Science.gov

Sample records for acid lna probes

  1. Diagnosis of Phytoplasmas by Real-Time PCR Using Locked Nucleic Acid (LNA) Probes.

    PubMed

    Palmano, Sabrina; Mulholland, Vincent; Kenyon, David; Saddler, Gerry S; Jeffries, Colin

    2015-01-01

    Phytoplasma infections are regularly reported worldwide, and concerns about their threats on agricultural production, especially in relation to global climate change, are increasing. Sensitive and reliable detection methods are important to ensure that propagation material is free of phytoplasma infection and for epidemiological studies that may provide information to limit the extent of phytoplasma diseases and to prevent large-scale crop losses. The detection method described here uses LNA chemistry in real-time PCR. It has been developed and validated for use on potatoes, and its sensitivity and specificity make it suitable for use in postentry potato quarantine and initiation of potato nuclear stocks to ensure that material is phytoplasma-free. PMID:25981250

  2. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  3. Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

    PubMed

    Vilas Boas, Diana; Almeida, Carina; Sillankorva, Sanna; Nicolau, Ana; Azeredo, Joana; Azevedo, Nuno F

    2016-01-01

    Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations. PMID:26813295

  4. Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

    PubMed

    Vilas Boas, Diana; Almeida, Carina; Sillankorva, Sanna; Nicolau, Ana; Azeredo, Joana; Azevedo, Nuno F

    2016-01-01

    Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

  5. Regulating the on-surface LNA probe density for the highest target recognition efficiency.

    PubMed

    Mishra, Sourav; Ghosh, Srabani; Mukhopadhyay, Rupa

    2014-09-01

    The recent emergence of on-surface LNA-based assays as potentially better alternatives over DNA-based approaches, due to enhanced sensitivity and target specificity, raises the need for the precise identification of the factors that control the performance of these assays. In this work, we investigated whether the probe density of fully modified ssLNA probes on the gold(111) surface could influence the target recognition capacity of the LNA sensing layer and illustrated simple means to control it, primarily by adjusting the salt concentration, nature of the cation, and pH of the immobilization buffer. It was observed that monovalent Na(+) could more effectively control the sensor probe density compared to bivalent Mg(2+), leading to better target recognition. Interestingly, unlike in the case of ssDNA sensor probes, the target recognition efficiency of the LNA layer at the optimum probe density was found to be almost spacer-independent, probably due to the rigidity of the LNA backbone. The optimized LNA sensor layer could discriminate single base mismatches, detect a minimum target DNA concentration of 5 nM, and sense a significant level of hybridization within a time scale of a few minutes. To our knowledge, for the first time, we identify the factors that control the on-surface LNA probe density for maximizing the performance of the LNA sensing layer.

  6. Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

    PubMed Central

    Moreno, Pedro M. D.; Geny, Sylvain; Pabon, Y. Vladimir; Bergquist, Helen; Zaghloul, Eman M.; Rocha, Cristina S. J.; Oprea, Iulian I.; Bestas, Burcu; Andaloussi, Samir EL; Jørgensen, Per T.; Pedersen, Erik B.; Lundin, Karin E.; Zain, Rula; Wengel, Jesper; Smith, C. I. Edvard

    2013-01-01

    In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes. PMID:23345620

  7. X-ray Crystal Structure of a Locked Nucleic Acid (LNA) Duplex Composed of a Palindromic 10-mer DNA Strand Containing One LNA Thymine Monomer

    SciTech Connect

    Egli, M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J.

    2010-03-05

    Locked nucleic acid (LNA), a recently introduced nucleic acid analogue with a bicyclic 2'-O,4'-C-methylene linked furanose sugar, exhibits enhanced affinities for DNA and RNA relative to the corresponding oligodeoxyribonucleotides and oligoribonucleotides; we report the first crystal structure of an LNA unit incorporated in an oligonucleotide duplex. The structure at 1.4 {angstrom} resolution of the DNA-LNA decamer duplex with one LNA thymine monomer per strand provides a detailed view of the conformation and hydration of locked nucleic acid residues in a duplex A-form. Our study provides a first look at the conformational properties of LNA in a crystal structure at relatively high resolution. The main characteristics of the structure are the standard A-type conformation induced by LNA residues and the capacity of the 2'-oxygen that is part of the bicyclic sugar framework to engage in a least two hydrogen bonds to water molecules. Circular dichroism spectra (CD) of LNA-LNA duplexes in solution indicated that such duplexes appear to adopt a conformation that closely resembles the A-form geometry of RNA-RNA duplexes. However, these spectra also manifested subtle differences between the two species (data not shown). The present analysis of a duplex with only a single LNA residue per strand does not provide any insight into potential conformational differences between LNA and RNA duplexes. Attempts to determine a crystal structure of a completely modified LNA-LNA duplex are underway.

  8. Optimization of an AMBER Force Field for the Artificial Nucleic Acid, LNA, and Benchmarking with NMR of L(CAAU)

    PubMed Central

    2013-01-01

    Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced. PMID:24377321

  9. Computational investigation of locked nucleic acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations.

    PubMed

    Poongavanam, Vasanthanathan; Madala, Praveen K; Højland, Torben; Veedu, Rakesh N

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5'-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3'-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  10. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    PubMed Central

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  11. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    PubMed Central

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-01-01

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

  12. A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    PubMed

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  13. Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

    PubMed Central

    Poongavanam, Vasanthanathan; Madala, Praveen K.; Højland, Torben; Veedu, Rakesh N.

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  14. Synthesis of selenomethylene-locked nucleic acid (SeLNA)-modified oligonucleotides by polymerases.

    PubMed

    Wheeler, Megan; Chardon, Antoine; Goubet, Astrid; Morihiro, Kunihiko; Tsan, Sze Yee; Edwards, Stacey L; Kodama, Tetsuya; Obika, Satoshi; Veedu, Rakesh N

    2012-11-18

    Enzymatic recognition of SeLNA nucleotides was investigated. KOD XL DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of SeLNA-modified DNA templates was also efficiently achieved by Phusion and KOD XL DNA polymerases. PMID:23042489

  15. Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe.

    PubMed

    Yoon, Jee-Hyun; Nam, Ji-Sun; Kim, Kyung-Jin; Ro, Young-Tae

    2013-03-01

    Single nucleotide polymorphisms in the codon 306 of embB gene are most frequently reported in ethambutol-resistant Mycobacterium tuberculosis clinical isolates. Here, we report a simple and rapid real-time PCR assay using a locked nucleic acid (LNA)-TaqMan probe for discriminating the embB306 mutations. The use of a 15-bp chimeric LNA/DNA probe led to a relatively higher level of sensitivity and fluorescence signal in the wild-type embB306 ATG codon. Therefore, the mutant alleles were easily distinguishable from the wild-type allele by their distinctive amplification curve shapes without a melting analysis of the PCR product. This system was fast and less than 0.1 pg of genomic DNA per reaction was needed for detection. Because the results from this real-time assay were absolutely consistent with those from DNA sequencing, it can be effectively applied as a simple and rapid method for primary screening of embB306 mutations that occur frequently in ethambutol-resistant and/or multidrug-resistant M. tuberculosis isolates.

  16. Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses.

    PubMed

    Chen, Ru; Gao, Xiao-Bo; Yu, Xiao-Lu; Song, Chang-Xu; Qiu, Yang

    2016-02-01

    A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay. PMID:26615807

  17. Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides.

    PubMed

    Højland, Torben; Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2012-01-01

    We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide. PMID:22679529

  18. Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

    PubMed

    Moschos, Sterghios A; Frick, Manfred; Taylor, Bruce; Turnpenny, Paul; Graves, Helen; Spink, Karen G; Brady, Kevin; Lamb, David; Collins, David; Rockel, Thomas D; Weber, Markus; Lazari, Ovadia; Perez-Tosar, Luis; Fancy, Sally A; Lapthorn, Chris; Green, Martin X; Evans, Steve; Selby, Matthew; Jones, Gareth; Jones, Lyn; Kearney, Sarah; Mechiche, Houria; Gikunju, Diana; Subramanian, Romesh; Uhlmann, Eugen; Jurk, Marion; Vollmer, Jörg; Ciaramella, Giuseppe; Yeadon, Michael

    2011-12-01

    Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

  19. Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

    PubMed Central

    Geny, Sylvain; Moreno, Pedro M. D.; Krzywkowski, Tomasz; Gissberg, Olof; Andersen, Nicolai K.; Isse, Abdirisaq J.; El-Madani, Amro M.; Lou, Chenguang; Pabon, Y. Vladimir; Anderson, Brooke A.; Zaghloul, Eman M.; Zain, Rula; Hrdlicka, Patrick J.; Jørgensen, Per T.; Nilsson, Mats; Lundin, Karin E.; Pedersen, Erik B.; Wengel, Jesper; Smith, C. I. Edvard

    2016-01-01

    Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson–Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson–Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2′-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context. PMID:26857548

  20. Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes.

    PubMed

    Geny, Sylvain; Moreno, Pedro M D; Krzywkowski, Tomasz; Gissberg, Olof; Andersen, Nicolai K; Isse, Abdirisaq J; El-Madani, Amro M; Lou, Chenguang; Pabon, Y Vladimir; Anderson, Brooke A; Zaghloul, Eman M; Zain, Rula; Hrdlicka, Patrick J; Jørgensen, Per T; Nilsson, Mats; Lundin, Karin E; Pedersen, Erik B; Wengel, Jesper; Smith, C I Edvard

    2016-03-18

    Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context. PMID:26857548

  1. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    PubMed

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  2. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  3. Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

    PubMed

    Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

    2008-06-25

    Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays. PMID:18494480

  4. Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

    PubMed

    Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

    2008-06-25

    Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.

  5. Application of Locked Nucleic Acid (LNA) Oligonucleotide–PCR Clamping Technique to Selectively PCR Amplify the SSU rRNA Genes of Bacteria in Investigating the Plant-Associated Community Structures

    PubMed Central

    Ikenaga, Makoto; Sakai, Masao

    2014-01-01

    The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide–PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3′ end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide–PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes. PMID:25030190

  6. Application of Locked Nucleic Acid (LNA) oligonucleotide-PCR clamping technique to selectively PCR amplify the SSU rRNA genes of bacteria in investigating the plant-associated community structures.

    PubMed

    Ikenaga, Makoto; Sakai, Masao

    2014-09-17

    The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide-PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3' end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide-PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes.

  7. Functionalized 2′-Amino-α-L-LNA - Directed Positioning of Intercalators for DNA Targeting

    PubMed Central

    Kumar, T. Santhosh; Madsen, Andreas S.; Østergaard, Michael E.; Sau, Sujay P.; Wengel, Jesper; Hrdlicka, Patrick J.

    2010-01-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (Locked Nucleic Acid) and its diastereomer α-L-LNA are two promising examples hereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small non-aromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases of circular dichroism signals, and molecular modeling studies suggest that pyrene functionalized 2′-amino-α-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety, allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development probes for emerging therapeutic and diagnostic applications focusing on DNA-targeting. PMID:19108636

  8. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    PubMed

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  9. Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents

    PubMed Central

    Torres, Adrian G.; Threlfall, Richard N.

    2011-01-01

    Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics. PMID:22567190

  10. Functional nucleic acid probes and uses thereof

    DOEpatents

    Nilsen-Hamilton, Marit

    2006-10-03

    The present invention provides functional nucleic acid probes, and methods of using functional nucleic acid probes, for binding a target to carry out a desired function. The probes have at least one functional nucleic acid, at least one regulating nucleic acid, and at least one attenuator. The functional nucleic acid is maintained in an inactive state by the attenuator and activated by the regulating nucleic acid only in the presence of a regulating nucleic acid target. In its activated state the functional nucleic acid can bind to its target to carry out a desired function, such as generating a signal, cleaving a nucleic acid, or catalyzing a reaction.

  11. Nucleic acid probes in diagnostic medicine

    NASA Technical Reports Server (NTRS)

    Oberry, Phillip A.

    1991-01-01

    The need for improved diagnostic procedures is outlined and variations in probe technology are briefly reviewed. A discussion of the application of probe technology to the diagnosis of disease in animals and humans is presented. A comparison of probe versus nonprobe diagnostics and isotopic versus nonisotopic probes is made and the current state of sequence amplification is described. The current market status of nucleic acid probes is reviewed with respect to their diagnostic application in human and veterinary medicine. Representative product examples are described and information on probes being developed that offer promise as future products is discussed.

  12. Simultaneous detection and differentiation of human rhino- and enteroviruses in clinical specimens by real-time PCR with locked nucleic Acid probes.

    PubMed

    Osterback, Riikka; Tevaluoto, Tuire; Ylinen, Tiina; Peltola, Ville; Susi, Petri; Hyypiä, Timo; Waris, Matti

    2013-12-01

    Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.

  13. Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.

    PubMed

    Seth, Punit P; Jazayeri, Ali; Yu, Jeff; Allerson, Charles R; Bhat, Balkrishen; Swayze, Eric E

    2012-01-01

    We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012. PMID:23344239

  14. In vitro incorporation of LNA nucleotides.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product. PMID:18058567

  15. An Exocyclic Methylene Group Acts As a Bioisostere of the 2′-Oxygen Atom in LNA

    SciTech Connect

    Seth, Punit P.; Allerson, Charles R.; Berdeja, Andres; Siwkowski, Andrew; Pallan, Pradeep S.; Gaus, Hans; Prakash, Thazha P.; Watt, Andrew T.; Egli, Martin; Swayze, Eric E.

    2010-12-07

    We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2{prime}-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2{prime}-position and a propynyl group at the C-4{prime} position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2{prime}-deoxyribose residue of the 5{prime}-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2{prime}-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.

  16. The LNA VPH characterization experiment

    NASA Astrophysics Data System (ADS)

    Ribeiro, Flávio F.; Katime-Santrich, Orlando J.; Gneiding, Clemens D.; Castilho, Bruno V.; Campos, Rodrigo P.; Nicolau, Rogério A.

