phage was determined almost exclusively by qualitative clinical assessment of patients, and details of dosages and clinical criteria were very sketchy. There were also six British reports describing controlled trials of phage in animal models (mice, guinea pigs and livestock), measuring survival rates and other objective criteria. All of the British studies raised phage against specific pathogens then used to create experimental infections. Demonstrable efficacy against Escherichia, Acinetobacter, Pseudomonas and Staphylococcus spp. was noted in these model systems. Two U.S. papers dealt with improving the bioavailability of phage. Phage is sequestered in the spleen and removed from circulation. This can be overcome by serial passage of phage through mice to isolate mutants that resist sequestration. In conclusion, bacteriophages may show promise for treating antibiotic resistant pathogens. To facilitate further progress, directions for future research are discussed and a directory of authors from the reviewed

  4. Mercury's Core Molten, Radar Study Shows

    NASA Astrophysics Data System (ADS)

    2007-05-01

    100 times, and showed that Mercury's spin axis is almost, but not exactly, perpendicular to the plane of its rotation around the Sun," Margot said. Margot worked with Stanton Peale of the University of California, Santa Barbara, Raymond Jurgens and Martin Slade of NASA's Jet Propulsion Laboratory, and Igor Holin of the Space Research Institute in Moscow. The National Radio Astronomy Observatory is a facility of the National Science Foundation, operated under cooperative agreement by Associated Universities, Inc. The Arecibo Observatory is part of the National Astronomy and Ionosphere Center, which is operated by Cornell University under a cooperative agreement with the NSF. Part of this work was supported by the Jet Propulsion Laboratory, operated by Caltech under contract with NASA.

  5. Show Horse Welfare: The Viewpoints of Judges, Stewards, and Show Managers.

    PubMed

    Voigt, Melissa; Hiney, Kristina; Croney, Candace; Waite, Karen; Borron, Abigail; Brady, Colleen

    2016-01-01

    The purpose of this study was to gain a better understanding of the current state of stock-type show horse welfare based on the perceptions of show officials and to identify potential means of preventing and intervening in compromises to show horse welfare. Thirteen horse show officials, including judges, stewards, and show managers, were interviewed. Findings revealed the officials had an incomplete understanding of nonhuman animal welfare and a high level of concern regarding the public's perception of show horse welfare. The officials attributed most of the frequently observed compromises to show horse welfare to (a) novices', amateurs', and young trainers' lack of experience or expertise, and (b) trainers' and owners' unrealistic expectations and prioritization of winning over horse welfare. The officials emphasized a need for distribution of responsibility among associations, officials, and individuals within the industry. Although the officials noted recent observable positive changes in the industry, they emphasized the need for continued improvements in equine welfare and greater educational opportunities for stakeholders. PMID:26742585

  6. Show Horse Welfare: Horse Show Competitors' Understanding, Awareness, and Perceptions of Equine Welfare.

    PubMed

    Voigt, Melissa A; Hiney, Kristina; Richardson, Jennifer C; Waite, Karen; Borron, Abigail; Brady, Colleen M

    2016-01-01

    The purpose of this study was to gain a better understanding of stock-type horse show competitors' understanding of welfare and level of concern for stock-type show horses' welfare. Data were collected through an online questionnaire that included questions relating to (a) interest and general understanding of horse welfare, (b) welfare concerns of the horse show industry and specifically the stock-type horse show industry, (c) decision-making influences, and (d) level of empathic characteristics. The majority of respondents indicated they agree or strongly agree that physical metrics should be a factor when assessing horse welfare, while fewer agreed that behavioral and mental metrics should be a factor. Respondent empathy levels were moderate to high and were positively correlated with the belief that mental and behavioral metrics should be a factor in assessing horse welfare. Respondents indicated the inhumane practices that most often occur at stock-type shows include excessive jerking on reins, excessive spurring, and induced excessive unnatural movement. Additionally, respondents indicated association rules, hired trainers, and hired riding instructors are the most influential regarding the decisions they make related to their horses' care and treatment. PMID:27029609

  7. Flashlight und Lichtorgel: Englischunterricht als Show (Flashlight and Lighting Console: English Teaching as a Show).

    ERIC Educational Resources Information Center

    Boettcher, Karl-Heinz

    1978-01-01

    Reports on using amateur theatricals in fifth-grade English classes as a motivating device. The project developed into a "show," which was performed publicly. Practical problems are discussed. The project is evaluated and suggestions are offered for other teachers. (IFS/WGA)

  8. Showing and Telling Farming: Agricultural Shows and Re-Imaging British Agriculture

    ERIC Educational Resources Information Center

    Holloway, Lewis

    2004-01-01

    Some actors in the ''mainstream'' agricultural sector are beginning to engage in strategies of influencing public perceptions of farming, responding to public anxieties over industrialised agriculture and to a supposed separation of non-farming publics from food production. This paper focuses on agricultural shows as sites and events central to…

  9. Best in show but not best shape: a photographic assessment of show dog body condition.

    PubMed

    Such, Z R; German, A J

    2015-08-01

    Previous studies suggest that owners often wrongly perceive overweight dogs to be in normal condition. The body shape of dogs attending shows might influence owners' perceptions, with online images of overweight show winners having a negative effect. This was an observational in silico study of canine body condition. 14 obese-prone breeds and 14 matched non-obese-probe breeds were first selected, and one operator then used an online search engine to identify 40 images, per breed, of dogs that had appeared at a major national UK show (Crufts). After images were anonymised and coded, a second observer subjectively assessed body condition, in a single sitting, using a previously validated method. Of 1120 photographs initially identified, 960 were suitable for assessing body condition, with all unsuitable images being from longhaired breeds. None of the dogs (0 per cent) were underweight, 708 (74 per cent) were in ideal condition and 252 (26 per cent) were overweight. Pugs, basset hounds and Labrador retrievers were most likely to be overweight, while standard poodles, Rhodesian ridgebacks, Hungarian vizslas and Dobermanns were least likely to be overweight. Given the proportion of show dogs from some breeds that are overweight, breed standards should be redefined to be consistent with a dog in optimal body condition. PMID:26169655

  10. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity

    PubMed Central

    Mohan, Vijee; Gupta, Soni; Thomas, Sherinmol; Mickey, Hanjabam; Charakana, Chaitanya; Chauhan, Vineeta Singh; Sharma, Kapil; Kumar, Rakesh; Tyagi, Kamal; Sarma, Supriya; Gupta, Suresh Kumar; Kilambi, Himabindu Vasuki; Nongmaithem, Sapana; Kumari, Alka; Gupta, Prateek; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima’D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future. PMID:27077652

  11. Development of triplex real-time PCR and detection of Toxoplasma gondii DNA in infected mice tissues and spiked human samples.

    PubMed

    Rahumatullah, A; Khoo, B Y; Noordin, R

    2015-06-01

    Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples. PMID:26691266

  12. Development of a triplex real-time PCR assay for the simultaneous detection of Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum in milk.

    PubMed

    Morandi, Stefano; Cremonesi, Paola; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

    2015-08-01

    Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R(2) = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species. PMID:25870135

  13. Gem and Mineral Shows as Geologic Teaching Opportunities.

    ERIC Educational Resources Information Center

    Cordua, William Sinclair

    1988-01-01

    Gem and mineral shows are excellent nontraditional opportunities for community education and outreach by geology teachers. Discusses initial club contacts, displays, shows, and the advantages of show participation to academic geologists. (CW)

  14. 32 CFR 552.78 - “Show cause” hearing.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ....78 “Show cause” hearing. Before suspending the solicitation privilege, the company and the agent will have a chance to show cause why the action should not be taken. “Show cause” is an opportunity for...

  15. 29 CFR 34.41 - Notice to Show Cause.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Notice to Show Cause. 34.41 Section 34.41 Labor Office of... Show Cause. (a) The Director may issue a Notice to Show Cause to a recipient failing to comply with the... compliance review. (b) The Notice to Show Cause shall contain: (1) A description of the violation and...

  16. 42 CFR 456.655 - Validation of showings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Validation of showings. 456.655 Section 456.655... Showing of an Effective Institutional Utilization Control Program § 456.655 Validation of showings. (a) The Administrator will periodically validate showings submitted under § 456.654. Validation...

  17. 42 CFR 456.655 - Validation of showings.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Validation of showings. 456.655 Section 456.655... Showing of an Effective Institutional Utilization Control Program § 456.655 Validation of showings. (a) The Administrator will periodically validate showings submitted under § 456.654. Validation...

  18. "The George Lopez Show": The Same Old Hispano?