    2008-07-01

    The use of Volume Phase Holographic (VPH) gratings in astronomy is increasing worldwide due to its high efficiency, flexibility in manufacturing and lower costs. For example 3 of 4 SOAR Telescope spectrographs are based on VPH gratings. Following the growth in this technology use, tools are needed to characterize these gratings for their physical and diffraction efficiency properties. We developed, at Laboratorio Nacional de Astrofisica / MCT (LNA), Brazil, an assembly for characterization of VPH gratings. The relative efficiency of the gratings can be measured for specific angles or scanned through the grating operation angles. Furthermore surface flatness and mounting stress effects are measured using interferometric techniques. We present the experiment design and characteristics, describe the measurement procedures and show the characterization results for some gratings of the SOAR Telescope spectrograph STELES.

  17. Pharmacokinetics and Pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in Mice and Non-human Primates

    PubMed Central

    Gallo Cantafio, Maria Eugenia; Nielsen, Boye Schnack; Mignogna, Chiara; Arbitrio, Mariamena; Botta, Cirino; Frandsen, Niels M; Rolfo, Christian; Tagliaferri, Pierosandro; Tassone, Pierfrancesco; Di Martino, Maria Teresa

    2016-01-01

    Locked nucleic acid (LNA) oligonucleotides have been successfully used to efficiently inhibit endogenous small noncoding RNAs in vitro and in vivo. We previously demonstrated that the direct miR-221 inhibition by the novel 13-mer LNA-i-miR-221 induces significant antimyeloma activity and upregulates canonical miR-221 targets in vitro and in vivo. To evaluate the LNA-i-miR-221 pharmacokinetics and pharmacodynamics, novel assays for oligonucleotides quantification in NOD.SCID mice and Cynomolgus monkeys (Macaca fascicularis) plasma, urine and tissues were developed. To this aim, a liquid chromatography/mass spectrometry method, after solid-phase extraction, was used for the detection of LNA-i-miR-221 in plasma and urine, while a specific in situ hybridization assay for tissue uptake analysis was designed. Our analysis revealed short half-life, optimal tissue biovailability and minimal urine excretion of LNA-i-miR-221 in mice and monkeys. Up to 3 weeks, LNA-i-miR-221 was still detectable in mice vital organs and in xenografted tumors, together with p27 target upregulation. Importantly, no toxicity in the pilot monkey study was observed. Overall, our findings indicate the suitability of LNA-i-miR-221 for clinical use and we provide here pilot data for safety analysis and further development of LNA-miRNA-based therapeutics for human cancer. PMID:27327137

  18. Arrays of nucleic acid probes on biological chips

    DOEpatents

    Chee, Mark; Cronin, Maureen T.; Fodor, Stephen P. A.; Huang, Xiaohua X.; Hubbell, Earl A.; Lipshutz, Robert J.; Lobban, Peter E.; Morris, MacDonald S.; Sheldon, Edward L.

    1998-11-17

    DNA chips containing arrays of oligonucleotide probes can be used to determine whether a target nucleic acid has a nucleotide sequence identical to or different from a specific reference sequence. The array of probes comprises probes exactly complementary to the reference sequence, as well as probes that differ by one or more bases from the exactly complementary probes.

  19. Vibrational Stark Effect Probes for Nucleic Acids

    PubMed Central

    Silverman, Lisa N.; Pitzer, Michael E.; Ankomah, Peter O.; Boxer, Steven G.; Fenlon, Edward E.

    2008-01-01

    The vibrational Stark effect (VSE) has proven to be an effective method for the study of electric fields in proteins via the use of infrared probes. In order to explore the use of VSE in nucleic acids, the Stark spectroscopy of nine structurally diverse nucleosides was investigated. These nucleosides contained nitrile or azide probes in positions that correspond to both the major and minor grooves of DNA. The nitrile probes showed better characteristics and exhibited absorption frequencies over a broad range; i.e., from 2253 cm−1 for 2′-O-cyanoethyl ribonucleosides 8 and 9 to 2102 cm−1 for a 13C-labeled 5-thiocyanatomethyl-2’-deoxyuridine 3c. The largest Stark tuning rate observed was |Δµ| = 1.1 cm−1/(MV/cm) for both 5-cyano-2′-deoxyuridine 1 and N2-nitrile-2′-deoxyguanosine 7. The latter is a particularly attractive probe because of its high extinction coefficient (ε = 412 M−1cm−1) and ease of incorporation into oligomers. PMID:17877390

  20. Probing protein stability with unnatural amino acids

    SciTech Connect

    Mendel, D.; Ellman, J.A.; Zhiyuh Chang; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G. )

    1992-06-26

    Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (1) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (2) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (3) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

  1. Quantum mechanical studies of DNA and LNA.

    PubMed

    Koch, Troels; Shim, Irene; Lindow, Morten; Ørum, Henrik; Bohr, Henrik G

    2014-04-01

    Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs.

  2. Quantum Mechanical Studies of DNA and LNA

    PubMed Central

    Shim, Irene; Lindow, Morten; Ørum, Henrik

    2014-01-01

    Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs. PMID:24491259

  3. Scaffolding along nucleic acid duplexes using 2'-amino-locked nucleic acids.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2014-06-17

    CONSPECTUS: Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2'-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable

  4. Scaffolding along nucleic acid duplexes using 2'-amino-locked nucleic acids.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2014-06-17

    CONSPECTUS: Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2'-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable

  5. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    SciTech Connect

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  6. Kit for detecting nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2001-01-01

    A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the

  7. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Fontenete, Silvia; Silvia, Fontenete; Barros, Joana; Joana, Barros; Madureira, Pedro; Pedro, Madureira; Figueiredo, Céu; Céu, Figueiredo; Wengel, Jesper; Jesper, Wengel; Azevedo, Nuno Filipe; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

  8. Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

    PubMed

    Dean, Zachary S; Elias, Paul; Jamilpour, Nima; Utzinger, Urs; Wong, Pak Kin

    2016-09-01

    Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

  9. Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes.

    PubMed

    Ahn, Jeong Jin; Kim, Youngjoo; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2016-07-01

    Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

  10. A new general model for predicting melting thermodynamics of complementary and mismatched B-form duplexes containing locked nucleic acids: application to probe design for digital PCR detection of somatic mutations.

    PubMed

    Hughesman, Curtis; Fakhfakh, Kareem; Bidshahri, Roza; Lund, H Louise; Haynes, Charles

    2015-02-17

    Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a

  11. Enzymatic polymerisation involving 2'-amino-LNA nucleotides.

    PubMed

    Johannsen, Marie W; Veedu, Rakesh N; Madsen, Andreas Stahl; Wengel, Jesper

    2012-05-15

    The triphosphate of the thymine derivative of 2'-amino-LNA (2'-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2'-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2'-Amino-LNA-T in the template and 2'-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2'-amino-LNA-T nucleotides. PMID:22503454

  12. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  13. PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

    PubMed

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels

    2012-02-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

  14. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  15. LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

    PubMed

    Darfeuille, Fabien; Hansen, Jens Bo; Orum, Henrik; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2004-01-01

    One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets. PMID:15181175

  16. Monitoring HCV RNA viral load by locked nucleic acid molecular beacons real time PCR.

    PubMed

    Morandi, Luca; Ferrari, Daniela; Lombardo, Claudia; Pession, Annalisa; Tallini, Giovanni

    2007-03-01

    Locked nucleic acids (LNA) based real time PCR was used in particular situations where there are difficulties in primer design due to sequence complexity. In this study a new real time RT-PCR assay was developed using LNA modified primers and LNA molecular beacon probes to monitor hepatitis C virus (HCV) viral load in plasma and serum samples. The technique did not suffer from an heterogeneity of the HCV genome and, in addition, an internal RNA control was amplified in the same reaction tube with different short primers and beacon probe. Due to the short consensus LNA primers length, the PCR efficiency was close to 100% with no formation of hairpin loop structures. In summary a new LNA molecular beacon based real time RT-PCR assay was used successfully to measure quantitatively the total level of HCV RNA in both experimental and clinical specimens. The high sensitivity (50 IU/ml), the wide range of genotype detection, increased specificity and robustness obtained with this test are particularly useful for screening large number of specimens and measuring viral loads to monitor the progress of the disease.

  17. Polymerase-directed synthesis of C5-ethynyl locked nucleic acids.

    PubMed

    Veedu, Rakesh N; Burri, Harsha V; Kumar, Pawan; Sharma, Pawan K; Hrdlicka, Patrick J; Vester, Birte; Wengel, Jesper

    2010-11-15

    Modified nucleic acids have considerable potential in nanobiotechnology for the development of nanomedicines and new materials. Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far and we herein for the first time report the enzymatic incorporation of LNA-U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5'-triphosphate as substrate for RNA transcripts. PMID:20932755

  18. Hybrid molecular probe for nucleic acid analysis in biological samples.

    PubMed

    Yang, Chaoyong James; Martinez, Karen; Lin, Hui; Tan, Weihong

    2006-08-01

    The ability to detect changes in gene expression, especially in real-time and with sensitivity sufficient enough to monitor small variations in a single-cell, will have considerable value in biomedical research and applications. Out of the many available molecular probes for intracellular monitoring of nucleic acids, molecular beacon (MB) is the most frequently used probe with the advantages of high sensitivity and selectivity. However, any processes in which the MB stem-loop structure is broken will result in a restoration of the fluorescence in MB. This brings in a few possibilities for false positive signal such as nuclease degradation, protein binding, thermodynamic fluctuation, solution composition variations (such as pH, salt concentration) and sticky-end pairing. These unwanted processes do exist inside living cells, making nucleic acid monitoring inside living cells difficult. We have designed and synthesized a hybrid molecular probe (HMP) for intracellular nucleic acid monitoring to overcome these problems. HMP has two DNA probes, one labeled with a donor and the other an acceptor. The two DNA probes are linked by a poly(ethylene glycol) (PEG) linker, with each DNA being complementary to adjacent areas of a target sequence. Target binding event brings the donor and acceptor in proximity, resulting in quenching of the donor fluorescence and enhancement of the acceptor emission. The newly designed HMP has high sensitivity, selectivity, and fast hybridization kinetics. The probe is easy to design and synthesize. HMP does not generate any false positive signal upon digestion by nuclease, binding by proteins, forming complexes by sticky-end pairing, or by other molecular interaction processes. HMP is capable of selectively detecting nucleic acid targets from cellular samples.

  19. PEI-complexed LNA antiseeds as miRNA inhibitors

    PubMed Central

    Thomas, Maren; Lange-Grünweller, Kerstin; Dayyoub, Eyas; Bakowsky, Udo; Weirauch, Ulrike; Aigner, Achim; Hartmann, Roland K.; Grünweller, Arnold

    2012-01-01

    Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17–5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3′-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications. PMID:22894918

  20. Designer nucleic acids to probe and program the cell.

    PubMed

    Krishnan, Yamuna; Bathe, Mark

    2012-12-01

    Recent advances in nucleic acid sequencing, structural, and computational technologies have resulted in dramatic progress in our understanding of nucleic acid structure and function in the cell. This knowledge, together with the predictable base-pairing of nucleic acids and powerful synthesis and expression capabilities now offers the unique ability to program nucleic acids to form precise 3D architectures with diverse applications in synthetic and cell biology. The unique modularity of structural motifs that include aptamers, DNAzymes, and ribozymes, together with their well-defined construction rules, enables the synthesis of functional higher-order nucleic acid complexes from these subcomponents. As we illustrate here, these highly programmable, smart complexes are increasingly enabling researchers to probe and program the cell in a sophisticated manner that moves well beyond the use of nucleic acids for conventional genetic manipulation alone.

  1. Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology.

    PubMed

    Diaz, Mara R; Jacobson, James W; Goodwin, Kelly D; Dunbar, Sherry A; Fell, Jack W

    2010-06-01

    Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

  2. Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

    PubMed

    Dean, Zachary S; Elias, Paul; Jamilpour, Nima; Utzinger, Urs; Wong, Pak Kin

    2016-09-01

    Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future. PMID:27529634

  3. Luminescent Probes for Ultrasensitive Detection of Nucleic Acids

    PubMed Central

    Krasnoperov, Lev N.; Marras, Salvatore A.E.; Kozlov, Maxim; Wirpsza, Laura; Mustaev, Arkady

    2010-01-01

    Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of complementary DNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications. PMID:20085336

  4. Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology

    PubMed Central

    Diaz, Mara R.; Jacobson, James W.; Goodwin, Kelly D.; Dunbar, Sherry A.; Fell, Jack W.

    2010-01-01

    Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid–modified oligonucleotides (LNA) and Mirus Label IT® nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5′ terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions. PMID:21165155

  5. Interplay of LNA and 2'-O-methyl RNA in the structure and thermodynamics of RNA hybrid systems: a molecular dynamics study using the revised AMBER force field and comparison with experimental results.

    PubMed

    Yildirim, Ilyas; Kierzek, Elzbieta; Kierzek, Ryszard; Schatz, George C

    2014-12-11

    When used in nucleic acid duplexes, locked nucleic acid (LNA) and 2'-O-methyl RNA residues enhance the duplex stabilities, and this makes it possible to create much better RNA aptamers to target specific molecules in cells. Thus, LNA and 2'-O-methyl RNA residues are finding increasingly widespread use in RNA-based therapeutics. Herein, we utilize molecular dynamics (MD) simulations and UV melting experiments to investigate the structural and thermodynamic properties of 13 nucleic acid duplexes, including full DNA, RNA, LNA, and 2'-O-methyl RNA duplexes as well as hybrid systems such as LNA:RNA, 2'-O-methyl RNA:RNA, LNA/2'-O-methyl RNA:RNA, and RNA/2'-O-methyl RNA:RNA duplexes. The MD simulations are based on a version of the Amber force field revised specifically for RNA and LNA residues. Our results indicate that LNA and 2'-O-methyl RNA residues have two different hybridization mechanisms when included in hybrid duplexes with RNA wherein the former underwinds while the latter overwinds the duplexes. These computational predictions are supported by X-ray structures of LNA and 2'-O-methyl RNA duplexes that were recently presented by different groups, and there is also good agreement with the measured thermal stabilities of the duplexes. We find out that the "underwinding" phenomenon seen in LNA and LNA:RNA hybrid duplexes happens due to expansion of the major groove widths (Mgw) of the duplexes that is associated with decrease in the slide and twist values in base-pair steps. In contrast, 2'-O-methyl RNA residues in RNA duplexes slightly overwind the duplexes while the backbone is forced to stay in C3'-endo. Moreover, base-pair stacking in the LNA and LNA:RNA hybrid systems is gradually reduced with the inclusion of LNA residues in the duplexes while no such effect is seen in the 2'-O-methyl RNA systems. Our results show how competition between base stacking and structural rigidity in these RNA hybrid systems influences structures and stabilities. Even though both

  6. Synthesis of kojic acid derivatives as secondary binding site probes of D-amino acid oxidase

    PubMed Central

    Raje, Mithun; Hin, Niyada; Duvall, Bridget; Ferraris, Dana V.; Berry, James F.; Thomas, Ajit G.; Alt, Jesse; Rojas, Camilo; Slusher, Barbara S.; Tsukamoto, Takashi

    2013-01-01

    A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and tested for their ability to inhibit D-amino acid oxidase (DAAO). Various substituents were incorporated into kojic acid at its 2-hydroxymethyl group. These analogs serve as useful molecular probes to explore the secondary binding site, which can be exploited in designing more potent DAAO inhibitors. PMID:23683589

  7. Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system.

    PubMed

    Yin, Huanshun; Zhou, Yunlei; Zhang, Haixia; Meng, Xiaomeng; Ai, Shiyun

    2012-03-15

    MicroRNAs (miRNAs), a kind of small, endogenous, noncoding RNAs (∼22 nucleotides), might play a crucial role in early cancer diagnose due to its abnormal expression in many solid tumors. As a result, label-free and PCR-amplification-free assay for miRNAs is of great significance. In this work, a highly sensitive biosensor for sequence specific miRNA-21 detection without miRNA-21 labeling and enrichment was constructed based on the substrate electrode of dendritic gold nanostructure (DenAu) and graphene nanosheets modified glassy carbon electrode. Sulfydryl functionalized locked nucleic acid (LNA) integrated hairpin molecule beacon (MB) probe was used as miRNA-21 capture probe. After hybridized with miRNA-21 and reported DNA loading in gold nanoparticles (AuNPs) and biotin multi-functionalized bio bar codes, streptavidin-HRP was brought to the electrode through the specific interaction with biotin to catalyze the chemical oxidation of hydroquinone by H(2)O(2) to form benzoquinone. The electrochemical reduction signal of benzoquinone was utilized to monitor the miRNA-21 hybridization event. The effect of experimental variables on the amperometric response was investigated and optimized. Based on the specific confirmation of probe and signal amplification, the biosensor showed excellent selectivity and high sensitivity with low detection limit of 0.06 pM. Successful attempts are made in miRNA-21 expression analysis of human hepatocarcinoma BEL-7402 cells and normal human hepatic L02 cells.

  8. The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA.

    PubMed

    Dong, Haifeng; Ding, Lin; Yan, Feng; Ji, Hanxu; Ju, Huangxian

    2011-05-01

    A simple nanocarrier of polyethylenimine-grafted graphene nanoribbon (PEI-g-GNR) was proposed as an effective gene vector. The GNR was formed by longitudinally unzipping multiwalled carbon nanotubes (MWCNTs), and treated with strong acids and sonication to obtain surface carboxylic acid groups for graft of PEI via electrostatic assembly. The PEI-g-GNR appeared to protect locked nucleic acid modified molecular beacon (LNA-m-MB) probes from nuclease digestion or single-strand binding protein interaction, thus could be used as a nanocarrier of the probes for more efficient transfection of cells than PEI or PEI-g-MWCNTs due to the large surface area of the GNR and high charge density of PEI. The cytotoxicity and apoptosis induced by the PEI-g-GNR were negligible under optimal transfection conditions. Combining with the remarkable affinity and specificity of LNA to microRNA (miRNA), a delivery system by the LNA-m-MB/PEI-g-GNR was proposed for effectively transferring LNA-m-MB into the cells to recognize the target miRNA. Using HeLa cells as model, a method for detection of miRNA in single cell was developed. These results suggested that PEI-g-GNR would be a promising nonviral vector for in situ detection of gene in cytoplasm and gene therapy in clinical application.

  9. Pyrene excimer signaling molecular beacons for probing nucleic acids.

    PubMed

    Conlon, Patrick; Yang, Chaoyong James; Wu, Yanrong; Chen, Yan; Martinez, Karen; Kim, Youngmi; Stevens, Nathan; Marti, Angel A; Jockusch, Steffen; Turro, Nicholas J; Tan, Weihong

    2008-01-01

    Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.

  10. Pyrene Excimer Signaling Molecular Beacons for Probing Nucleic Acids

    PubMed Central

    Conlon, Patrick; Yang, Chaoyong James; Wu, Yanrong; Chen, Yan; Martinez, Karen; Kim, Youngmi; Stevens, Nathan; Marti, Angel A.; Jockusch, Steffen

    2008-01-01

    Molecular beacon DNA probes, containing one to four pyrene monomers on the 5′ end and the quencher DABCYL on the 3′ end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a sub-nanomolar limit of detection in buffer, while time-resolved signaling enabled low-nanomolar target detection in cell growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5′ terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime (~40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes. In addition, this excimer complex serves as an efficient FRET donor for red-emitting fluorophores, such as TMR, for further extending the Stokes shift of the fluorescent complex. PMID:18078339

  11. Oligonucleotide-conjugated thiazole orange probes as "light-up" probes for messenger ribonucleic acid molecules in living cells.

    PubMed

    Privat, E; Melvin, T; Asseline, U; Vigny, P

    2001-10-01

    "Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

  12. Dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the marine finfish Larimichthys crocea: dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the large yellow croaker.

    PubMed

    Zuo, Rantao; Mai, Kangsen; Xu, Wei; Turchini, Giovanni M; Ai, Qinghui

    2015-02-01

    Whilst aquaculture feed is increasingly formulated with the inclusion of plant oils replacing fish oil, and increasing research effort has been invested in understanding the metabolic effects of reduced dietary n-3 long chain poly unsaturated fatty acids (n-3 LC-PUFA), relatively little information is available on the potential direct metabolic roles of dietary alpha-linolenic acid (ALA, 18:3n-3) and alpha-linolenic acid/linoleic acid (LNA, 18:2n-6) ratio in cultured marine finfish species. In this study, four plant oil based diets, with varying ALA/LNA ratio (0.0, 0.5, 1.0 and 1.5) were fed to juvenile large yellow croakers (Larimichthys crocea) and compared to a fish oil-based control diet (CD) to evaluate the resulting effects on growth, nonspecific immunity, anti-oxidant capacity and related gene expression. High dietary LNA negatively impacted fish growth performance, nonspecific immunity and antioxidant capacity, but growth and immunity were maintained to levels comparable to CD by increasing the ratio of dietary ALA/LNA. The over-expression of genes associated with inflammation (cyclooxygenase-2 and interleukin-1β) and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in croakers fed high concentrations of LNA were reduced to levels comparable to those fed CD by increasing dietary ALA/LNA. This study showed that dietary ALA, by increasing the overall n-3/n-6 PUFA ratio, exerts direct anti-inflammatory and antioxidant effects, similar to those exerted by dietary n-3 LC-PUFA.

  13. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-L-LNA Adenine Monomers: High-affinity Targeting of Single-Stranded DNA

    PubMed Central

    Andersen, Nicolai K.; Anderson, Brooke A.; Wengel, Jesper

    2014-01-01

    Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications. PMID:24304240

  14. Crystallization and preliminary X-ray diffraction data of an LNA 7-mer duplex derived from a ricin aptamer

    PubMed Central

    Förster, Charlotte; Oberthuer, Dominik; Gao, Jiang; Eichert, André; Quast, Frederick G.; Betzel, Christian; Nitsche, Andreas; Erdmann, Volker A.; Fürste, Jens P.

    2009-01-01

    Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as β-d-2′-O,4′-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The β-d-2′-O,4′-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability. A 7-­mer helix derived from the terminal part of an aptamer that was targeted against ricin was chosen. The ricin aptamer originally consisted of natural RNA building blocks and showed high affinity in ricin binding. For future stabilization of the aptamer, the terminal helix has been constructed as an ‘all-locked’ LNA and was successfully crystallized in order to investigate its structural properties. Optimization of crystal growth succeeded by the use of different metal salts as additives, such as CuCl2, MgCl2, MnCl2, CaCl2, CoCl2 and ZnSO4. Preliminary X-ray diffraction data were collected and processed to 2.8 Å resolution. The LNA crystallized in space group P65, with unit-cell parameters a = 50.11, b = 50.11, c = 40.72 Å. The crystals contained one LNA helix per asymmetric unit with a Matthews coefficient of 3.17 Å3 Da−1, which implies a solvent content of 70.15%. PMID:19724123

  15. Method for producing labeled single-stranded nucleic acid probes

    DOEpatents

    Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

    1999-10-19

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

  16. Probing the Specificity Determinants of Amino Acid Recognition by Arginase

    SciTech Connect

    Shishova, E.; Di Costanzo, L; Emig, F; Ash, D; Christianson, D

    2009-01-01

    Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the ?-amino and ?-carboxylate groups) of substrate l-arginine and inhibitors in the active site of arginase I. Specifically, we focus on (1) a water-mediated hydrogen bond between the substrate ?-carboxylate and T135, (2) a direct hydrogen bond between the substrate ?-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate ?-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased KM values but have less pronounced effects on catalytic function as reflected by minimal variations of kcat. As with substrate KM values, inhibitor Kd values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis.

  17. Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.

    2003-12-09

    A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

  18. Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.

    2002-01-01

    A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

  19. The effect of linoleic acid on the whole body synthesis rates of polyunsaturated fatty acids from α-linolenic acid and linoleic acid in free-living rats.

    PubMed

    Domenichiello, Anthony F; Kitson, Alex P; Chen, Chuck T; Trépanier, Marc-Olivier; Stavro, P Mark; Bazinet, Richard P

    2016-04-01

    Docosahexaenoic acid (DHA) is thought to be important for brain function. The main dietary source of DHA is fish, however, DHA can also be synthesized from precursor omega-3 polyunsaturated fatty acids (n-3 PUFA), the most abundantly consumed being α-linolenic acid (ALA). The enzymes required to synthesize DHA from ALA are also used to synthesize longer chain omega-6 (n-6) PUFA from linoleic acid (LNA). The large increase in LNA consumption that has occurred over the last century has led to concern that LNA and other n-6 PUFA outcompete n-3 PUFA for enzymes involved in DHA synthesis, and therefore, decrease overall DHA synthesis. To assess this, rats were fed diets containing LNA at 53 (high LNA diet), 11 (medium LNA diet) or 1.5% (low LNA diet) of the fatty acids with ALA being constant across all diets (approximately 4% of the fatty acids). Rats were maintained on these diets from weaning for 8 weeks, at which point they were subjected to a steady-state infusion of labeled ALA and LNA to measure DHA and arachidonic acid (ARA) synthesis rates. DHA and ARA synthesis rates were generally highest in rats fed the medium and high LNA diets, while the plasma half-life of DHA was longer in rats fed the low LNA diet. Therefore, increasing dietary LNA, in rats, did not impair DHA synthesis; however, low dietary LNA led to a decrease in DHA synthesis with tissue concentrations of DHA possibly being maintained by a longer DHA half-life.

  20. Method for replicating an array of nucleic acid probes

    DOEpatents

    Cantor, Charles R.; Przetakiewicz, Marek; Smith, Cassandra L.; Sano, Takeshi

    1998-01-01

    The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

  1. Method for replicating an array of nucleic acid probes

    DOEpatents

    Cantor, C.R.; Przetakiewicz, M.; Smith, C.L.; Sano, T.

    1998-08-18

    The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5{prime}- and/or 3{prime}-overhangs. 16 figs.

  2. Novel applications of locked nucleic acids.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    Locked Nucleic Acid (LNA) nucleoside triphosphates were prepared and their substrate properties for different polymerases during primer extension and PCR experiments investigated. Phusion High Fidelity DNA polymerase and 9( degrees )Nm(TM) DNA polymerase readily accept LNA nucleoside 5'-triphosphates as substrates in primer extension assays. However, in PCR assays, However, in PCR assays, DNA 9oN(m) polymerase proved to be the best for amplification employing the LNA-A nucleotide. PMID:18029570

  3. Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution.

    PubMed

    Svanvik, N; Westman, G; Wang, D; Kubista, M

    2000-05-15

    We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

  4. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes.

    PubMed

    Junager, Nina P L; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. PMID:27472344

  5. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

    PubMed Central

    Junager, Nina P. L.; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. PMID:27472344

  6. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes.

    PubMed

    Junager, Nina P L; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells.

  7. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1997-01-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

  8. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1997-04-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

  9. Energetic aspects of locked nucleic acids quadruplex association and dissociation.

    PubMed

    Petraccone, Luigi; Erra, Eva; Randazzo, Antonio; Giancola, Concetta

    2006-12-15

    The design of modified nucleic acid aptamers is improved by considering thermodynamics and kinetics of their association/dissociation processes. Locked Nucleic Acids (LNA) is a promising class of nucleic acid analogs. In this work the thermodynamic and kinetic properties of a LNA quadruplex formed by the TGGGT sequence, containing only conformationally restricted LNA residues, are reported and compared to those of 2'-OMe-RNA (O-RNA) and DNA quadruplexes. The thermodynamic analysis indicates that the sugar-modified quadruplexes (LNA and O-RNA) are stabilized by entropic effects. The kinetic analysis shows that LNA and O-RNA quadruplexes are characterized by a slower dissociation and a faster association with respect to DNA quadruplex. Interestingly, the LNA quadruplex formation process shows a second-order kinetics with respect to single strand concentration and has a negative activation energy. To explain these data, a mechanism for tetramer formation with two intermediate states was proposed.