    ERIC Educational Resources Information Center

    Markert, John

    2007-01-01

    "The George Lopez Show" is the first successful television show with a Latino in a leading role that features Hispanic material since Freddie Prinze's thirty-year-old sitcom, "Chico and the Man." This study seeks to assess how Latinos are presented on "The George Lopez Show." A content analysis reveals that the show perpetuates some of the…

  19. UEDGE Simulation of Triple-X Divertors

    NASA Astrophysics Data System (ADS)

    Wiley, J.; Kotschenreuther, M.; Valanju, P.; Pekker, M.; Rognlien, T.

    2006-04-01

    Novel magnetic divertors with additional X-points downstream from the main plasma X-point have been proposed to overcome reactor heat flux limitations. These divertor designs may allow a fully detached state at the divertor plate - without the poor confinement and disruptive tendencies by avoiding x-point MARFEs found in conventional divertor magnetic geometries. These new configurations are examined using UEDGE for existing machines that are considering experimental implementation of these divertors: PEGASUS, MAST, and EAST(China's new long-pulse, superconducting tokamak) as well as proposed reactor designs.

  20. View looking southwest showing northeast corner, with northwest corner of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View looking southwest showing northeast corner, with northwest corner of building no. 1015 (ice house/storage shed) showing at the left. - McGraw Ranch, Eagle's Nest Cabin, McGraw Ranch Road, Estes Park, Larimer County, CO

  1. Stem Cell Therapy Shows Promise Against Heart Failure

    MedlinePlus

    ... nlm.nih.gov/medlineplus/news/fullstory_158122.html Stem Cell Therapy Shows Promise Against Heart Failure A second ... 4, 2016 MONDAY, April 4, 2016 (HealthDay News) -- Stem cell therapy shows promise for people battling heart failure, ...

  2. 2. VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. Shows 499-501, Munoz House (AZ-73-37) on far right. - Antonio Bustamente House, 485-489 South Meyer Avenue & 186 West Kennedy Street, Tucson, Pima County, AZ

  3. TRMM Satellite Shows Bertha's Heavy Rain Pushed From Wind Shear

    NASA Video Gallery

    TRMM Satellite Shows Bertha's Heavy Rain Pushed From Wind Shear This 3-D flyby of Tropical Storm Bertha on Aug. 1 was created from TRMM satellite data. It shows (from the south) intense thunderstor...

  4. 78. VIEW OF UNCOMPLETED RESERVOIR, SHOWING FOREBAY AND FLUME; ALSO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    78. VIEW OF UNCOMPLETED RESERVOIR, SHOWING FOREBAY AND FLUME; ALSO SHOWING POOL ARRANGEMENT FOR TEMPORARILY UTILIZING WATER WITHOUT FILLING THE RESERVOIR, Print No. 232, April 1904 - Electron Hydroelectric Project, Along Puyallup River, Electron, Pierce County, WA

  5. 22. DETAIL EXTERIOR VIEW LOOKING NORTHWEST, SHOWING FISH LADDER AT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. DETAIL EXTERIOR VIEW LOOKING NORTHWEST, SHOWING FISH LADDER AT NORTH END OF DAM/SPILLWAY; VIEW SHOWS SECTION OF FISH LADDER NEAR WHERE IT ENTERS THE COLUMBIA RIVER. - Bonneville Project, Bonneville Dam, Columbia River, Bonneville, Multnomah County, OR

  6. 1. AERIAL VIEW, LOOKING NORTH, SHOWING COVERED BARGE (VESSEL 37) ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. AERIAL VIEW, LOOKING NORTH, SHOWING COVERED BARGE (VESSEL 37) IN CENTER OF PICTURE WITH FOUR HATCHES SHOWING IN SUPERSTRUCTURE Charles Wisniewski, photographer, January 1985 - Shooters Island, Ships Graveyard, Vessel No. 37, Newark Bay, Staten Island (subdivision), Richmond County, NY

  7. Southeast upstairs room of northwest wing showing southeast masonry wall ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Southeast upstairs room of northwest wing showing southeast masonry wall shared with Federal addition. note angled cracks showing original 1 1/2 story gable. - Scheetz Farm, House, 7161 Camp Hill Road, Fort Washington, Montgomery County, PA

  8. 77. GENERAL VIEW SHOWING RIVERFRONT SIDE OF THE PROPERTY, FROM ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    77. GENERAL VIEW SHOWING RIVERFRONT SIDE OF THE PROPERTY, FROM WEST 77. GENERAL VIEW SHOWING RIVERFRONT SIDE OF THE PROPERTY, FROM WEST 77. GENERAL VIEW SHOWING RIVERFRONT SIDE OF THE PROPERTY, FROM WEST - Westover, State Route 633, Westover, Charles City, VA

  9. 3. Historic American Buildings Survey UNDATED EXTERIOR SHOWING NORTH AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Historic American Buildings Survey UNDATED EXTERIOR SHOWING NORTH AND EAST FACADES (View B) This view shows clapboards on gable end and therefore, predates View A which shows shingles in upper gable From the collection of Mrs. L. T. Hazall, descendent of Rev. Woodward - Reverend John Woodward House, 409 Forbes Avenue, New Haven, New Haven County, CT

  10. 20. MEMBER 'A' SHOWS TENON AS USED IN POST 'A' ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. MEMBER 'A' SHOWS TENON AS USED IN POST 'A' (TN-159A-19), MEMBER 'B' IS BEAM 'B' IN TN-159A-19 AND SHOWS METHOD OF JOINING THESE MEMBERS. MEMBER 'C' SHOWS MORTISE IN BEAM 'B'. - Caleb Crosby Threshing Barn, Noeton (moved to Norris Dam State Park, Lake City), Morristown, Hamblen County, TN

  11. 47 CFR 1.701 - Show cause orders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Show cause orders. 1.701 Section 1.701..., and Reports Involving Common Carriers General § 1.701 Show cause orders. (a) The Commission may... order to show cause. The order shall contain a statement of the particulars and matters concerning...

  12. 41 CFR 60-1.28 - Show cause notices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Show cause notices. 60-1... Procedure § 60-1.28 Show cause notices. When the Deputy Assistant Secretary has reasonable cause to believe... contractor to show cause, within 30 days, why monitoring, enforcement proceedings or other appropriate...

  13. 25 CFR 141.56 - Show cause procedures.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Show cause procedures. 141.56 Section 141.56 Indians... NAVAJO, HOPI AND ZUNI RESERVATIONS Enforcement Powers, Procedures and Remedies § 141.56 Show cause... from the date of receipt of notice in which to show cause why the contemplated remedial action...

  14. 21 CFR 1309.46 - Order to show cause.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Order to show cause. 1309.46 Section 1309.46 Food... Or Suspension of Registration § 1309.46 Order to show cause. (a) If, upon examination of the... upon the applicant an order to show cause why the application for registration should not be denied....

  15. 46 CFR 502.66 - Order to show cause.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 9 2010-10-01 2010-10-01 false Order to show cause. 502.66 Section 502.66 Shipping... Proceedings; Pleadings; Motions; Replies § 502.66 Order to show cause. The Commission may institute a proceeding by order to show cause. The order shall be served upon all persons named therein, shall...

  16. 21 CFR 1301.37 - Order to show cause.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Order to show cause. 1301.37 Section 1301.37 Food... Suspension of Registration § 1301.37 Order to show cause. (a) If, upon examination of the application for... Administrator shall serve upon the applicant an order to show cause why the registration should not be...

  17. 21 CFR 1314.150 - Order To show cause.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Order To show cause. 1314.150 Section 1314.150 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE RETAIL SALE OF SCHEDULED LISTED CHEMICAL PRODUCTS Order to Show Cause § 1314.150 Order To show cause. (a) If, upon information gathered...

  18. 22 CFR 1421.16 - Showing of interest.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Showing of interest. 1421.16 Section 1421.16... TERMS AS USED IN THIS SUBCHAPTER § 1421.16 Showing of interest. Showing of interest means evidence of... forms executed by an employee and the labor organization's authorized official; current dues...

  19. 56. View looking east. Detail showing an end of three ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    56. View looking east. Detail showing an end of three crib side walls, being flown by the derrick. The upper part of the fourth member, marked with an identifying tag, shows evidence of burning. Notice how well the lower members fit together. This view shows that individual timber members were worked, in part, after assembly. - Wabash & Erie Canal, Lock No. 2, 8 miles east of Fort Wayne, adjacent to U.S. Route 24, New Haven, Allen County, IN

  20. 52. View from ground level showing lower radar scanner switch ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    52. View from ground level showing lower radar scanner switch with open port door in radar scanner building 105 showing emanating waveguides from lower switch in vertical run; photograph also shows catwalk to upper scanner switch in upper left side of photograph and structural supports. - Clear Air Force Station, Ballistic Missile Early Warning System Site II, One mile west of mile marker 293.5 on Parks Highway, 5 miles southwest of Anderson, Anderson, Denali Borough, AK

  1. 34 CFR 300.193 - Request to show cause.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 2 2010-07-01 2010-07-01 false Request to show cause. 300.193 Section 300.193... show cause. An SEA, LEA or other public agency in receipt of a notice under § 300.192 that seeks an opportunity to show cause why a by-pass should not be implemented must submit a written request for a...