  10. Fluorescence probe for the convenient and sensitive detection of ascorbic acid.

    PubMed

    Matsuoka, Yuta; Yamato, Mayumi; Yamada, Ken-Ichi

    2016-01-01

    Ascorbic acid is an important antioxidant that plays an essential role in the biosynthesis of numerous bioactive substances. The detection of ascorbic acid has traditionally been achieved using high-performance liquid chromatography and absorption spectrophotometry assays. However, the development of fluorescence probes for this purpose is highly desired because they provide a much more convenient and highly sensitive technique for the detection of this material. OFF-ON-type fluorescent probes have been developed for the detection of non-fluorescent compounds. Photo-induced electron transfer and fluorescence resonance energy transfer are the two main fluorescence quenching mechanisms for the detection of ascorbic acid, and several fluorescence probes have been reported based on redox-responsive metals and quantum dots. Profluorescent nitroxide compounds have also been developed as non-metal organic fluorescence probes for ascorbic acid. These nitroxide systems have a stable unpaired electron and can therefore react with ascorbic acid and a strong fluorescence quencher. Furthermore, recent synthetic advances have allowed for the synthesis of α-substituted nitroxides with varying levels of reactivity towards ascorbic acid. In this review, we have discussed the design strategies used for the preparation of fluorescent probes for ascorbic acid, with particular emphasis on profluorescent nitroxides, which are unique radical-based redox-active fluorescent probes.

  11. Fluorescence probe for the convenient and sensitive detection of ascorbic acid

    PubMed Central

    Matsuoka, Yuta; Yamato, Mayumi; Yamada, Ken-ichi

    2016-01-01

    Ascorbic acid is an important antioxidant that plays an essential role in the biosynthesis of numerous bioactive substances. The detection of ascorbic acid has traditionally been achieved using high-performance liquid chromatography and absorption spectrophotometry assays. However, the development of fluorescence probes for this purpose is highly desired because they provide a much more convenient and highly sensitive technique for the detection of this material. OFF-ON-type fluorescent probes have been developed for the detection of non-fluorescent compounds. Photo-induced electron transfer and fluorescence resonance energy transfer are the two main fluorescence quenching mechanisms for the detection of ascorbic acid, and several fluorescence probes have been reported based on redox-responsive metals and quantum dots. Profluorescent nitroxide compounds have also been developed as non-metal organic fluorescence probes for ascorbic acid. These nitroxide systems have a stable unpaired electron and can therefore react with ascorbic acid and a strong fluorescence quencher. Furthermore, recent synthetic advances have allowed for the synthesis of α-substituted nitroxides with varying levels of reactivity towards ascorbic acid. In this review, we have discussed the design strategies used for the preparation of fluorescent probes for ascorbic acid, with particular emphasis on profluorescent nitroxides, which are unique radical-based redox-active fluorescent probes. PMID:26798193

  12. A phosphomolybdic acid anion probe-based label-free, stable and simple electrochemical biosensing platform.

    PubMed

    Wei, Tianxiang; Chen, Yuyun; Tu, Wenwen; Lan, Yaqian; Dai, Zhihui

    2014-08-25

    A versatile label-free, stable, low-cost and simple electrochemical biosensing platform has been developed based on a phosphomolybdic acid anion probe by jointly taking advantages of its native electronegativity, electrochemical activity and chemisorption with graphene oxide.

  13. Probe kit for identifying a base in a nucleic acid

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2001-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  14. Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2000-01-01

    A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

  15. Synthesis of nucleic acid probes on membrane supports: a procedure for the removal of unincorporated precursors.

    PubMed

    Bhat, S P

    1990-01-01

    We have used DNA bound to small pieces of nylon membrane for the synthesis of radioactive probes. The DNA to be used for generating the probe(s) is first bound to nylon membranes and then introduced into the reaction mix. The labeling reaction takes place on the membrane and therefore allows easy removal of unincorporated precursors by simple washing for 1-2 min. The clean labeled probe is eluted from the membrane in formamide or in water and is ready for use. This DNA-membrane can be stored for reuse. Synthesis of probes on a solid support such as nylon membrane thus circumvents problems associated with chromatographic manipulations needed for the separation of labeled DNA from unicorporated precursors. Probes synthesized in this manner are as efficient in detecting nucleic acid sequences as those synthesized in solution. PMID:2321760

  16. Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

    2006-10-03

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  17. Acid properties of solid acid catalysts characterized by solid-state 31P NMR of adsorbed phosphorous probe molecules.

    PubMed

    Zheng, Anmin; Huang, Shing-Jong; Liu, Shang-Bin; Deng, Feng

    2011-09-01

    A brief review is presented on acidity characterization of solid acid catalysts by means of solid-state phosphor-31 magic-angle-spinning nuclear magnetic resonance ((31)P MAS NMR) spectroscopy using phosphor-containing molecules as probes. It is emphasized that such a simple approach using (31)P MAS NMR of adsorbed phosphorous probe molecules, namely trimethylphosphine (TMP) and trialkylphosphine oxides (R(3)PO), represents a unique technique in providing detailed qualitative and quantitative features, viz. type, strength, distribution, and concentration of acid sites in solid acid catalysts. In particular, it will be shown that when applied with a proper choice of probe molecules with varied sizes and results obtained from elemental analysis, the amounts and locations (intracrystalline vs. extracrystalline) of different types (Brønsted vs. Lewis) of acid sites may be determined. In addition, by incorporating the NMR results with that obtained from theoretical density functional theory (DFT) calculations, correlations between the (31)P chemical shifts (δ(31)P) and acidic strengths of Brønsted and Lewis acid sites may also be derived, facilitating a suitable acidity scale for solid acid catalysts.

  18. Terbium fluorescence as a sensitive, inexpensive probe for UV-induced damage in nucleic acids.

    PubMed

    El-Yazbi, Amira F; Loppnow, Glen R

    2013-07-01

    Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb(3+)). Single-stranded oligonucleotides greatly enhance the Tb(3+) emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb(3+)/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb(3+), producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb(3+)/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb(3+)/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36±1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.

  19. Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids.

    PubMed

    Nguyen, Camha; Grimes, Jeffrey; Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2011-11-11

    Hybridization probes are often inefficient in the analysis of single-stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real-time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T(m) > 80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure-folded analytes, whereas a MB-based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.

  20. A single molecular beacon probe is sufficient for the analysis of multiple nucleic acid sequences.

    PubMed

    Gerasimova, Yulia V; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M

    2010-08-16

    Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping.

  1. Design and synthesis of an activity-based protein profiling probe derived from cinnamic hydroxamic acid.

    PubMed

    Ai, Teng; Qiu, Li; Xie, Jiashu; Geraghty, Robert J; Chen, Liqiang

    2016-02-15

    In our continued effort to discover new anti-hepatitis C virus (HCV) agents, we validated the anti-replicon activity of compound 1, a potent and selective anti-HCV hydroxamic acid recently reported by us. Generally favorable physicochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties exhibited by 1 made it an ideal parent compound from which activity-based protein profiling (ABPP) probe 3 was designed and synthesized. Evaluation of probe 3 revealed that it possessed necessary anti-HCV activity and selectivity. Therefore, we have successfully obtained compound 3 as a suitable ABPP probe to identify potential molecular targets of compound 1. Probe 3 and its improved analogs are expected to join a growing list of ABPP probes that have made important contributions to not only the studies of biochemical and cellular functions but also discovery of selective inhibitors of protein targets.

  2. Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

    PubMed Central

    Song, Young K.; Evangelista, Jennifer; Aschenbach, Konrad; Johansson, Peter; Wen, Xinyu; Chen, Qingrong; Lee, Albert; Hempel, Heidi; Gheeya, Jinesh S.; Getty, Stephanie; Gomez, Romel; Khan, Javed

    2013-01-01

    Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes. PMID:23593123

  3. Signal-on electrochemical Y or junction probe detection of nucleic acid.

    PubMed

    Shen, Zuliang; Nakayama, Shizuka; Semancik, Steve; Sintim, Herman O

    2012-08-01

    A methylene-blue (MB)-labeled molecular beacon junction probe allows for a signal-on electrochemical detection of nucleic acids via target recycling using endonucleases. Electron transfer is reduced when the MB is intercalated in the stem of the molecular beacon, but then electron transfer from MB to a gold electrode is enhanced upon cleavage of the junction probe due to increased probability of MB approaching the electrode when attached to the more flexible ssDNA.

  4. NMR structure of a kissing complex formed between the TAR RNA element of HIV-1 and a LNA-modified aptamer

    PubMed Central

    Lebars, Isabelle; Richard, Tristan; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2007-01-01

    The trans-activating responsive (TAR) RNA element located in the 5′ untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605–1614]. These aptamers display a 5′-GUCCCAGA-3′ consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR–aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2′-O,4′-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR–LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G•A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson–Crick base pairing with a loop–loop conformation close to an A-type. PMID:17768146

  5. Acidity characterization of heterogeneous catalysts by solid-state NMR spectroscopy using probe molecules.

    PubMed

    Zheng, Anmin; Liu, Shang-Bin; Deng, Feng

    2013-01-01

    Characterization of the surface acidic properties of solid acid catalysts is a key issue in heterogeneous catalysis. Important acid features of solid acids, such as their type (Brønsted vs. Lewis acid), distribution and accessibility (internal vs. external sites), concentration (amount), and strength of acid sites are crucial factors dictating their reactivity and selectivity. This short review provides information on different solid-state NMR techniques used for acidity characterization of solid acid catalysts. In particular, different approaches using probe molecules containing a specific nucleus of interest, such as pyridine-d5, 2-(13)C-acetone, trimethylphosphine, and trimethylphosphine oxide, are compared. Incorporation of valuable information (such as the adsorption structure, deprotonation energy, and NMR parameters) from density functional theory (DFT) calculations can yield explicit correlations between the chemical shift of adsorbed probe molecules and the intrinsic acid strength of solid acids. Methods that combine experimental NMR data with DFT calculations can therefore provide both qualitative and quantitative information on acid sites.

  6. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

    PubMed Central

    van Ravesteyn, Thomas W.; Dekker, Marleen; Fish, Alexander; Sixma, Titia K.; Wolters, Astrid; Dekker, Rob J.; te Riele, Hein P. J.

    2016-01-01

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype. PMID:26951689

  7. Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids.

    PubMed

    Beld, Joris; Abbriano, Raffaela; Finzel, Kara; Hildebrand, Mark; Burkart, Michael D

    2016-04-01

    In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes. We fed various odd and short chain fatty acids to the bacterium Escherichia coli, cyanobacterium Synechocystis sp. PCC 6803, green microalga Chlamydomonas reinhardtii and diatom Thalassiosira pseudonana. Major differences were observed, specifically in the ability among species to incorporate and elongate short chain fatty acids. We demonstrate that E. coli, C. reinhardtii, and T. pseudonana can produce longer fatty acid products from short chain precursors (C3 and C5), while Synechocystis sp. PCC 6803 lacks this ability. However, Synechocystis can incorporate and elongate longer chain fatty acids due to acyl-acyl carrier protein synthetase (AasS) activity, and knockout of this protein eliminates the ability to incorporate these fatty acids. In addition, expression of a characterized AasS from Vibrio harveyii confers a similar capability to E. coli. The ability to desaturate exogenously added fatty acids was only observed in Synechocystis and C. reinhardtii. We further probed fatty acid metabolism of these organisms by feeding desaturase inhibitors to test the specificity of long-chain fatty acid desaturases. In particular, supplementation with thia fatty acids can alter fatty acid profiles based on the location of the sulfur in the chain. We show that coupling sensitive gas chromatography mass spectrometry to supplementation of unnatural fatty acids can reveal major differences between fatty acid metabolism in various organisms. Often unnatural fatty acids have antibacterial or even therapeutic properties. Feeding of short

  8. Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids.

    PubMed

    Beld, Joris; Abbriano, Raffaela; Finzel, Kara; Hildebrand, Mark; Burkart, Michael D

    2016-04-01

    In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes. We fed various odd and short chain fatty acids to the bacterium Escherichia coli, cyanobacterium Synechocystis sp. PCC 6803, green microalga Chlamydomonas reinhardtii and diatom Thalassiosira pseudonana. Major differences were observed, specifically in the ability among species to incorporate and elongate short chain fatty acids. We demonstrate that E. coli, C. reinhardtii, and T. pseudonana can produce longer fatty acid products from short chain precursors (C3 and C5), while Synechocystis sp. PCC 6803 lacks this ability. However, Synechocystis can incorporate and elongate longer chain fatty acids due to acyl-acyl carrier protein synthetase (AasS) activity, and knockout of this protein eliminates the ability to incorporate these fatty acids. In addition, expression of a characterized AasS from Vibrio harveyii confers a similar capability to E. coli. The ability to desaturate exogenously added fatty acids was only observed in Synechocystis and C. reinhardtii. We further probed fatty acid metabolism of these organisms by feeding desaturase inhibitors to test the specificity of long-chain fatty acid desaturases. In particular, supplementation with thia fatty acids can alter fatty acid profiles based on the location of the sulfur in the chain. We show that coupling sensitive gas chromatography mass spectrometry to supplementation of unnatural fatty acids can reveal major differences between fatty acid metabolism in various organisms. Often unnatural fatty acids have antibacterial or even therapeutic properties. Feeding of short

  9. LNA with wide range of gain control and wideband interference rejection

    NASA Astrophysics Data System (ADS)

    Wang, Jhen-Ji; Chen, Duan-Yu

    2016-10-01

    This work presents a low-noise amplifier (LNA) design with a wide-range gain control characteristic that integrates adjustable current distribution and output impedance techniques. For a given gain characteristic, the proposed LNA provides better wideband interference rejection performance than conventional LNA. Moreover, the proposed LNA also has a wider gain control range than conventional LNA. Therefore, it is suitable for satellite communications systems. The simulation results demonstrate that the voltage gain control range is between 14.5 and 34.2 dB for such applications (2600 MHz); the input reflection coefficient is less than -18.9 dB; the noise figure (NF) is 1.25 dB; and the third-order intercept point (IIP3) is 4.52 dBm. The proposed LNA consumes 23.85-28.17 mW at a supply voltage of 1.8 V. It is implemented by using TSMC 0.18-um RF CMOS process technology.