  2. 1. Southeast elevation of Oil House showing loading platform. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. Southeast elevation of Oil House showing loading platform. - Delaware, Lackawanna & Western Railroad, Scranton Yards, Oil House, 650 feet Southeast of Cliff & Mechanic Streets, Scranton, Lackawanna County, PA

  3. 11. Exterior detail view of northeast corner, showing stucco finish ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. Exterior detail view of northeast corner, showing stucco finish and woodwork details - American Railway Express Company Freight Building, 1060 Northeast Division Street, Bend, Deschutes County, OR

  4. 3. Photocopy of 1932 photograph showing another general view of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Photocopy of 1932 photograph showing another general view of the mansion, looking northwest. Original photograph at the Philadelphia Museum of Art. - Strawberry Mansion, Philadelphia, Philadelphia County, PA

  5. RIVERSIDE AVE. FROM SOUTH, SOUTHEAST OF BUILDINGS #433 SHOWING BUILDINGS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    RIVERSIDE AVE. FROM SOUTH, SOUTHEAST OF BUILDINGS #433 SHOWING BUILDINGS #434 AND #435, LOOKING EAST-SOUTHEAST - Fort Leavenworth, Metropolitan Avenue & Seventh Street, Leavenworth, Leavenworth County, KS

  6. 1. DELAWARE AVE. (right, looking north) AND WASHINGTON AVE. SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. DELAWARE AVE. (right, looking north) AND WASHINGTON AVE. SHOWING GLORIA DEI CHURCH (note steeple) - Independence National Historical Park, Walnut, Sixth, Chestnut & Second Streets, Philadelphia, Philadelphia County, PA

  7. 10. VIEW, LOOKING NORTHEAST, OF MAIN LOBBY, FIRST FLOOR, SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. VIEW, LOOKING NORTHEAST, OF MAIN LOBBY, FIRST FLOOR, SHOWING PORTALS TO NEW LOBBY - Pennsylvania Railroad, Harrisburg Station & Trainshed, Market & South Fourth Streets, Harrisburg, Dauphin County, PA

  8. 2. VIEW SOUTH SHOWING NORTHEAST ELEVATION; BRICK CORBELLING, BUTTRESSES AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. VIEW SOUTH SHOWING NORTHEAST ELEVATION; BRICK CORBELLING, BUTTRESSES AND ART DECO STAINED GLASS - Poletown Historic District, St. Michael's Greek Catholic Church, 2390 East Grand Boulevard, Detroit, MI

  9. 6. SOUTHEAST ABUTMENT AT CALVERT STREET, SHOWING LEON HERMANT ALLEGORICAL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. SOUTHEAST ABUTMENT AT CALVERT STREET, SHOWING LEON HERMANT ALLEGORICAL RELIEF OF TRANSPORTATION BY AUTOMOBILE - Calvert Street Bridge, Spanning Rock Creek & Potomac Parkway, Washington, District of Columbia, DC

  10. 24. CONSTRUCTION PROGRESS VIEW TO NORTHWEST, SHOWING BLOWER BUILDING. INEEL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. CONSTRUCTION PROGRESS VIEW TO NORTHWEST, SHOWING BLOWER BUILDING. INEEL PHOTO NUMBER NRTS-60-4407. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

  11. 26. VIEW, LOOKING NORTHWEST INSIDE TRANSFORMER ROOM, SHOWING OIL FILLED ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    26. VIEW, LOOKING NORTHWEST INSIDE TRANSFORMER ROOM, SHOWING OIL- FILLED TRANSFORMER POTS - Sacramento River Bridge, Spanning Sacramento River at California State Highway 275, Sacramento, Sacramento County, CA

  12. 25. VIEW, LOOKING SOUTHWEST INSIDE TRANSFORMER ROOM, SHOWING TRANSFORMERS AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    25. VIEW, LOOKING SOUTHWEST INSIDE TRANSFORMER ROOM, SHOWING TRANSFORMERS AND KNIFE SWITCHES - Sacramento River Bridge, Spanning Sacramento River at California State Highway 275, Sacramento, Sacramento County, CA

  13. 10. View along North Plant Southwest Elevation, Showing Loading Dock ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. View along North Plant Southwest Elevation, Showing Loading Dock and Powerhouse (right rear) - Atwater Kent Manufacturing Company, North Plant, 5000 Wissahickon Avenue, Philadelphia, Philadelphia County, PA

  14. 7. Photocopy of photograph (from Broome County Historical Society) showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Photocopy of photograph (from Broome County Historical Society) showing SWIMMERS, PHOTOGRAPH TAKEN FACING NORTHEAST - Charles F. Johnson Pool, Charles F. Johnson Park, Johnson City, Broome County, NY

  15. 13. VIEW INTO BLOCK AREA SHOWING KEY MECHANISM, NOTE FLOOR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. VIEW INTO BLOCK AREA SHOWING KEY MECHANISM, NOTE FLOOR SEPARATION AT THRESHOLD AND KEY-WINDING MECHANISM - Montgomery County Jail, Washington & Spring Streets, Crawfordsville, Montgomery County, IN

  16. Interior view, detail of the staircase to show the burnished ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior view, detail of the staircase to show the burnished aluminum and brass balustrade - Departmental Auditorium, Constitution Avenue between Twelfth and Fourteenth Streets, Washington, District of Columbia, DC

  17. INTERIOR VIEW OF ENTRANCE TO LABORATORY, SHOWING HANDHAMMERED ALUMINUM DOORS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    INTERIOR VIEW OF ENTRANCE TO LABORATORY, SHOWING HAND-HAMMERED ALUMINUM DOORS AND MARBLE. NOTE ALUMINUM LIGHT FIXTURE - Alcoa Research Laboratory, Freeport Road, New Kensington, Westmoreland County, PA

  18. INTERIOR OF COLD STORAGE ROOM, SHOWING MOVABLE HANGING RACKS. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    INTERIOR OF COLD STORAGE ROOM, SHOWING MOVABLE HANGING RACKS. - Naval Air Station Barbers Point, Aircraft Storehouse, Between Midway & Card Streets at Enterprise Avenue intersection, Ewa, Honolulu County, HI

  19. 6. INTERIOR VIEW SHOWING STRUCTURAL DESIGN AND SAWN REPLACEMENT EYEBROW ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. INTERIOR VIEW SHOWING STRUCTURAL DESIGN AND SAWN REPLACEMENT EYEBROW RAFTERS - Camp Cleawox, Adirondack Sleeping Shelter, Oregon Dunes National Recreation Area, Siuslaw National Forest, Florence, Lane County, OR

  20. 54. VIEW SHOWING THE PLACEMENT OF SPIDER WEB BRACING, SHOOFLY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    54. VIEW SHOWING THE PLACEMENT OF SPIDER WEB BRACING, SHOOFLY BRIDGE, January 1935 - Sacramento River Bridge, Spanning Sacramento River at California State Highway 275, Sacramento, Sacramento County, CA

  1. 2. GENERAL VIEW FROM SOUTH SHOWING SOUTHWEST AND SOUTHEAST SIDES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. GENERAL VIEW FROM SOUTH SHOWING SOUTHWEST AND SOUTHEAST SIDES AND CLERESTORY ARRANGEMENT - Sulphur Springs Methodist Campground, Sulphur Springs Road (Sulphur Springs), Sulphur Springs, Washington County, TN

  2. New rain shed (Building No. 241) interior showing posts, braces, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    New rain shed (Building No. 241) interior showing posts, braces, and roof structure. - Hawaii Volcanoes National Park Water Collection System, Hawaii Volcanoes National Park, Volcano, Hawaii County, HI

  3. North elevation, showing watergate and bridge plaza at left. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    North elevation, showing watergate and bridge plaza at left. - Arlington Memorial Bridge, Spanning Potomac River between Lincoln Memorial & Arlington National Cemetery, Washington, District of Columbia, DC

  4. General view, showing eastern view from atop hemi circle. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    General view, showing eastern view from atop hemi circle. - Arlington Memorial Bridge, Spanning Potomac River between Lincoln Memorial & Arlington National Cemetery, Washington, District of Columbia, DC

  5. Interior room within eastern lift span, showing auxiliary electric generator. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior room within eastern lift span, showing auxiliary electric generator. - Arlington Memorial Bridge, Spanning Potomac River between Lincoln Memorial & Arlington National Cemetery, Washington, District of Columbia, DC

  6. 29. Detail view north showing amperage and voltage meters, operator's ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    29. Detail view north showing amperage and voltage meters, operator's room, west operator's house. - Yellow Mill Bridge, Spanning Yellow Mill Channel at Stratford Avenue, Bridgeport, Fairfield County, CT

  7. Experimental Therapy Shows Promise for Type 1 Diabetes

    MedlinePlus

    ... Relief Health Capsules Experimental Therapy Shows Promise for Type 1 Diabetes Complementary Approaches for Depression Featured Website: Prescription Drug Abuse Past Issues Most Viewed June 2016 Print RSS ...

  8. Detail view to show northwestern section of the building with ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail view to show northwestern section of the building with its modified mansard roof - Internal Revenue Service Headquarters Building, 1111 Constitution Avenue Northwest, Washington, District of Columbia, DC

  9. 3. GENERAL VIEW OF COMPLEX, LOOKING SOUTHWEST, SHOWING THE 1959 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. GENERAL VIEW OF COMPLEX, LOOKING SOUTHWEST, SHOWING THE 1959 GENERATING STATION. - Commonwealth Electric Company, Fisk Street Electrical Generating Station, 1111 West Cermak Avenue, Chicago, Cook County, IL

  10. Contextual view showing northeastern eucalyptus windbreak and portion of citrus ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Contextual view showing northeastern eucalyptus windbreak and portion of citrus orchard. Camera facing 118" east-southeast. - Goerlitz House, 9893 Highland Avenue, Rancho Cucamonga, San Bernardino County, CA

  11. 12. VIEW LOOKING SOUTH FROM PAVILION, SHOWING SOUTH ENTRANCE HOUSE, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. VIEW LOOKING SOUTH FROM PAVILION, SHOWING SOUTH ENTRANCE HOUSE, SOUTH WING, AND ENGINE HOUSE - Fairmount Waterworks, East bank of Schuylkill River, Aquarium Drive, Philadelphia, Philadelphia County, PA

  12. View looking south from pavilion, showing south entrance house, south ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View looking south from pavilion, showing south entrance house, south wing, and engine house - Fairmount Waterworks, East bank of Schuylkill River, Aquarium Drive, Philadelphia, Philadelphia County, PA

  13. View looking south, showing portico of north wing with pavilion ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View looking south, showing portico of north wing with pavilion and engine house in background - Fairmount Waterworks, East bank of Schuylkill River, Aquarium Drive, Philadelphia, Philadelphia County, PA

  14. 11. SECOND FLOOR INTERIOR, SHOWING EAST BALCONY AND BASKETBALL COURT. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. SECOND FLOOR INTERIOR, SHOWING EAST BALCONY AND BASKETBALL COURT. VIEW TO NORTHEAST. - Fort David A. Russell, Gymnasium, Randall Avenue between Fourth & Fifth Streets, Cheyenne, Laramie County, WY

  15. 13. FOURTH FLOOR ROASTING ROOM, SHOWING CLERESTORY. VIEW TO SOUTH. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. FOURTH FLOOR ROASTING ROOM, SHOWING CLERESTORY. VIEW TO SOUTH. - Commercial & Industrial Buildings, McFadden Coffee & Spice Company, Factory & Warehouse, 145 First Street, Dubuque, Dubuque County, IA

  16. 23. VIEW SHOWING SALT RIVER PROJECT CREWS SLIPFORMING LATERAL DURING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    23. VIEW SHOWING SALT RIVER PROJECT CREWS SLIPFORMING LATERAL DURING REHABILITATION AND BETTERMENT PROGRAM Photographer: unknown. April 1968 - Arizona Canal, North of Salt River, Phoenix, Maricopa County, AZ

  17. 8. GENERAL INTERIOR VIEW OF FISH HATCHERY BUILDING, SHOWING INCUBATION ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. GENERAL INTERIOR VIEW OF FISH HATCHERY BUILDING, SHOWING INCUBATION TANKS. - Bonneville Project, Fish Hatchery, On Columbia River bordered on South by Union Pacific, Bonneville, Multnomah County, OR

  18. 1. GENERAL EXTERIOR VIEW LOOKING SOUTH, SHOWING FRONT FACADE OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. GENERAL EXTERIOR VIEW LOOKING SOUTH, SHOWING FRONT FACADE OF ADMINISTRATION BUILDING. - Bonneville Project, Administration Building, South side of main entrance, Bonneville Project, Bonneville, Multnomah County, OR

  19. 5. GENERAL EXTERIOR VIEW LOOKING SOUTHWEST, SHOWING EAST FACADE OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. GENERAL EXTERIOR VIEW LOOKING SOUTHWEST, SHOWING EAST FACADE OF ADMINISTRATION BUILDING. - Bonneville Project, Administration Building, South side of main entrance, Bonneville Project, Bonneville, Multnomah County, OR

  20. 4. GENERAL EXTERIOR VIEW LOOKING WEST, SHOWING REAR WING OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. GENERAL EXTERIOR VIEW LOOKING WEST, SHOWING REAR WING OF ADMINISTRATION BUILDING. - Bonneville Project, Administration Building, South side of main entrance, Bonneville Project, Bonneville, Multnomah County, OR

  1. 1. Aerial view, looking northeast up Newark Bay, showing entire ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. Aerial view, looking northeast up Newark Bay, showing entire island Charles Wisniewski, photographer, January 1985 - Shooters Island, Ships Graveyard, Newark Bay, Staten Island (subdivision), Richmond County, NY

  2. Underside of span over Pickering Creek, showing highly skewed piers, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Underside of span over Pickering Creek, showing highly skewed piers, looking south. - Pennsylvania Railroad, Pickering Creek Trestle, Spanning Pickering Creek, south of Buckwalter Road, Pickering, Chester County, PA

  3. 10. Detail, northerly spans, west side, showing deterioration; note spalls, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. Detail, northerly spans, west side, showing deterioration; note spalls, cracks, and efflorescence; view to southeast. - Fordway Bridge, Spanning Concord River at Pollard Street, Billerica, Middlesex County, MA

  4. 4. View facing southwest showing the Silvertop Diner, Providence Fruit ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. View facing southwest showing the Silvertop Diner, Providence Fruit & Produce Building, and Merchants' Cold Storage Warehouse. - Provisions Warehouse Historic District, Kinsley & Harris Avenues, Providence, Providence County, RI

  5. Contextual view showing west elevation; camera facing southeast. Mare ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Contextual view showing west elevation; camera facing southeast. - Mare Island Naval Shipyard, Ordnance Warehouse, Blake Avenue, northeast corner of Blake Avenue & Railroad Avenue, Vallejo, Solano County, CA

  6. 7. SOUTHEAST REAR DETAIL, SHOWING WINDOWS. VIEW TO NORTHWEST. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. SOUTHEAST REAR DETAIL, SHOWING WINDOWS. VIEW TO NORTHWEST. - Commercial & Industrial Buildings, Key City Electric Street Railroad, Powerhouse & Storage Barn, Eighth & Washington Streets, Dubuque, Dubuque County, IA

  7. 15. FIRST FLOOR WAREHOUSE SPACE, SHOWING COLUMN / BEAM CONNECTION. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. FIRST FLOOR WAREHOUSE SPACE, SHOWING COLUMN / BEAM CONNECTION. VIEW TO SOUTHWEST. - Commercial & Industrial Buildings, Dubuque Seed Company Warehouse, 169-171 Iowa Street, Dubuque, Dubuque County, IA

  8. 12. TRANSMISSION GEARING SHOWING RELATION TO SEGMENT GEAR ON WATERWHEEL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. TRANSMISSION GEARING SHOWING RELATION TO SEGMENT GEAR ON WATERWHEEL william E. Barrett, photographer, 1973 (copy negative) - Thomas Shepherd's Grist Mill, High Street Vicinity, Shepherdstown, Jefferson County, WV

  9. Time dependent patient no-show predictive modelling development.

    PubMed

    Huang, Yu-Li; Hanauer, David A

    2016-05-01

    Purpose - The purpose of this paper is to develop evident-based predictive no-show models considering patients' each past appointment status, a time-dependent component, as an independent predictor to improve predictability. Design/methodology/approach - A ten-year retrospective data set was extracted from a pediatric clinic. It consisted of 7,291 distinct patients who had at least two visits along with their appointment characteristics, patient demographics, and insurance information. Logistic regression was adopted to develop no-show models using two-thirds of the data for training and the remaining data for validation. The no-show threshold was then determined based on minimizing the misclassification of show/no-show assignments. There were a total of 26 predictive model developed based on the number of available past appointments. Simulation was employed to test the effective of each model on costs of patient wait time, physician idle time, and overtime. Findings - The results demonstrated the misclassification rate and the area under the curve of the receiver operating characteristic gradually improved as more appointment history was included until around the 20th predictive model. The overbooking method with no-show predictive models suggested incorporating up to the 16th model and outperformed other overbooking methods by as much as 9.4 per cent in the cost per patient while allowing two additional patients in a clinic day. Research limitations/implications - The challenge now is to actually implement the no-show predictive model systematically to further demonstrate its robustness and simplicity in various scheduling systems. Originality/value - This paper provides examples of how to build the no-show predictive models with time-dependent components to improve the overbooking policy. Accurately identifying scheduled patients' show/no-show status allows clinics to proactively schedule patients to reduce the negative impact of patient no-shows. PMID:27142954