  10. Role of the heat capacity change in understanding and modeling melting thermodynamics of complementary duplexes containing standard and nucleobase-modified LNA.

    PubMed

    Hughesman, Curtis B; Turner, Robin F B; Haynes, Charles A

    2011-06-14

    Melting thermodynamic data obtained by differential scanning calorimetry (DSC) are reported for 43 duplexed oligonucleotides containing one or more locked nucleic acid (LNA) substitutions. The measured heat capacity change (ΔC(p)) for the helix-to-coil transition is used to compute the changes in enthalpy and entropy for melting of an LNA-bearing duplex at the T(m) of its corresponding isosequential unmodified DNA duplex to allow rigorous thermodynamic analysis of the stability enhancements provided by LNA substitutions. Contrary to previous studies, our analysis shows that the origin of the improved stability is almost exclusively a net reduction (ΔΔS° < 0) in the entropy gain accompanying the helix-to-coil transition, with the magnitude of the reduction dependent on the type of nucleobase and its base pairing properties. This knowledge and our average measured value for ΔC(p) of 42 ± 11 cal mol(-1) K(-1) bp(-1) are then used to derive a new model that accurately predicts melting thermodynamics and the increased melting temperature (ΔT(m)) of heteroduplexes formed between an unmodified DNA strand and a complementary strand containing any number and configuration of standard LNA nucleotides A, T, C, and G. This single-base thermodynamic (SBT) model requires only four entropy-related parameters in addition to ΔC(p). Finally, DSC data for 20 duplexes containing the nucleobase-modified LNAs 2-aminoadenine (D) and 2-thiothymine (H) are reported and used to determine SBT model parameters for D and H. The data and model suggest that along with the greater stability enhancement provided by D and H bases relative to their corresponding A and T analogues, the unique pseudocomplementary properties of D-H base pairs may make their use appealing for in vitro and in vivo applications.

  11. Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

    PubMed Central

    Kim, Hyun-Joong

    2014-01-01

    Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM. PMID:25428160

  12. Intra-albumin migration of bound fatty acid probed by spin label ESR

    SciTech Connect

    Gurachevsky, Andrey . E-mail: a.gurachevsky@medinnovation.de; Shimanovitch, Ekaterina; Gurachevskaya, Tatjana; Muravsky, Vladimir

    2007-09-07

    Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains.

  13. Signal turn-on probe for nucleic acid detection based on (19)F nuclear magnetic resonance.

    PubMed

    Sakamoto, Takashi; Shimizu, Yu-ki; Sasaki, Jun; Hayakawa, Hikaru; Fujimoto, Kenzo

    2011-01-01

    To image gene expression in vivo, we designed and synthesized a novel signal turn-on probe for (19)F nuclear magnetic resonance (MR) imaging based on paramagnetic relaxation enhancement. The stem-loop structured oligodeoxyribonucleotide (ODN) having a molecular beacon sequence for point mutated K-ras mRNA was doubly labeled with bis(trifluoromethyl)benzene moiety and Gd-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelate moiety at the each termini of the ODN probe, respectively. We found that the (19)F MR signal of the bis(trifluoromethyl)benzene moiety tethered at the 5' termini of the probe turned on by the addition of complementary ODN. The probe has the potential to image gene expressions in vivo.

  14. Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation.

    PubMed

    Igarashi, Miki; DeMar, James C; Ma, Kaizong; Chang, Lisa; Bell, Jane M; Rapoport, Stanley I

    2007-05-01

    Rates of conversion of alpha-linolenic acid (alpha-LNA, 18:3n-3) to docosahexaenoic acid (DHA, 22:6n-3) by the mammalian brain and the brain's ability to upregulate these rates during dietary deprivation of n-3 polyunsaturated fatty acids (PUFAs) are unknown. To answer these questions, we measured conversion coefficients and rates in post-weaning rats fed an n-3 PUFA deficient (0.2% alpha-LNA of total fatty acids, no DHA) or adequate (4.6% alpha-LNA, no DHA) diet for 15 weeks. Unanesthetized rats in each group were infused intravenously with [1-(14)C]alpha-LNA, and their arterial plasma and microwaved brains collected at 5 minutes were analyzed. The deficient compared with adequate diet reduced brain DHA by 37% and increased brain arachidonic (20:4n-6) and docosapentaenoic (22:5n-6) acids. Only 1% of plasma [1-(14)C]alpha-LNA entering brain was converted to DHA with the adequate diet, and conversion coefficients of alpha-LNA to DHA were unchanged by the deficient diet. In summary, the brain's ability to synthesize DHA from alpha-LNA is very low and is not altered by n-3 PUFA deprivation. Because the liver's reported ability is much higher, and can be upregulated by the deficient diet, DHA converted by the liver from circulating alphaLNA is the source of the brain's DHA when DHA is not in the diet.

  15. Probing the Binding Site of Bile Acids in TGR5.

    PubMed

    Macchiarulo, Antonio; Gioiello, Antimo; Thomas, Charles; Pols, Thijs W H; Nuti, Roberto; Ferrari, Cristina; Giacchè, Nicola; De Franco, Francesca; Pruzanski, Mark; Auwerx, Johan; Schoonjans, Kristina; Pellicciari, Roberto

    2013-12-12

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure-activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

  16. Probing the Binding Site of Bile Acids in TGR5

    PubMed Central

    2013-01-01

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure–activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

  17. [Study on recovery and its influencing factors of ferulic acid and tetramethylpyrazine in cerebral microdialysis probe].

    PubMed

    Liao, Wei-guo; Wang, Li-sheng; Fan, Wen-tao; Li, Zhou; Yu, Jian-ye; Liao, Feng-yun; Wu, Yin-ai; Ba, Wen-qiang; Wang, Ding

    2015-11-01

    To establish a method for detecting microdialysis recovery of tetramethylpyrazine (TMP) and ferulic acid (FA) and investigating the influencing factors, providing the basis for further in vivo microdialysis experiments. The concentration of FA and TMP in dialysates were determined by high pressure liquid chromatography ( HPLC) and probe recovery were calculated respectively. The influence of the flow rates, medium concentration, temperature and in vivo probe stability on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). The recovery obtained by incremental method were similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5 μL min(-1), and increased obviously with the temperature of 25-42 degrees C under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method was good. The recovery of brain microdialysis probes in vivo 8 h maintained a relatively stable, but certain differences existed between different brain microdialysis probes, demonstrating that each probe was required for recovery correction in vivo experiment. Microdialysis sampling can be used for the local brain pharmacokinetic study of FA and TMP, and retrodialysis method can be used in probe recovery of FA and TMP in vivo. PMID:27071270

  18. Ratiometric emission fluorescent pH probe for imaging of living cells in extreme acidity.

    PubMed

    Niu, Weifen; Fan, Li; Nan, Ming; Li, Zengbo; Lu, Dongtao; Wong, Man Shing; Shuang, Shaomin; Dong, Chuan

    2015-03-01

    A novel ratiometric emission fluorescent probe, 1,1-dimethyl-2-[2-(quinolin-4-yl)vinyl]-1H-benzo[e]indole (QVBI), is facilely synthesized via ethylene bridging of benzoindole and quinoline. The probe exhibits ratiometric fluorescence emission (F(522nm)/F(630nm)) characteristics with pKa 3.27 and linear response to extreme-acidity range of 3.8-2.0. Also, its high fluorescence quantum yield (Φ = 0.89) and large Stokes shift (110 nm) are favorable. Moreover, QVBI possesses highly selective response to H(+) over metal ions and some bioactive molecules, good photostability, and excellent reversibility. The probe has excellent cell membrane permeability and is further applied successfully to monitor pH fluctuations in live cells and imaging extreme acidity in Escherichia coli cells without influence of autofluorescence and native cellular species in biological systems. PMID:25664606

  19. Bioavailability of xenobiotics in unsaturated soils – implications for nucleic acid based stable isotope probing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of stable isotopes to label phylogenetically informative biomolecules (phospholipid fatty acids, DNA, or RNA), typically referred to as stable isotope probing (SIP) has the potential of providing definitive evidence that a detected population is active in a specific process, if that process ...

  20. Discovery of boronic acid-based fluorescent probes targeting amyloid-beta plaques in Alzheimer's disease.

    PubMed

    Jung, Seung-Jin; Lee, Jun Young; Kim, Tae Ho; Lee, Dong-Eun; Jeon, Jongho; Yang, Seung Dae; Hur, Min Goo; Min, Jung-Joon; Park, Yong Dae

    2016-04-01

    A boronic acid-based fluorescent probe was developed for diagnosis of amyloid-β (Aβ) plaques from Alzheimer's disease (AD). Probe 4c, which included boronic acid as a functional group, exhibited a significant increase (64.37-fold, FAβ/F0) in fluorescence intensity as a response to Aβ aggregates, with a blue shift (105nm) in the maximum emission wavelength. We found that boronic acid as a functional group improved the binding affinity (KD value=0.79±0.05μM for 4c) for Aβ aggregates and confirmed that 4c selectively stained Aβ plaques in brain sections from APP/PS1 mice. Ex vivo fluorescence imaging using mice (normal and APP/PS1) also revealed that 4c was able to penetrate the blood-brain barrier (BBB) and to stain Aβ plaques in the brain. From these results, we believe that 4c will be useful as a fluorescent probe in preclinical research related to AD. Furthermore, we believe that our results with boronic acid also provide valuable information for the development of a probe for Aβ plaques. PMID:26927427

  1. Ion trap collision-induced dissociation of locked nucleic acids.

    PubMed

    Huang, Teng-yi; Kharlamova, Anastasia; McLuckey, Scott A

    2010-01-01

    Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3'C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.

  2. Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

    PubMed Central

    Qin, Peter Z; Haworth, Ian S; Cai, Qi; Kusnetzow, Ana K; Grant, Gian Paola G; Price, Eric A; Sowa, Glenna Z; Popova, Anna; Herreros, Bruno; He, Honghang

    2008-01-01

    This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron–electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes. PMID:17947978

  3. Probing the structure of nucleic acids with Ni(II) complexes

    SciTech Connect

    Chen, Xiaoying.

    1992-01-01

    The structure of nucleic acids determines their biological function. Interest in the development of novel probes from structures of nucleic acid has led to the discovery of conformation-specific oxidation of guanine sites in DNA and RNA using Ni(II) complexes. The reaction is highly dependent upon the nature of Ni(II) complexes with the most important feature of a strong in-plane ligand field. The unique properties of Ni(II) complexes combining redox and coordination features provide sensitive probes for nucleic acid conformation. One of these nickel complexes, NiCR, has been shown to selectively promote cleavage of DNA at guanine sites held accessible through the formation of unusual secondary structures such as ends, mismatches, bulges and loops. An unique mechanism for the base and conformation-specific oxidation of DNA promoted by Ni(II) complexes is proposed, involving direct ligation of nickel to N-7 of guanine delivering a non-diffusible oxidizing species. NiCR has been proved to be a sensitive and predictable probe for the tertiary structure of RNAs. The specific sites of oxidation of tRNS[sup phe] promoted by NiCR correspond to the most accessible guanine residues determined by theoretic calculations. NiCR has also been successfully applied to probe the tertiary structure of self-splicing Tetrahymena pre-rRNA intron, and has provided important information about the folding of this intron, especially in the region of the catalytic core.

  4. Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

    PubMed

    Kim, Youngmi; Yang, Chaoyong James; Tan, Weihong

    2007-01-01

    Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2'-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

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

    PubMed

    Nåbo, Lina J; Madsen, Charlotte S; Jensen, Knud J; Kongsted, Jacob; Astakhova, Kira

    2015-05-26

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations.

  6. Forced intercalation probes (FIT Probes): thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection.

    PubMed

    Köhler, Olaf; Jarikote, Dilip Venkatrao; Seitz, Oliver

    2005-01-01

    Fluorescent base analogues in DNA are versatile probes of nucleic acid-nucleic acid and nucleic acid-protein interactions. New peptide nucleic acid (PNA) based probes are described in which the intercalator dye thiazole orange (TO) serves as a base surrogate. The investigation of six TO derivatives revealed that the linker length and the conjugation site decided whether a base surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. One TO derivative conferred universal PNA-DNA base pairing while maintaining duplex stability and hybridization selectivity. TO fluorescence increased up to 26-fold upon hybridization. In contrast to most other probes, in which fluorescence is invariant once hybridization had occurred, the emission of TO-containing PNA probes is attenuated when forced to intercalate next to a mismatched base pair. The specificity of DNA detection is therefore not limited by the selectivity of probe-target binding and a DNA target can be distinguished from its single-base mutant under nonstringent hybridization conditions. This property should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.

  7. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  8. Docosahexaenoic acid conjugated near infrared flourescence probe for in vivo early tumor diagnosis

    NASA Astrophysics Data System (ADS)

    Li, Siwen; Cao, Jie; Qin, Jingyi; Zhang, Xin; Achilefu, Samuel; Qian, Zhiyu; Gu, Yueqing

    2013-02-01

    Docosahexaenoic acid(DHA) is an omega-3 C22 natural fatty acid with six cis double bonds and as a constituent of membranes used as a precursor for metabolic and biochemical path ways. In this manuscript,we describe the synthesis of near-infrared(NIR) flourescence ICG-Der-01 labeled DHA for in vitro and vivo tumor targeting.The structure of the probe was intensively characterized by UV and MS. The in vitro and vivo tumor targeting abilities of the DHA-based NIR probes were investigeted in MCF-7 cells and MCF-7 xenograft mice model differently by confocal microscopy and CCD camera. The cell cytotoxicity were tested in tumor cells MCF-7 .The results shows that the DHA-based NIR probes have high affinity with the tumor both in vitro and vivo.In addition ,we also found that the DHA-based NIR probes have the apparent cytotoxicity on MCF-7 cells .which demonstrated that DHA was conjugated with other antitumor drug could increase the abilities of antirumor efficacy .So DHA-ICG-Der-01 is a promising optical agent for diagnosis of tumors especially in their early stage.

  9. A Locked Nucleic Acid Probe Based on Selective Salt-Induced Effect Detects Single Nucleotide Polymorphisms

    PubMed Central

    Zhang, Jing; Wu, Huizhe; Chen, Qiuchen; Zhao, Pengfei; Zhao, Haishan; Yao, Weifan; Wei, Minjie

    2015-01-01

    Detection of single based genetic mutation by using oligonucleotide probes is one of the common methods of detecting single nucleotide polymorphisms at known loci. In this paper, we demonstrated a hybridization system which included a buffer solution that produced selective salt-induced effect and a locked nucleic acid modified 12 nt oligonucleotide probe. The hybridization system is suitable for hybridization under room temperature. By using magnetic nanoparticles as carriers for PCR products, the SNPs (MDR1 C3435T/A) from 45 volunteers were analyzed, and the results were consistent with the results from pyrophosphoric acid sequencing. The method presented in this paper differs from the traditional method of using molecular beacons to detect SNPs in that it is suitable for research institutions lacking real-time quantitative PCR detecting systems, to detect PCR products at room temperature. PMID:26347880

  10. Visible sensing of nucleic acid sequences using a genetically encodable unmodified mRNA probe.

    PubMed

    Narita, Atsushi; Ogawa, Kazumasa; Sando, Shinsuke; Aoyama, Yasuhiro

    2006-01-01

    We previously reported a molecular beacon-mRNA (MB-mRNA) strategy for nucleic acid detection/sensing in a cell-free translation system using unmodified RNA as a probe. Here in this presentation, we report that a combination with RNase H activity, which induces an additional process of irreversible cleavage of MB-domain, achieves an improved sequence selectivity (one nucleotide selectivity) and an enhanced sensitivity. This improved system finally enabled visible sensing of target nucleic acid sequence at a single nucleotide resolution under isothermal conditions.

  11. Locked nucleic acid molecular beacons.

    PubMed

    Wang, Lin; Yang, Chaoyong James; Medley, Colin D; Benner, Steven A; Tan, Weihong

    2005-11-16

    A novel LNA-MB (molecular beacon based on locked nucleic acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.

  12. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated. PMID:26695270

  13. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

  14. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, Heinz-Ulrich G.; Gray, Joe W.

    1995-01-01

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

  15. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, H.U.G.; Gray, J.W.

    1995-06-27

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers and probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity. 18 figs.

  16. Fluorescence imaging of siRNA delivery by peptide nucleic acid-based probe.

    PubMed

    Sato, Takaya; Sato, Yusuke; Iwai, Kenta; Kuge, Shusuke; Teramae, Norio; Nishizawa, Seiichi

    2015-01-01

    We report on the use of a peptide nucleic acid (PNA)-based fluorescent probe for the analysis of siRNA delivery to living cells. The probe, Py-AA-TO, possesses thiazole orange (TO) and pyrene moieties in the C- and N-termini of PNA, and can function as a light-up probe capable of selective binding to 3'-overhanging nucleotides of target siRNAs. The affinity-labeling of the siRNAs with Py-AA-TO facilitates fluorescence imaging of cellular uptake of polymer-based carriers encapsulating the siRNAs (polyplexes) through endocytosis and subsequent sequestration into lysosome. In addition, flow cytometric measurements reveal that the monitoring of Py-AA-TO fluorescence inside the cells is successfully applicable to the analysis of the polyplex disassembly. These promising functions of Py-AA-TO are presented and discussed as a basis for the design of molecular probes for fluorescent imaging and quantitative analysis of the siRNA delivery process. PMID:25864675

  17. High-affinity DNA-targeting using Readily Accessible Mimics of N2′-Functionalized 2′-Amino-α-L-LNA

    PubMed Central

    Karmakar, Saswata; Anderson, Brooke A.; Rathje, Rie L.; Andersen, Sanne; Jensen, Troels B.; Nielsen, Poul; Hrdlicka, Patrick J.

    2011-01-01

    N2′-Pyrene-functionalized 2′-amino-α-L-LNAs (Locked Nucleic Acids) display extraordinary affinity toward complementary DNA targets due to favorable preorganization of the pyrene moieties for hybridization-induced intercalation. Unfortunately, the synthesis of these monomers is challenging (~20 steps, <3% overall yield), which has precluded full characterization of DNA-targeting applications based on these materials. Access to more readily accessible functional mimics would be highly desirable. Here we describe short synthetic routes toward a series of O2′-intercalator-functionalized uridine and N2′-intercalator-functionalized 2′-N-methyl-2′-aminouridine monomers and demonstrate – via thermal denaturation, UV-visible absorption and fluorescence spectroscopy experiments – that several of them mimic the DNA-hybridization properties of N2′-pyrene-functionalized 2′-amino-α-L-LNAs. For example, oligodeoxyribonucleotides (ONs) modified with 2′-O-(coronen-1-yl)methyluridine monomer Z, 2′-O-(pyren-1-yl)methyluridine monomer Y or 2′-N-(pyren-1-ylmethyl)-2′-N-methylaminouridine monomer Q, display prominent increases in thermal affinity toward complementary DNA relative to reference strands (average ΔTm/mod up to +12 °C), pronounced DNA-selectivity, and higher target specificity than 2′-amino-β-L-LNA benchmark probes. In contrast, ONs modified with 2′-O-(2-napthyl)uridine monomer W, 2′-O-(pyren-1-yl)uridine monomer X or 2′-N-(pyren-1-ylcarbonyl)-2′-N-methylaminouridine monomer S display very low affinity toward DNA targets. This demonstrates that even conservative alterations in linker chemistry, linker length and surface area of the appended intercalators have marked impact on DNA-hybridization characteristics. Straightforward access to high-affinity building blocks such as Q/Y/Z is likely to accelerate their use in DNA-targeting applications within nucleic acid based diagnostics, therapeutics, and material science. PMID:21827174

  18. Probing the structural dynamics of proteins and nucleic acids with optical tweezers.

    PubMed

    Ritchie, Dustin B; Woodside, Michael T

    2015-10-01

    Conformational changes are an essential feature of most molecular processes in biology. Optical tweezers have emerged as a powerful tool for probing conformational dynamics at the single-molecule level because of their high resolution and sensitivity, opening new windows on phenomena ranging from folding and ligand binding to enzyme function, molecular machines, and protein aggregation. By measuring conformational changes induced in a molecule by forces applied by optical tweezers, new insight has been gained into the relationship between dynamics and function. We discuss recent advances from studies of how structure forms in proteins and RNA, including non-native structures, fluctuations in disordered proteins, and interactions with chaperones assisting native folding. We also review the development of assays probing the dynamics of complex protein-nucleic acid and protein-protein assemblies that reveal the dynamic interactions between biomolecular machines and their substrates.

  19. Ascorbic acid-functionalized Ag NPs as a probe for colorimetric sensing of glutathione

    NASA Astrophysics Data System (ADS)

    D'souza, Stephanie L.; Pati, Ranjan; Kailasa, Suresh Kumar

    2015-08-01

    In this work, we report the use of ascorbic acid-capped silver nanoparticles (AA-Ag NPs) as a probe for selective colorimetric detection of glutathione (GSH) in aqueous solution. This detection system was based on the GSH-induced aggregation of AA-Ag NPs, resulting in drastic changes in the absorption spectra and color of the AA-Ag NPs system. The GSH-induced AA-Ag NPs aggregation was confirmed by UV-visible spectrometry, dynamic light scattering (DLS) and transmission electron microscopic (TEM) techniques. Under optimal conditions, this method exhibited good linearity over the concentration ranges from 5.0 to 50 µM, with the limit of detection 2.4 × 10-7 M. This method was successfully applied to detect GSH in the presence of other biomolecules, which confirms that this probe can be used for the detection of GSH in real samples.

  20. Development of a Radioiodinated Triazolopyrimidine Probe for Nuclear Medical Imaging of Fatty Acid Binding Protein 4

    PubMed Central

    Onoe, Satoru; Sampei, Sotaro; Kimura, Ikuo; Ono, Masahiro; Saji, Hideo

    2014-01-01

    Fatty acid binding protein 4 (FABP4) is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki = 44.5±9.8 nM, Kd = 69.1±12.3 nM). The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection). The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging. PMID:24732569

  1. Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics.

    PubMed

    Asanuma, Daisuke; Takaoka, Yousuke; Namiki, Shigeyuki; Takikawa, Kenji; Kamiya, Mako; Nagano, Tetsuo; Urano, Yasuteru; Hirose, Kenzo

    2014-06-10

    Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

  2. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration.

  3. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-03-24

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

  4. Folic acid-conjugated europium complexes as luminescent probes for selective targeting of cancer cells.

    PubMed

    Quici, Silvio; Casoni, Alessandro; Foschi, Francesca; Armelao, Lidia; Bottaro, Gregorio; Seraglia, Roberta; Bolzati, Cristina; Salvarese, Nicola; Carpanese, Debora; Rosato, Antonio

    2015-02-26

    We report the synthesis of three optical probes (Eu(3+)⊂1, Eu(3+)⊂2, and Eu(3+)⊂3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu(3+)⊂1 and Eu(3+)⊂2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu(3+)⊂2 > Eu(3+)⊂1 > Eu(3+)⊂3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu(3+)⊂2 and FA or Eu(3+)⊂2 versus (3)H-FA, indicate that internalization is receptor-mediated and that Eu(3+)⊂2 shows high selectivity and specificity for FR.

  5. Translational and rotational diffusion of flexible PEG and rigid dendrimer probes in sodium caseinate dispersions and acid gels.

    PubMed

    Salami, Souad; Rondeau-Mouro, Corinne; Barhoum, Myriam; van Duynhoven, John; Mariette, François

    2014-09-01

    The dynamics of rigid dendrimer and flexible PEG probes in sodium caseinate dispersions and acid gels, including both translational diffusion and rotational diffusion, were studied by NMR. Above the onset of the close-packing limit (C ∼ 10 g/100 g H2 O), translational diffusion of the probe depended on its flexibility and on the fluctuations of the matrix chains. The PEG probe diffused more rapidly than the spherical dendrimer probe of corresponding hydrodynamic radius. The greater conformational flexibility of PEG facilitated its motion through the crowded casein matrix. Rotational diffusion was, however, substantially less hindered than the translational diffusion and depended on the local protein-probe friction which became high when the casein concentration increased. The coagulation of the matrix led to the formation of large voids, which resulted in an increase in the translational diffusion of the probes, whereas the rotational diffusion of the probes was retarded in the gel, which could be attributed to the immobilized environment surrounding the probe. Quantitative information from PFG-NMR and SEM micrographs have been combined for characterizing microstructural details in SC acid gels.

  6. Boronic Acid: A Bio-Inspired Strategy To Increase the Sensitivity and Selectivity of Fluorescent NADH Probe.

    PubMed

    Wang, Lu; Zhang, Jingye; Kim, Beomsue; Peng, Juanjuan; Berry, Stuart N; Ni, Yong; Su, Dongdong; Lee, Jungyeol; Yuan, Lin; Chang, Young-Tae

    2016-08-24

    Fluorescent probes have emerged as an essential tool in the molecular recognition events in biological systems; however, due to the complex structures of certain biomolecules, it remains a challenge to design small-molecule fluorescent probes with high sensitivity and selectivity. Inspired by the enzyme-catalyzed reaction between biomolecule and probe, we present a novel combination-reaction two-step sensing strategy to improve sensitivity and selectivity. Based on this strategy, we successfully prepared a turn-on fluorescent reduced nicotinamide adenine dinucleotide (NADH) probe, in which boronic acid was introduced to bind with NADH and subsequently accelerate the sensing process. This probe shows remarkably improved sensitivity (detection limit: 0.084 μM) and selectivity to NADH in the absence of any enzymes. In order to improve the practicality, the boronic acid was further modified to change the measurement conditions from alkalescent (pH 9.5) to physiological environment (pH 7.4). Utilizing these probes, we not only accurately quantified the NADH weight in a health care product but also evaluated intracellular NADH levels in live cell imaging. Thus, these bio-inspired fluorescent probes offer excellent tools for elucidating the roles of NADH in biological systems as well as a practical strategy to develop future sensitive and selective probes for complicated biomolecules. PMID:27500425

  7. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe

    PubMed Central

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V.

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  8. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

    PubMed

    Liu, Jie; Hao, Zhenyu; Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river. PMID:27082986

  9. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River

    PubMed Central

    Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river. PMID:27082986

  10. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

    PubMed

    Liu, Jie; Hao, Zhenyu; Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.

  11. An aldehyde group-based P-acid probe for selective fluorescence turn-on sensing of cysteine and homocysteine.

    PubMed

    Yang, Chunlei; Wang, Xiu; Shen, Lei; Deng, Wenping; Liu, Haiyun; Ge, Shenguang; Yan, Mei; Song, Xianrang

    2016-06-15

    A highly sensitive and selective turn on fluorescent probe P-acid-aldehyde (P-CHO) is developed for the determination of cysteine (Cys) and homocysteine (Hcy). The probe is designed and synthesized by incorporating the specific functional group aldehyde group for thiols into a stable π-conjugated material 4,4'-(2,5-dimethoxy-1,4-phenylene) bis(ethyne-2,1-diyl) dibenzoic acid (P-acid). The probe fluorescence is quenched through donor photoinduced electron transfer (d-PET) between the fluorophore (P-acid) and the recognition group (aldehyde group). In the presence of thiols, Cys and Hcy can selectively react with aldehyde group of the probe because the inhibition of d-PET between fluorophore and recognition group. Therefore, a turn-on fluorescent sensor was established for the fluorescence recovery. Under the optimized conditions, the fluorescence response of probe is directly proportional to the concentration of Cys in the range of 4-95 NM L(-1), with a detection limit 3.0 nM. In addition, the sensing system exhibits good selectively toward Cys and Hcy in the presence of other amino acids. It has been successfully applied for bioimaging of Cys and Hcy in living cells with low cell toxicity.