  10. Survey Shows Blacks Not Concerned Enough about Kidney Disease

    ERIC Educational Resources Information Center

    Black Issues in Higher Education, 2004

    2004-01-01

    Health officials may have an uphill battle in educating Blacks about a disease that's being called a "silent killer," a recent survey shows. Kidney disease is an illness that's become more prevalent, especially in the nation's Black population, but a survey conducted in Jackson, Atlanta, Baltimore and Cleveland shows only 15 percent of those…

  11. The Easy Way to Create Computer Slide Shows.

    ERIC Educational Resources Information Center

    Anderson, Mary Alice

    1995-01-01

    Discusses techniques for creating computer slide shows. Topics include memory; format; color use; HyperCard and CD-ROM; font styles and sizes; graphs and graphics; the slide show option; special effects; and tips for effective presentation. (Author/AEF)

  12. 2. DETAIL VIEW SHOWING WOODEN CRIBBING WITH LOWERED LAKE LEVEL, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. DETAIL VIEW SHOWING WOODEN CRIBBING WITH LOWERED LAKE LEVEL, EAST DAM, LOOKING NORTHEAST (View is middle of the perimeter showing in MT-88-A-1 above.) - Three Bears Lake & Dams, East Dam, North of Marias Pass, East Glacier Park, Glacier County, MT

  13. 13. VIEW SHOWING MOST OF THE PERIMETER FROM SPILLWAY BOX ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. VIEW SHOWING MOST OF THE PERIMETER FROM SPILLWAY BOX TO END OF EAST DAM. FOREGROUND VIEW SHOWS TRIPLE WALL CONSTRUCTION OF TONGUE AND GROOVE PLANKING USED IN CRIBBING - Three Bears Lake & Dams, North of Marias Pass, East Glacier Park, Glacier County, MT

  14. 15. BALD MOUNTAIN MILL, INTERIOR SHOWING PRECIPITATION AREA FROM NORTH, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. BALD MOUNTAIN MILL, INTERIOR SHOWING PRECIPITATION AREA FROM NORTH, c. 1934. SHOWS PRECIPITATION TANK No. 1 (NOTE LOCKS), ZINC FEEDER WITH MIXING CONE, VACUUM RECEIVER AND PIPING. CREDIT WR. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  15. 28. VIEW SOUTHWEST, EAST ABUTMENT SHOWING BASCULE LEAF IN RAISED ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    28. VIEW SOUTHWEST, EAST ABUTMENT SHOWING BASCULE LEAF IN RAISED POSITION WITH THE EXTREME UPPER PORTION OF THE BASCULE RACK GEAR SHOWING IN THE LOWER RIGHT PORTION OF THE PHOTOGRAPH - Tomlinson Bridge, Spanning Quinnipiac River at Forbes Street (U.S. Route 1), New Haven, New Haven County, CT

  16. 27. GENERAL INTERIOR VIEW SHOWING SOUTH CORNER OF SHED WITH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    27. GENERAL INTERIOR VIEW SHOWING SOUTH CORNER OF SHED WITH ONE-STORY OFFICES, SHOWING TYPICAL COLUMN BASE WITH TIMBER BOLTED TO STEEL 'L' SHOE - Oakland Army Base, Transit Shed, East of Dunkirk Street & South of Burma Road, Oakland, Alameda County, CA

  17. The Daily Show with Jon Stewart: Part 2

    ERIC Educational Resources Information Center

    Trier, James

    2008-01-01

    "The Daily Show With Jon Stewart" is one of the best critical literacy programs on television, and in this Media Literacy column the author suggests ways that teachers can use video clips from the show in their classrooms. (For Part 1, see EJ784683.)

  18. 8. INTERIOR OF KERN RIVER No. 1 POWERHOUSE BUILDING SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. INTERIOR OF KERN RIVER No. 1 POWERHOUSE BUILDING SHOWING EXCITER No 1. SIDE VIEW OF LOMBARD GOVERNOR SHOWING BELT ATTACHMENT TO EXCITER SHAFT. GENERATOR UNIT No. 2 IN BACKGROUND. VIEW TO NORTHWEST. - Kern County No. 1 Hydroelectric System, Powerhouse Exciters, Kern River Canyon, Bakersfield, Kern County, CA

  19. 35. Ca. 1930 historic view facing northeast, showing Yellow Mill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    35. Ca. 1930 historic view facing northeast, showing Yellow Mill Bridge and inset showing former ca. 1901 Yellow Mill Bridge. Photo located at the Bridgeport Public Library, Bridgeport, CT. - Yellow Mill Bridge, Spanning Yellow Mill Channel at Stratford Avenue, Bridgeport, Fairfield County, CT

  20. 21 CFR 1314.150 - Order To show cause.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Order To show cause. 1314.150 Section 1314.150 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE RETAIL SALE OF SCHEDULED LISTED... should not be prohibited from selling scheduled listed chemical products. (b) The order to show...

  1. 21 CFR 1314.150 - Order To show cause.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 9 2014-04-01 2014-04-01 false Order To show cause. 1314.150 Section 1314.150 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE RETAIL SALE OF SCHEDULED LISTED... should not be prohibited from selling scheduled listed chemical products. (b) The order to show...

  2. 21 CFR 1314.150 - Order To show cause.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 9 2011-04-01 2011-04-01 false Order To show cause. 1314.150 Section 1314.150 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE RETAIL SALE OF SCHEDULED LISTED... should not be prohibited from selling scheduled listed chemical products. (b) The order to show...

  3. 21 CFR 1314.150 - Order To show cause.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Order To show cause. 1314.150 Section 1314.150 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE RETAIL SALE OF SCHEDULED LISTED... should not be prohibited from selling scheduled listed chemical products. (b) The order to show...

  4. 108. View showing storage yard where material is received and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    108. View showing storage yard where material is received and sorted: also shows derrick framed to raise material from tracks and land on deck of approach. Material is then moved by narrow gage locomotive out to erection traveler. - Carquinez Bridge, Spanning Carquinez Strait at Interstate 80, Vallejo, Solano County, CA

  5. 7. Photocopy of house, circa 1910. It shows small original ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Photocopy of house, circa 1910. It shows small original details which have been changed and also shows how little the exterior appearence has been altered. Original photograph at Nebraska State Historical Society. - Chambers-Mayberry House, Northwest corner of Oak Street & Fifth Avenue (moved to Crofton, NE), Niobrara, Knox County, NE

  6. Showing and Telling: The Difference That Makes a Difference.

    ERIC Educational Resources Information Center

    Lewis, David

    2001-01-01

    Attempts to clarify an essential difference between the ways in which pictures and words convey meaning. Examines one attempt to differentiate and characterize various types of picture books and concludes by showing how Anthony Browne exploits the distinction between showing and telling to create the atmosphere of uncertainty and mystery in his…

  7. 14 CFR 314.14 - Show-cause order.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Show-cause order. 314.14 Section 314.14 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) PROCEDURAL REGULATIONS EMPLOYEE PROTECTION PROGRAM Determination of Qualifying Dislocation § 314.14 Show-cause...

  8. 28. DETAIL: View looking southwest from inside the chamber, showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    28. DETAIL: View looking southwest from inside the chamber, showing the west gate sill, south corner. This photograph shows the most complete gate corner assembly in Lock No. 2. - Wabash & Erie Canal, Lock No. 2, 8 miles east of Fort Wayne, adjacent to U.S. Route 24, New Haven, Allen County, IN

  9. 31. Photographic copy of drawing showing profile of bridge after ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. Photographic copy of drawing showing profile of bridge after the 1888-1889 and 1899-1900 reconstructions; also shows profile of bridge before 1888 (Martin Sigvart Grytbak, Wabasha St. Bridge, Formerly St. Paul Bridge, 1919); profile of Wabasha street bridge - Wabasha Street Bridge, Spanning Mississippi River at Wabasha Street, Saint Paul, Ramsey County, MN

  10. 47 CFR 73.33 - Antenna systems; showing required.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 4 2012-10-01 2012-10-01 false Antenna systems; showing required. 73.33... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.33 Antenna systems; showing required. (a) An application for authority to install a broadcast antenna shall specify a definite site and include...

  11. 47 CFR 73.33 - Antenna systems; showing required.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 4 2011-10-01 2011-10-01 false Antenna systems; showing required. 73.33... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.33 Antenna systems; showing required. (a) An application for authority to install a broadcast antenna shall specify a definite site and include...

  12. 47 CFR 73.33 - Antenna systems; showing required.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Antenna systems; showing required. 73.33... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.33 Antenna systems; showing required. (a) An application for authority to install a broadcast antenna shall specify a definite site and include...

  13. 47 CFR 73.33 - Antenna systems; showing required.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 4 2014-10-01 2014-10-01 false Antenna systems; showing required. 73.33... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.33 Antenna systems; showing required. (a) An application for authority to install a broadcast antenna shall specify a definite site and include...

  14. 47 CFR 73.33 - Antenna systems; showing required.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 4 2013-10-01 2013-10-01 false Antenna systems; showing required. 73.33... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.33 Antenna systems; showing required. (a) An application for authority to install a broadcast antenna shall specify a definite site and include...

  15. 77 FR 1513 - Air Show and Air Races; Public Hearing

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-10

    ... From the Federal Register Online via the Government Publishing Office NATIONAL TRANSPORTATION SAFETY BOARD Air Show and Air Races; Public Hearing TIME AND DATE: 9 a.m., Tuesday, January 10, 2012... hearing is to examine current regulations and oversight practices for air shows and air races,...

  16. 15. Photocopy of historic view No. 31001 showing expansion of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. Photocopy of historic view No. 31001 showing expansion of Building 461 (9/4/56) showing Building 463 tanks to the right of the photo. Original is on file Mare Island Naval Shipyard-Photography Lab. Photographer unknown. - Mare Island Naval Shipyard, Acid Mixing Facility, California Avenue & E Street, Vallejo, Solano County, CA

  17. 47 CFR 101.411 - Supplementary showing required.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Supplementary showing required. 101.411 Section 101.411 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES FIXED MICROWAVE SERVICES Developmental Authorizations § 101.411 Supplementary showing required....

  18. 47 CFR 101.411 - Supplementary showing required.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Supplementary showing required. 101.411 Section 101.411 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES FIXED MICROWAVE SERVICES Developmental Authorizations § 101.411 Supplementary showing required....

  19. 16 CFR 5.57 - Order to show cause.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Practice. 16 CFR 4.7. ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Order to show cause. 5.57 Section 5.57... CONDUCT Disciplinary Actions Concerning Postemployment Conflict of Interest § 5.57 Order to show cause....

  20. 41 CFR 60-300.64 - Show cause notices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Show cause notices. 60... VETERANS General Enforcement and Complaint Procedures § 60-300.64 Show cause notices. When the Deputy Assistant Secretary has reasonable cause to believe that the contractor has violated the Act or this...

  1. 14 CFR 303.44 - Show cause proceedings.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Show cause proceedings. 303.44 Section 303... cause proceedings. If the Assistant Secretary determines that an application, or review of a previously granted application, will be considered in a show cause proceeding, a tentative decision shall be...

  2. 10 CFR 110.62 - Order to show cause.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Order to show cause. 110.62 Section 110.62 Energy NUCLEAR... Enforcement § 110.62 Order to show cause. (a) In response to an alleged violation, described in § 110.60, the... cause: (1) Stating the alleged violation and proposed enforcement action; and (2) Informing the...

  3. 41 CFR 60-250.64 - Show cause notices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Show cause notices. 60... VETERANS General Enforcement and Complaint Procedures § 60-250.64 Show cause notices. When the Deputy Assistant Secretary has reasonable cause to believe that the contractor has violated the Act or this...

  4. 18 CFR 808.15 - Show cause proceeding.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Show cause proceeding... HEARINGS AND ENFORCEMENT ACTIONS Compliance and Enforcement § 808.15 Show cause proceeding. (a) The... cause why a penalty should not be assessed in accordance with the provisions of this chapter and...

  5. 41 CFR 60-741.64 - Show cause notices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Show cause notices. 60... INDIVIDUALS WITH DISABILITIES General Enforcement and Complaint Procedures § 60-741.64 Show cause notices. When the Deputy Assistant Secretary has reasonable cause to believe that the contractor has...

  6. The Presentation of Science in Everyday Life: The Science Show

    ERIC Educational Resources Information Center

    Watermeyer, Richard

    2013-01-01

    This paper constitutes a case-study of the "science show" model of public engagement employed by a company of science communicators focused on the popularization of science, technology, engineering and mathematics (STEM) subject disciplines with learner constituencies. It examines the potential of the science show to foster the interest…

  7. View of Lake Sabrina Dam showing wooden planks along the ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of Lake Sabrina Dam showing wooden planks along the upstream face and concrete base added in 1916/1917 and showing the iron grating covering upstream side of outlet structure is visible at lower photo center, view northeast - Bishop Creek Hydroelectric System, Plant 2, Lake Sabrina Dam, Bishop Creek, Bishop, Inyo County, CA

  8. "The Daily Show with Jon Stewart": Part 1

    ERIC Educational Resources Information Center

    Trier, James

    2008-01-01

    Comedy Central's popular program "The Daily Show With Jon Stewart" is the best critical media literacy program on television, and it can be used in valuable ways in the classroom as part of a media literacy pedagogy. This Media Literacy column provides an overview of the show and its accompanying website and considers ways it might be used in the…

  9. TV shows on Light Pollution Education for the Public

    NASA Astrophysics Data System (ADS)

    Grigore, Valentin

    2015-03-01

    TV shows have the biggest impact for the public, so we can use them to inform and educate the public about light pollution and the importance of the dark sky for humanity and for the contemporary society. Some examples used in the TV show Us and the Sky at Columna TV, Romania, are presented.

  10. 36 CFR 14.24 - Showing as to citizenship required.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 36 Parks, Forests, and Public Property 1 2013-07-01 2013-07-01 false Showing as to citizenship... INTERIOR RIGHTS-OF-WAY Procedures § 14.24 Showing as to citizenship required. (a) Individuals. An...-955), as amended, must state whether he is native born or naturalized, and, if naturalized, the...

  11. 36 CFR 14.24 - Showing as to citizenship required.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Showing as to citizenship... INTERIOR RIGHTS-OF-WAY Procedures § 14.24 Showing as to citizenship required. (a) Individuals. An...-955), as amended, must state whether he is native born or naturalized, and, if naturalized, the...

  12. 36 CFR 14.24 - Showing as to citizenship required.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 1 2011-07-01 2011-07-01 false Showing as to citizenship... INTERIOR RIGHTS-OF-WAY Procedures § 14.24 Showing as to citizenship required. (a) Individuals. An...-955), as amended, must state whether he is native born or naturalized, and, if naturalized, the...

  13. Computer Slide Shows: A Trap for Bad Teaching

    ERIC Educational Resources Information Center

    Klemm, W. R.

    2007-01-01

    Slide shows presented with software such as PowerPoint or WordPerfect Presentations can trap instructors into bad teaching practices. Research on memory suggests that slide-show instruction can actually be less effective than traditional lecturing when the teacher uses a blackboard or overhead projector. The author proposes a model of classroom…

  14. 5. INTERIOR VIEW OF THE CLUB MODERNE LOUNGE SHOWING EXAMPLES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. INTERIOR VIEW OF THE CLUB MODERNE LOUNGE SHOWING EXAMPLES OF THE LEATHER COVERED BOOTHS INSTALLED IN 19486. FACADE OF THE CLUB MODERNE, SHOWING THE ORIGINAL CURVED CORNER PROFILE AND TRI-COLOR CARRERE GLASS FACADE. - Anaconda Historic District, Club Moderne, 801 East Park Avenue, Anaconda, Deer Lodge County, MT

  15. 47 CFR 101.411 - Supplementary showing required.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Supplementary showing required. 101.411 Section 101.411 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES FIXED MICROWAVE SERVICES Developmental Authorizations § 101.411 Supplementary showing required....

  16. Dolphin shows and interaction programs: benefits for conservation education?

    PubMed

    Miller, L J; Zeigler-Hill, V; Mellen, J; Koeppel, J; Greer, T; Kuczaj, S

    2013-01-01

    Dolphin shows and dolphin interaction programs are two types of education programs within zoological institutions used to educate visitors about dolphins and the marine environment. The current study examined the short- and long-term effects of these programs on visitors' conservation-related knowledge, attitude, and behavior. Participants of both dolphin shows and interaction programs demonstrated a significant short-term increase in knowledge, attitudes, and behavioral intentions. Three months following the experience, participants of both dolphin shows and interaction programs retained the knowledge learned during their experience and reported engaging in more conservation-related behaviors. Additionally, the number of dolphin shows attended in the past was a significant predictor of recent conservation-related behavior suggesting that repetition of these types of experiences may be important in inspiring people to conservation action. These results suggest that both dolphin shows and dolphin interaction programs can be an important part of a conservation education program for visitors of zoological facilities. PMID:22622768

  17. ShowFlow: A practical interface for groundwater modeling

    SciTech Connect

    Tauxe, J.D.