  12. Probe design rules and effective enzymes for endonuclease-based detection of nucleic acids.

    PubMed

    Yan, Lei; Nakayama, Shizuka; Sintim, Herman O

    2013-10-15

    Junction probe (JP) platform is an isothermal endonuclease-based detection assay for both RNA and DNA. Herein, we screen 31 REAse and identify effective restriction endonucleases that can be used for JP detection. Secondly, we investigate how different probe architectures affect JP cleavage rates and conclude that although molecular beacon (MB) JP probes give less background noise than linear JP probes, the cleavage of MB JP probes are slower than linear JP probes.

  13. Nd:LNA laser optical pumping of He-4 - Application to space magnetometers

    NASA Astrophysics Data System (ADS)

    Slocum, R. E.; Schearer, L. D.; Tin, P.; Marquedant, R.

    1988-12-01

    Results obtained from laser pumping in a helium magnetometer sensor, using a tunable Nd:LNA laser pumped with a high-power diode laser, are reported. It is shown that it was possible to observe both the Hanle signals and the n = 0, p = 1 parametric resonance by monitoring the pumping radiation passing through the cell. As the diode laser-pumped Nd:LNA laser was tuned through the D0, D1, and D2 transitions, three distinct resonance signals were produced. A comparison of the slope of lamp-pumped signals and laser-pumped D1 signals showed that, under otherwise identical conditions, the slope of the D1 laser signal was 45 times greater than the lamp-pumped signal.

  14. Exploring the Hybridization Thermodynamics of Spherical Nucleic Acids to Tailor Probes for Diagnostic and Therapeutic Applications

    NASA Astrophysics Data System (ADS)

    Randeria, Pratik Shailesh

    Spherical nucleic acids (SNAs), three-dimensional nanoparticle conjugates composed of densely packed and highly oriented oligonucleotides around organic or inorganic nanoparticles, are an emergent class of nanostructures that show promise as single-entity agents for intracellular messenger RNA (mRNA) detection and gene regulation. SNAs exhibit superior biocompatibility and biological properties compared to linear oligonucleotides, enabling them to overcome many of the limitations of linear oligonucleotides for use in biomedical applications. However, the origins of these biologically attractive properties are not well understood. In this dissertation, the chemistry underlying one such property is studied in detail, and the findings are applied towards the rational design of more effective SNAs for diagnostic and therapeutic applications. Chapter 1 introduces the synthesis of SNAs, the unique properties that make them superior to linear nucleic acids for biomedicine, and previously studied applications of these structures. Chapter 2 focuses on quantitatively studying the impact of the chemical structure of the SNA on its ability to hybridize multiple complementary nucleic acids. This chapter lays the groundwork for understanding the factors that govern SNA hybridization thermodynamics and how to tailor SNAs to increase their binding affinity to target mRNA strands. Chapters 3 and 4 capitalize on this knowledge to engineer probes for intracellular mRNA detection and gene regulation applications. Chapter 3 reports the development of an SNA-based probe that can simultaneously report the expression level of two different mRNA transcripts in live cells and differentiate diseased cells from non-diseased cells. Chapter 4 investigates the use of topically-applied SNAs to down-regulate a critical mediator of impaired wound healing in diabetic mice to accelerate wound closure. This study represents the first topical therapeutic application of SNA nanotechnology to treat open

  15. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    PubMed

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  16. Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.

    PubMed

    Bockstahler, L E; Li, Z; Nguyen, N Y; Van Houten, K A; Brennan, M J; Langone, J J; Morris, S L

    2002-03-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h. PMID:11926172

  17. Neutrophil chemotaxis and arachidonic acid metabolism are not linked: evidence from metal ion probe studies

    SciTech Connect

    Turner, S.R.; Turner, R.A.; Smith, D.M.; Johnson, J.A.

    1986-03-05

    Heavy metal ions can inhibit arachidonic acid (AA) metabolism protect against ionophore cytotoxicity (ibid) and inhibit neutrophil chemotaxis. In this study they used Au/sup 3 +/, Zn/sup 2 +/, Cr/sup 3 +/, Mn/sup 2 +/ and Cu/sup 2 +/ as probes of the interrelationships among AA metabolism, ionophore-mediated cytotoxicity, and chemotaxis. Phospholipid deacylation was measured in ionophore-treated cells prelabeled with /sup 3/H-AA. Eicosanoid release from ionophore-treated cells was monitored by radioimmunoassay. Cytoprotection was quantitated as ability to exclude trypan blue. Chemotaxis toward f-met-leu-phe was measured by leading front analysis. The results imply that metal ions attenuate ionophore cytotoxicity by blocking phospholipid deacylation and eicosanoid release. In contrast to previous reports, no correlation between AA metabolism and chemotaxis was demonstrated, suggesting that these 2 processes are not linked.

  18. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

    NASA Astrophysics Data System (ADS)

    Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

    2002-08-01

    The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, "real-time" DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated.

  19. Probing the Specificity Determinants of Amino Acid Recognition by Arginase†,‡

    PubMed Central

    Shishova, Ekaterina Y.; Di Costanzo, Luigi; Emig, Francis A.; Ash, David E.; Christianson, David W.

    2009-01-01

    Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the α-amino and α-carboxylate groups) of substrate l-arginine and inhibitors in the active site of human arginase I. Specifically, we focus on: (1) a water-mediated hydrogen bond between the substrate α-carboxylate and T135, (2) a direct hydrogen bond between the substrate α-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate α-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased KM values, but have less pronounced effects on catalytic function as reflected by minimal variations of kcat. As with substrate KM values, inhibitor Kd values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is: water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis. PMID:19093830

  20. Extraordinary adhesion of phenylboronic acid derivatives of polyvinylamine to wet cellulose: a colloidal probe microscopy investigation.

    PubMed

    Notley, Shannon M; Chen, Wei; Pelton, Robert

    2009-06-16

    Typically, the adhesion between cellulose surfaces under aqueous conditions is very poor. Often, adsorbed polymers such as polyvinylamine (PVAm) are used to increase the wet strength; however, this provides only a minimal increase in the adhesion energy. Here, the adhesion between cellulose surfaces with adsorbed layers of phenylboronic acid derivatized polyvinylamine has been studied using colloidal probe microscopy as a function of pH. The adhesion due to the phenylboronic acid (PBA) groups grafted on the polyvinylamine backbone is almost 30 times greater, providing a new, exciting class of polymers using covalent linkages to improve the strength of the joint between cellulose surfaces. The measured surface forces on approach provided key information on the molecular conformation of the polymers at the cellulose-solution interface. At low pH, the three polymers tested, PVAm, PVAm-Ph (with pendant phenol groups), and PVAm-PBA (with phenylboronic acid groups) all had a relatively flat conformation at the interface, which is in agreement with the predictions based upon theory for highly charged polyelectrolytes adsorbing to an oppositely charged interface. With increasing pH, the charge on the polymers is reduced, eventually resulting in a more expansive conformation at the interface at pH 10 and above with the development of a steric interaction force. The onset of this steric force correlates well with the observed significant increase in the pull-off force upon separation of the cellulose surfaces. Furthermore, a greater increase in the adhesion was observed for PVAm-PBA in agreement with previous studies using macroscopic cellulose surfaces. This is attributed to the formation of boronic acid esters between the polymer and the cis diol groups on the cellulose surface.

  1. Inhibition of Gastric Tumor Cell Growth Using Seed-targeting LNA as Specific, Long-lasting MicroRNA Inhibitors.

    PubMed

    Staedel, Cathy; Varon, Christine; Nguyen, Phu Hung; Vialet, Brune; Chambonnier, Lucie; Rousseau, Benoît; Soubeyran, Isabelle; Evrard, Serge; Couillaud, Franck; Darfeuille, Fabien

    2015-07-07

    MicroRNAs regulate eukaryotic gene expression upon pairing onto target mRNAs. This targeting is influenced by the complementarity between the microRNA "seed" sequence at its 5' end and the seed-matching sequences in the mRNA. Here, we assess the efficiency and specificity of 8-mer locked nucleic acid (LNA)-modified oligonucleotides raised against the seeds of miR-372 and miR-373, two embryonic stem cell-specific microRNAs prominently expressed in the human gastric adenocarcinoma AGS cell line. Provided that the pairing is perfect over all the eight nucleotides of the seed and starts at nucleotide 2 or 1 at the microRNA 5' end, these short LNAs inhibit miR-372/373 functions and derepress their common target, the cell cycle regulator LATS2. They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations. Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation. Their therapeutic potential is confirmed in fast-growing, miR-372-positive, primary human gastric adenocarcinoma xenografts in mice. Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies.

  2. A highly specific ferrocene-based fluorescent probe for hypochlorous acid and its application to cell imaging.

    PubMed

    Chen, Suming; Lu, Jinxin; Sun, Chengdong; Ma, Huimin

    2010-03-01

    A highly specific ferrocene-based fluorescent probe, (9-anthryl)ethenylferrocene, has been designed, synthesized and characterized for fluorescence imaging of hypochlorous acid (HOCl) in live cells. The design strategy for the probe is based on the strong quenching effect of electron-donor ferrocene on anthracene fluorescence via an intramolecular charge transfer process, and is accomplished through constructing the conjugated molecule by using a cleavable double bond as a linker. The double bond in the probe reacts selectively with HOCl rather than the other reactive oxygen species (e.g., *OH, *O(2)(-), (1)O(2), and H(2)O(2)) in pH 7.4, accompanied by more than 100-fold fluorescence enhancement. Moreover, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for fluorescence imaging of HOCl in HeLa cells.

  3. Real time RNA transcription monitoring by Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes: norovirus detection.

    PubMed

    Tonelli, Alessandro; Tedeschi, Tullia; Germini, Andrea; Sforza, Stefano; Corradini, Roberto; Medici, Maria Cristina; Chezzi, Carlo; Marchelli, Rosangela

    2011-05-01

    Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes have been reported as a valuable strategy for DNA analysis; however, no investigations targeting RNA molecules and no comparisons between different derivatization approaches have been reported so far. In this work, two TO-conjugated PNAs for genogroup II noroviruses (NoV GII) detection were designed and synthesized. Both the probes target the most conserved stretch of nucleotides identified in the open reading frame 1-2 (ORF1-ORF2) junction region and differ for the dye conjugation strategy: one PNA is end-labelled with the TO molecule tethered by a linker; the other probe bears the TO molecule directly linked to the PNA backbone, replacing a conventional nucleobase. The spectroscopic properties of the two PNA probes were studied and their applicability to NoVs detection, using an isothermal assay, was investigated. Both probes showed good specificity and high fluorescence enhancement upon hybridization, especially targeting RNA molecules. Moreover, the two probes were successfully employed for NoVs detection from stool specimens in an isothermal-based amplification assay targeting RNA 'amplicons'. The probes showed to be specific even in the presence of high concentrations of non-target RNA.

  4. A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening.

    PubMed

    Xu, Yongqian; Li, Benhao; Xiao, Liangliang; Ouyang, Jia; Sun, Shiguo; Pang, Yi

    2014-08-14

    A dual-channel including a colorimetric and fluorescent probe based on the aggregation-caused quenching (ACQ) and enzymolysis approach has been presented to screen acid phosphatase (ACP) and its inhibitor. Moreover, the ACP activity was determined by real time assay. PMID:24957006

  5. Probe depth matters in dermal microdialysis sampling of benzoic acid after topical application: an ex vivo study in human skin.

    PubMed

    Holmgaard, R; Benfeldt, E; Bangsgaard, N; Sorensen, J A; Brosen, K; Nielsen, F; Nielsen, J B

    2012-01-01

    Microdialysis (MD) in the skin - dermal microdialysis (DMD) - is a unique technique for sampling of topically as well as systemically administered drugs at the site of action, e.g. sampling of dermatological drug concentrations in the dermis. Debate has concerned the existence of a correlation between the depth of the sampling device - the probe - in the dermis and the amount of drug sampled following topical drug administration. This study evaluates the relation between probe depth and drug sampling using dermal DMD sampling ex vivo in human skin. We used superficial (<1 mm), intermediate (1-2 mm) and deep (>2 mm) positioning of the linear MD probe in the dermis of human abdominal skin, followed by topical application of 4 mg/ml of benzoic acid (BA) in skin chambers overlying the probes. Dialysate was sampled every hour for 12 h and analysed for BA content by high-performance liquid chromatography. Probe depth was measured by 20-MHz ultrasound scanning. The area under the time-versus-concentration curve (AUC) describes the drug exposure in the tissue during the experiment and is a relevant parameter to compare for the 3 dermal probe depths investigated. The AUC(0-12) were: superficial probes: 3,335 ± 1,094 μg·h/ml (mean ± SD); intermediate probes: 2,178 ± 1,068 μg·h/ml, and deep probes: 1,159 ± 306 μg·h/ml. AUC(0-12) sampled by the superficial probes was significantly higher than that of samples from the intermediate and deeply positioned probes (p value <0.05). There was a significant inverse correlation between probe depth and AUC(0-12) sampled by the same probe (p value <0.001, r(2) value = 0.5). The mean extrapolated lag-times (±SD) for the superficial probes were 0.8 ± 0.1 h, for the intermediate probes 1.7 ± 0.5 h, and for the deep probes 2.7 ± 0.5 h, which were all significantly different from each other (p value <0.05). In conclusion, this paper demonstrates that there is an inverse relationship between the depth of the probe in the dermis

  6. Probe depth matters in dermal microdialysis sampling of benzoic acid after topical application: an ex vivo study in human skin.

    PubMed

    Holmgaard, R; Benfeldt, E; Bangsgaard, N; Sorensen, J A; Brosen, K; Nielsen, F; Nielsen, J B

    2012-01-01

    Microdialysis (MD) in the skin - dermal microdialysis (DMD) - is a unique technique for sampling of topically as well as systemically administered drugs at the site of action, e.g. sampling of dermatological drug concentrations in the dermis. Debate has concerned the existence of a correlation between the depth of the sampling device - the probe - in the dermis and the amount of drug sampled following topical drug administration. This study evaluates the relation between probe depth and drug sampling using dermal DMD sampling ex vivo in human skin. We used superficial (<1 mm), intermediate (1-2 mm) and deep (>2 mm) positioning of the linear MD probe in the dermis of human abdominal skin, followed by topical application of 4 mg/ml of benzoic acid (BA) in skin chambers overlying the probes. Dialysate was sampled every hour for 12 h and analysed for BA content by high-performance liquid chromatography. Probe depth was measured by 20-MHz ultrasound scanning. The area under the time-versus-concentration curve (AUC) describes the drug exposure in the tissue during the experiment and is a relevant parameter to compare for the 3 dermal probe depths investigated. The AUC(0-12) were: superficial probes: 3,335 ± 1,094 μg·h/ml (mean ± SD); intermediate probes: 2,178 ± 1,068 μg·h/ml, and deep probes: 1,159 ± 306 μg·h/ml. AUC(0-12) sampled by the superficial probes was significantly higher than that of samples from the intermediate and deeply positioned probes (p value <0.05). There was a significant inverse correlation between probe depth and AUC(0-12) sampled by the same probe (p value <0.001, r(2) value = 0.5). The mean extrapolated lag-times (±SD) for the superficial probes were 0.8 ± 0.1 h, for the intermediate probes 1.7 ± 0.5 h, and for the deep probes 2.7 ± 0.5 h, which were all significantly different from each other (p value <0.05). In conclusion, this paper demonstrates that there is an inverse relationship between the depth of the probe in the dermis

  7. Wavelength-ratiometric near-physiological pH sensors based on 6-aminoquinolinium boronic acid probes.

    PubMed

    Badugu, Ramachandram; Lakowicz, Joseph R; Geddes, Chris D

    2005-04-30

    We describe the pH response of a set of isomeric water-soluble fluorescent probes based on both the 6-aminoquinolinium and boronic acid moieties. These probes show spectral shifts and intensity changes with pH, in a wavelength-ratiometric and colorimetric manner. Subsequently, changes in pH can readily be determined around the physiological level. Although boronic acid containing probes are known to exhibit pH sensitivity along with an ability for saccharide binding/chelating, the new probes reported here are considered to be unique and show an unperturbed pH response, even in the presence of high concentrations of background saccharide, such as with glucose and fructose, allowing for the predominant pH sensitivity. The response of the probes is based on the ability of the boronic acid group to interact with strong bases like OH(-), changing from the neutral form of the boronic acid group, R-B(OH)(2), to the anionic ester, R-B(-)(OH)(3), form, which is an electron donating group. The presence of an electron deficient quaternary heterocyclic nitrogen center and a strong electron donating amino group in the 6-position of the quinolinium backbone, provides for the spectral changes observed upon OH(-) complexation. In addition, by comparing the results obtained with systems separately incorporating 6-methoxy or 6-methyl substituents, the suppressed response towards monosaccharides, such as with glucose and fructose, can clearly be observed for these systems. Finally we compare our results to those of a control compound, BAQ, which does not contain the boronic acid group, allowing a rationale of the spectral changes to be made.

  8. Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

    PubMed Central

    Öhrmalm, Christina; Jobs, Magnus; Eriksson, Ronnie; Golbob, Sultan; Elfaitouri, Amal; Benachenhou, Farid; Strømme, Maria; Blomberg, Jonas

    2010-01-01

    One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes. PMID:20864443

  9. Quantification of syntrophic fatty acid-{beta}-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

    SciTech Connect

    Hansen, K.H.; Ahring, B.K.; Raskin, L.

    1999-11-01

    Small-subunit rRNA sequences were obtained for two saturated fatty acid-{beta}-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S.wolfei LYB was closely related to S.wolfei subsp. solfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-{beta}-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-{beta}-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, or which the majority was accounted for by the genus Syntrophomonas.

  10. Benzimidazole-based ratiometric two-photon fluorescent probes for acidic pH in live cells and tissues.

    PubMed

    Kim, Hyung Joong; Heo, Cheol Ho; Kim, Hwan Myung

    2013-11-27

    Many aspects of cell metabolism are controlled by acidic pH. We report a new family of small molecule and ratiometric two photon (TP) probes derived from benzimidazole (BH1-3 and BH1L) for monitoring acidic pH values. These probes are characterized by a strong two-photon excited fluorescence, a marked blue-to-green emission color change in response to pH, pKa values ranging from 4.9 to 6.1, a distinctive isoemissive point, negligible cytotoxicity, and high photostability, thereby allowing quantitative analysis of acidic pH. Moreover, we show that BH1L optimized as a lysosomal-targeted probe allows for direct, real-time estimation of the pH values inside lysosomal compartments in live cells as well as in living mouse brain tissues through the use of two-photon microscopy. These findings demonstrate that these probes will find useful applications in biomedical research.

  11. Characterization of Aqueous Oleic Acid/Oleate Dispersions by Fluorescent Probes and Raman Spectroscopy.

    PubMed

    Suga, Keishi; Kondo, Dai; Otsuka, Yoko; Okamoto, Yukihiro; Umakoshi, Hiroshi

    2016-08-01

    Oleic acid (OA) and oleates form self-assembled structures dispersible in aqueous media. Herein, the physicochemical properties of OA/oleate assemblies were characterized using fluorescent probes and Raman spectroscopy, under relatively high dilution (<100 mM of total amphiphile) at 25 °C. Anisotropy analysis using 1,6-diphenyl-1,3,5-hexatriene showed that the microviscosity of the OA/oleate assembly was highest at pH 7.5 (the pH range of 6.9-10.6 was investigated). The fluorescence spectra of 6-lauroyl-2-dimethylaminonaphthalene revealed the dehydrated environments on membrane surfaces at pH < 7.7. The pH-dependent Raman peak intensity ratios, chain torsion (S = I1124/I1096) and chain packing (R = I2850/I2930), showed local maxima, indicating the occurrence of metastable phases, such as dispersed cubic phase (pH = 7.5), vesicle (pH = 8.5), and dispersed cylindrical micelle (pH = 9.7). These results suggest that large-scale OA/oleate assemblies could possess particular membrane properties in a narrow pH region, e.g., at pH 7.5, and 9.7.

  12. Two-photon probes for intracellular free metal ions, acidic vesicles, and lipid rafts in live tissues.

    PubMed

    Kim, Hwan Myung; Cho, Bong Rae

    2009-07-21

    Optical imaging with fluorescence microscopy is a vital tool in the study of living systems. The most common method for cell imaging, one-photon microscopy (OPM), uses a single photon of higher energy to excite the fluorophore. However, two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is growing in popularity among biologists because of several distinct advantages. Using TPM, researchers can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts, self-absorption, autofluorescence, photobleaching, and photodamage. However, to make TPM a more versatile tool in biology, researchers need a wider variety of two-photon probes for specific applications. In this Account, we describe a series of two-photon probes that we developed that can visualize the distribution of intracellular metal ions, acidic vesicles, and lipid rafts in living cells and tissues. The development of these probes requires a significant two-photon cross section for the bright image and receptors (sensing moieties) that triggers the emission of the two-photon excited fluorescence upon binding with the ions or membrane in the living system. These probes also must be sensitive to the polarity of the environment to allow selective detection of cytosolic and membrane-bound probes. In addition, they need to be cell-permeable, water-soluble for the staining of cells and tissues, and highly photostable for long-term imaging. The resulting probes-AMg1 (Mg(2+)), ACa1-ACa3 (Ca(2+)), AZn1 and AZn2 (Zn(2+)), AH1, AH2, and AL1 (acidic vesicles), and CL2 (membrane)-use 2-acetyl-6-aminonaphthalene as the fluorophore and receptors for the target ions or membrane. All of these two-photon turn-on probes can detect the intracellular free metal ions, acidic vesicles, and lipid rafts at 100-300 microm depth in live tissues. Moreover, with ACa1-AM, we could simultaneously visualize the spontaneous Ca(2+) waves in the somas of

  13. Acid-Activatable Michael-Type Fluorescent Probes for Thiols and for Labeling Lysosomes in Live Cells.

    PubMed

    Dai, Chun-Guang; Du, Xiao-Jiao; Song, Qin-Hua

    2015-12-18

    A Michael addition is usually taken as a base-catalyzed reaction. Most fluorescent probes have been designed to detect thiols in slightly alkaline solutions (pH 7-9). The sensing reactions of almost all Michael-type fluorescent probes for thiols are faster in a high pH solution than in a low pH solution. In this work, we synthesized a series of 7-substituted 2-(quinolin-2-ylmethylene)malonic acids (QMAs, substituents: NEt2, OH, H, Cl, or NO2) and their ethyl esters (QMEs) as Michael-type fluorescent probes for thiols. The sensing reactions of QMAs and QMEs occur in distinct pH ranges, pH < 7 for QMAs and pH > 7 for QMEs. On the basis of experimental and theoretic studies, we have clarified the distinct pH effects on the sensing reactivity between QMAs and QMEs and demonstrated that two QMAs (NEt2, OH) are highly sensitive and selective fluorescent probes for thiols in acidic solutions (pH < 7) and promising dyes that can label lysosomes in live cells.

  14. A Differential CMOS Common-Gate LNA Linearized by Cross-Coupled Post Distortion Technique

    NASA Astrophysics Data System (ADS)

    Guo, Benqing; Yang, Guomin; Bin, Xiexian

    2014-05-01

    A linearized differential common-gate CMOS low noise amplifier is proposed. The linearity is improved by a cross-coupled post distortion technique, employing auxiliary PMOS transistors in weak inversion region to cancel the third-order nonlinear currents of common-gate LNA and impair the second-order nonlinear currents of that. The negative conductance characteristic of cross-coupled auxiliary PMOS transistors improves the gain while the resulted NF is little affected. Furthermore, noise contribution and linearity deterioration from the cascode stage is eliminated by an inductor resonating with the parasitic capacitance observed at the source net of the cascode transistor. The LNA implemented in a 0.18 μm CMOS technology demonstrates that IIP3 and gain have about 8.2 dB and 1.4 dB improvements in the designed frequency band, respectively. The noise figure of 3.4 dB is obtained with a power dissipation of 6.8 mW under a 1.8 V power supply.

  15. EGFR Mutation Analysis of Circulating Tumor DNA Using an Improved PNA-LNA PCR Clamp Method

    PubMed Central

    Watanabe, Kana; Fukuhara, Tatsuro; Tsukita, Yoko; Morita, Mami; Suzuki, Aya; Tanaka, Nobuyuki; Terasaki, Hiroshi; Nukiwa, Toshihiro

    2016-01-01

    Introduction. Rebiopsies have become more crucial in non-small cell lung cancer (NSCLC). Instead of invasive biopsies, development of collecting biological data of the tumor from blood samples is expected. We conducted a prospective study to assess the feasibility of detection of epidermal growth factor receptor (EGFR) mutation in plasma samples. Method. NSCLC patients harboring EGFR activating mutations, who were going to receive EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatment, were enrolled in this study. Plasma EGFR activating mutations and the T790M resistance mutation were analyzed by an improved PNA-LNA PCR clamp method, characterized by a 10-fold or more sensitivity compared with the original methods. Result. Six patients with wild-type EGFR and 24 patients with EGFR mutations were enrolled in this study. Pretreatment plasma samples achieved sensitivity of 79%. The 6 patients with wild-type EGFR were all negative for plasma EGFR mutations. At the time of disease progression, plasma T790M mutation was detected in 8 of 16 cases. Absence of T790M before and during TKI treatment and disappearance of activating mutations during TKI treatment were considered as predictors of EGFR-TKIs efficacy. Conclusion. We were able to detect EGFR mutations in plasma samples by using an improved PNA-LNA PCR clamp method. PMID:27478396

  16. Intracellular surface-enhanced Raman scattering probe based on gold nanorods functionalized with mercaptohexadecanoic acid with reduced cytotoxicity.

    PubMed

    Liu, Min; Wang, Zhuyuan; Zong, Shenfei; Zhang, Ruohu; Yang, Jing; Cui, Yiping

    2012-01-01

    A surface-enhanced Raman scattering (SERS) probe for intracellular detection was demonstrated by utilizing gold nanorods (GNRs) coated with p-aminothiophenol as the Raman reporters. In this probe, to reduce the cytotoxicity of GNRs, cetyltrimethylammonium bromide (CTAB) molecules adsorbed on the surfaces of GNRs as ligands were replaced by mercaptohexadecanoic acid via a "round-trip" phase change method. Such a ligand exchange can reduce the toxicity of the probe compared to the original CTAB-stabilized GNRs, which were confirmed by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bright field view of HeLa cells. Meanwhile, the transmission electron microscopy images indicated that there is no significant morphologic change of GNRs before and after the ligand exchange. Moreover, its SERS performance was adequately retained after the incorporation of the probe into living HeLa cells. This new type of SERS probe is expected to have great potential in intracellular imaging or sensing applications.

  17. Efficient detection of secondary structure folded nucleic acids related to Alzheimer's disease based on junction probes.

    PubMed

    Li, Juan; Qi, Xiu-Juan; Du, Yan-Yan; Fu, Hua-E; Chen, Guo-Nan; Yang, Huang-Hao

    2012-01-01

    Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples.

  18. A high-resolution mitochondria-targeting ratiometric fluorescent probe for detection of the endogenous hypochlorous acid

    NASA Astrophysics Data System (ADS)

    Zhou, Liyi; Lu, Dan-Qing; Wang, Qianqian; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Hypochlorite anion, one of the biologically important reactive oxygen species, plays an essential role in diverse normal biochemical functions and abnormal pathological processes. Herein, an efficient high-resolution mitochondria-targeting ratiometric fluorescent probe for hypochlorous acid detection has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (Cdbnd C) of a 2-(2-hydroxyphenyl) quinazolin-4(3H)-one fluorophore and