    1990-12-01

    ShowFlow was created to provide a user-friendly, intuitive environment for researchers and students who use computer modeling software. What traditionally has been a workplace available only to those familiar with command-line based computer systems is now within reach of almost anyone interested in the subject of modeling. In the case of this edition of ShowFlow, the user can easily experiment with simulations using the steady state gaussian plume groundwater pollutant transport model SSGPLUME, though ShowFlow can be rewritten to provide a similar interface for any computer model. Included in this thesis is all the source code for both the ShowFlow application for Microsoft{reg sign} Windows{trademark} and the SSGPLUME model, a User's Guide, and a Developer's Guide for converting ShowFlow to run other model programs. 18 refs., 13 figs.

  18. Snacking on Television: A Content Analysis of Adolescents’ Favorite Shows

    PubMed Central

    Larson, Nicole I.; Gollust, Sarah E.; Neumark-Sztainer, Dianne

    2016-01-01

    Introduction Snacking is a complex behavior that may be influenced by entertainment media. Research suggests that snacking and unhealthy foods are commonly shown in programming that targets young audiences, but shows selected for study have been limited. We conducted a content analysis on shows that were named as favorites by adolescents to characterize portrayals of snacking on popular television. Methods A diverse sample of 2,130 adolescents (mean age, 14.3 y) listed 3 favorite television shows in a 2010 school-based survey. Three episodes each of the 25 most popular shows were coded for food-related content, including healthfulness, portion size, screen time use, setting, and social context. We also analyzed the characteristics of characters involved in eating incidents, the show type, and the show rating. We used χ2 tests, binomial tests, and multilevel regression models to compare incidence of snacks versus meals, the characteristics of those involved, and snacking across show characteristics. Results Almost half of food incidents on television shows were snacks. Snacks were significantly more likely than meals to be “mostly unhealthy” (69.3% vs 22.6%, P < .001) and were more likely to include screen time use (25.0% of snacking incidents vs 4.0% of meals, P < .001). Young characters and those coded as being of low socioeconomic status or overweight were overrepresented in snacking incidents. Sitcoms and shows rated for a youth audience were significantly more likely to portray snacking than were shows for adult audiences. Conclusion Media awareness and literacy programs should include foods and snacking behaviors among the issues they address. More healthful portrayals of food and dietary intake in entertainment shows’ content would create a healthier media environment for youth. PMID:27197079

  19. Ultraviolet imaging spectroscopy shows an active saturnian system.

    PubMed

    Esposito, Larry W; Colwell, Joshua E; Larsen, Kristopher; McClintock, William E; Stewart, A Ian F; Hallett, Janet Tew; Shemansky, Donald E; Ajello, Joseph M; Hansen, Candice J; Hendrix, Amanda R; West, Robert A; Keller, H Uwe; Korth, Axel; Pryor, Wayne R; Reulke, Ralf; Yung, Yuk L

    2005-02-25

    Neutral oxygen in the saturnian system shows variability, and the total number of oxygen atoms peaks at 4 x 10(34). Saturn's aurora brightens in response to solar-wind forcing, and the auroral spectrum resembles Jupiter's. Phoebe's surface shows variable water-ice content, and the data indicate it originated in the outer solar system. Saturn's rings also show variable water abundance, with the purest ice in the outermost A ring. This radial variation is consistent with initially pure water ice bombarded by meteors, but smaller radial structures may indicate collisional transport and recent renewal events in the past 10(7) to 10(8) years. PMID:15604361

  20. Satellite Movie Shows Three Tropical Cyclones in Eastern Pacific

    NASA Video Gallery

    This animation of NOAA's GOES-Wast satellite imagery from August 2 through 4 shows the movement of Tropical Depression Genevieve (left) southwest of Hawaii, Hurricane Iselle (center) in the Eastern...

  1. 2. AERIAL VIEW LOOKING WEST SOUTHWEST SHOWING DOLPHIN MANUFACTURING CO., ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. AERIAL VIEW LOOKING WEST SOUTHWEST SHOWING DOLPHIN MANUFACTURING CO., BARBOUR FLAX SPINNING CO. -- SPRUCE ST. MILL, ROGERS LOCOMOTIVE AND MACHINE WORKS -- MILLWRIGHT SHOP AND FITTING SHOP. - Great Falls S. U. M. Historic District, Oliver Street, Paterson, Passaic County, NJ

  2. 6. VIEW SHOWING DOWNSTREAM FACE AND TOE OF DAM, LOOKING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. VIEW SHOWING DOWNSTREAM FACE AND TOE OF DAM, LOOKING SOUTHWEST - High Mountain Dams in Upalco Unit, Kidney Lake Dam, Ashley National Forest, 4.7 miles North of Miners Gulch Campground, Mountain Home, Duchesne County, UT

  3. 2. VIEW SHOWING NATURAL SAND BEACH ON KIDNEY LAKE, LOOKING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. VIEW SHOWING NATURAL SAND BEACH ON KIDNEY LAKE, LOOKING WEST - High Mountain Dams in Upalco Unit, Kidney Lake Dam, Ashley National Forest, 4.7 miles North of Miners Gulch Campground, Mountain Home, Duchesne County, UT

  4. 4. VIEW SHOWING UPSTREAM FACE OF DAM, LOOKING NORTHEAST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. VIEW SHOWING UPSTREAM FACE OF DAM, LOOKING NORTHEAST - High Mountain Dams in Upalco Unit, Kidney Lake Dam, Ashley National Forest, 4.7 miles North of Miners Gulch Campground, Mountain Home, Duchesne County, UT

  5. 3. OVERALL VIEW OF DAM, SHOWING UPSTREAM FACE, LOOKING EAST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. OVERALL VIEW OF DAM, SHOWING UPSTREAM FACE, LOOKING EAST - High Mountain Dams in Upalco Unit, Kidney Lake Dam, Ashley National Forest, 4.7 miles North of Miners Gulch Campground, Mountain Home, Duchesne County, UT

  6. 5. VIEW SHOWING DOWNSTREAM FACE AND TOE OF DAM, LOOKING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. VIEW SHOWING DOWNSTREAM FACE AND TOE OF DAM, LOOKING SOUTHWEST - High Mountain Dams in Upalco Unit, Kidney Lake Dam, Ashley National Forest, 4.7 miles North of Miners Gulch Campground, Mountain Home, Duchesne County, UT

  7. 5. VIEW NORTHWEST SHOWING AQUEDUCT PRISM. NOTE INTERIOR STONE WORK ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. VIEW NORTHWEST SHOWING AQUEDUCT PRISM. NOTE INTERIOR STONE WORK OF THE PARAPET WALL AND REMAINS OF 1920 TIMBER AND CONCRETE FLOORING SYSTEM. - Chesapeake & Ohio Canal, Conococheague Creek Aqueduct, Milepost 99.80, Williamsport, Washington County, MD

  8. 5. INTERIOR VIEW OF WEST END OF BUILDING, SHOWING CEILING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. INTERIOR VIEW OF WEST END OF BUILDING, SHOWING CEILING BRIDGE CRANE. - Oakland Army Base, Vehicle Maintenance Shop, Tobruk Street, between Warehouse Road & Ukraine Street, Oakland, Alameda County, CA

  9. 8. DETAIL VIEW, LOOKING NORTHEAST, SHOWING OUTRIGGERS FOR LATERAL BRACING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. DETAIL VIEW, LOOKING NORTHEAST, SHOWING OUTRIGGERS FOR LATERAL BRACING FOR TRUSSES AND BOTTOM CHORD CONNECTIONS. - White Bowstring Arch Truss Bridge, Spanning Yellow Creek at Cemetery Drive (Riverside Drive), Poland, Mahoning County, OH

  10. 5. DETAIL VIEW OF TWO PANEL POINTS OF TRUSS, SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. DETAIL VIEW OF TWO PANEL POINTS OF TRUSS, SHOWING OVAL, TUBULAR UPPER CHORD MEMBER, VERTICALS, DIAGONALS, AND LOWER CHORD. - White Bowstring Arch Truss Bridge, Spanning Yellow Creek at Cemetery Drive (Riverside Drive), Poland, Mahoning County, OH

  11. 9. DETAIL OF UNITEDTOD TWINTANDEM STEAM ENGINE, SHOWING HIGHPRESSURE CYLINDER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. DETAIL OF UNITED-TOD TWIN-TANDEM STEAM ENGINE, SHOWING HIGH-PRESSURE CYLINDER AND EXTENSION OF HOUSING. - Republic Iron & Steel Company, Youngstown Works, Blooming Mill & Blooming Mill Engines, North of Poland Avenue, Youngstown, Mahoning County, OH

  12. 14. BALD MOUNTAIN MILL, INTERIOR SHOWING GOLD TANKS FROM WEST, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. BALD MOUNTAIN MILL, INTERIOR SHOWING GOLD TANKS FROM WEST, c. 1937. DATE BASED ON USE IN PUBLICATION. CREDIT WR. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  13. 53. VIEW SHOWING GRAFFITI ON EAST WALL OF STAIRWELL INSIDE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    53. VIEW SHOWING GRAFFITI ON EAST WALL OF STAIRWELL INSIDE 'CATFISH' SILO Everett Weinreb, photographer, April 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  14. 55. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    55. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett Weinreb, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  15. 41. OVERALL VIEW SHOWING ENTRANCE TO 'CATFISH' SILO, LOOKING SOUTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    41. OVERALL VIEW SHOWING ENTRANCE TO 'CATFISH' SILO, LOOKING SOUTH Marilyn Ziemer, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  16. 38. VIEW SHOWING 'CATFISH' SILO ELEVATOR IN UP POSITION, LOOKING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    38. VIEW SHOWING 'CATFISH' SILO ELEVATOR IN UP POSITION, LOOKING NORTHWEST Everett Weinreb, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  17. 57. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    57. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett Weinreb, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  18. 56. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    56. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett Weinreb, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  19. 40. VIEW SHOWING TOP OF 'CATFISH' SILO, LOOKING SOUTH Everett ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    40. VIEW SHOWING TOP OF 'CATFISH' SILO, LOOKING SOUTH Everett Weinreb, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  20. 58. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    58. VIEW SHOWING GRAFFITI IN STAIRWELL INSIDE 'CATFISH' SILO Everett Weinreb, photographer, March 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  1. 54. VIEW SHOWING GRAFFITI ON WEST WALL OF STAIRWELL INSIDE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    54. VIEW SHOWING GRAFFITI ON WEST WALL OF STAIRWELL INSIDE 'CATFISH' SILO Everett Weinreb, photographer, April 1988 - Mount Gleason Nike Missile Site, Angeles National Forest, South of Soledad Canyon, Sylmar, Los Angeles County, CA

  2. 12. VIEW OF ELEVATOR MACHINE ROOM, SHOWING LOW CABINET TEAT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. VIEW OF ELEVATOR MACHINE ROOM, SHOWING LOW CABINET TEAT HOUSES HYDRAULIC CYLINDER AND CLOSET AT LEFT THAT HOUSES OPERATING VALVE, LOOKING NORTHEAST - 72 Marlborough Street, Residential Hydraulic Elevator, Boston, Suffolk County, MA

  3. Interior view of space in north wing, showing main entry; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior view of space in north wing, showing main entry; camera facing east. - Mare Island Naval Shipyard, Smithery, California Avenue, west side at California Avenue & Eighth Street, Vallejo, Solano County, CA

  4. View of west elevation showing connection to building 521; camera ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of west elevation showing connection to building 521; camera facing south. - Mare Island Naval Shipyard, Old Administrative Offices, Eighth Street, north side between Railroad Avenue & Walnut Avenue, Vallejo, Solano County, CA

  5. Interior detail of main entry lobby showing arched hallways and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior detail of main entry lobby showing arched hallways and staircase; camera facing north. - Mare Island Naval Shipyard, Old Administrative Offices, Eighth Street, north side between Railroad Avenue & Walnut Avenue, Vallejo, Solano County, CA

  6. View of east side north elevation from across courtyard, showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of east side north elevation from across courtyard, showing building 47A at left; camera facing south. - Mare Island Naval Shipyard, Old Administrative Offices, Eighth Street, north side between Railroad Avenue & Walnut Avenue, Vallejo, Solano County, CA

  7. Contextual view showing new wing and north portion of building ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Contextual view showing new wing and north portion of building 46, looking down California Avenue; camera facing northwest. - Mare Island Naval Shipyard, Smithery, California Avenue, west side at California Avenue & Eighth Street, Vallejo, Solano County, CA

  8. Contextual view showing northwest corner of building 46, looking down ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Contextual view showing northwest corner of building 46, looking down California Avenue; camera facing southwest. - Mare Island Naval Shipyard, Smithery, California Avenue, west side at California Avenue & Eighth Street, Vallejo, Solano County, CA

  9. Detail of front south wall section showing pediment with triangular ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail of front south wall section showing pediment with triangular inset molding; camera facing north. - Mare Island Naval Shipyard, Old Administrative Offices, Eighth Street, north side between Railroad Avenue & Walnut Avenue, Vallejo, Solano County, CA

  10. 84. VIEW FROM CAMERA TOWER LOOKING SOUTHWEST SHOWING VAL FIRING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    84. VIEW FROM CAMERA TOWER LOOKING SOUTHWEST SHOWING VAL FIRING RANGE WITH OVERHEAD CAMERA AND CABLES, Date unknown, circa 1949. - Variable Angle Launcher Complex, Variable Angle Launcher, CA State Highway 39 at Morris Reservior, Azusa, Los Angeles County, CA

  11. Interior detail of first floor room on north side showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior detail of first floor room on north side showing wainscoting and decorative grillwork; camera facing southwest. - Mare Island Naval Shipyard, Old Administrative Offices, Eighth Street, north side between Railroad Avenue & Walnut Avenue, Vallejo, Solano County, CA

  12. 17. DETAIL OF STILLCAMERA CONTROL PANEL SHOWING PATCHBOARD FOR SELECTING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    17. DETAIL OF STILL-CAMERA CONTROL PANEL SHOWING PATCHBOARD FOR SELECTING TIMING OF CAMERAS - Vandenberg Air Force Base, Space Launch Complex 3, Launch Operations Building, Napa & Alden Roads, Lompoc, Santa Barbara County, CA

  13. Interior view of north wall showing roof trusses & monitor; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior view of north wall showing roof trusses & monitor; camera facing northeast. - Mare Island Naval Shipyard, Smithery, California Avenue, west side at California Avenue & Eighth Street, Vallejo, Solano County, CA

  14. 12. DETAIL VIEW OF NORTHEAST CORNER, SHOWING HIP VERTICAL, INCLINED ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. DETAIL VIEW OF NORTHEAST CORNER, SHOWING HIP VERTICAL, INCLINED END POST, AND UPPER CHORD - Kennan-Jump River Bridge, Spanning South fork of Jump River on County Highway "N", Kennan, Price County, WI

  15. MAGAZINE # B10. OBLIQUE VIEW FROM RIGHT SIDE SHOWING LOADING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    MAGAZINE # B-10. OBLIQUE VIEW FROM RIGHT SIDE SHOWING LOADING PLATFORM AND PART OF MAGAZINE B-9 IN BACKGROUND. - Naval Magazine Lualualei, Waikele Branch, Tunnel Magazine Type, Waikakalaua & Kipapa Gulches, Pearl City, Honolulu County, HI

  16. E SERIES MAGAZINES FROM HASTINGS ST. SHOWING ACCESS DRIVE AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    E SERIES MAGAZINES FROM HASTINGS ST. SHOWING ACCESS DRIVE AND LOADING PLATFORMS. E 103 MAGAZINES IN FORGROUND. - Naval Magazine Lualualei, Headquarters Branch, Magazine Type, Eleventh, Thirteenth, Fifteenth, Sixteenth, & Seventeenth Streets, Pearl City, Honolulu County, HI

  17. MAGAZINE # B11. OBLIQUE VIEW FROM RIGHT SIDE SHOWING LOADING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    MAGAZINE # B-11. OBLIQUE VIEW FROM RIGHT SIDE SHOWING LOADING PLATFORM AND ENTRY. - Naval Magazine Lualualei, Waikele Branch, Tunnel Magazine Type, Waikakalaua & Kipapa Gulches, Pearl City, Honolulu County, HI

  18. 27. View within machine room showing water tank, tool chest ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    27. View within machine room showing water tank, tool chest and oil/grease cans used for maintenance. (Nov. 25, 1988) - University Heights Bridge, Spanning Harlem River at 207th Street & West Harlem Road, New York County, NY

  19. 113. INTERIOR COMMUNICATIONS COMPARTMENT PORT LOOKING TO STARBOARD SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    113. INTERIOR COMMUNICATIONS COMPARTMENT - PORT LOOKING TO STARBOARD SHOWING MASTER GYRO AND INTERIOR COMMUNICATIONS SWITCHBOARD. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  20. 35. PILOT HOUSE PORT LOOKING TO STARBOARD SHOWING HELM, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    35. PILOT HOUSE - PORT LOOKING TO STARBOARD SHOWING HELM, ENGINE ORDER TELEGRAPH, SPEED INDICATOR, AFT GYRO REPEATER AND FORWARD GYRO REPEATER. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA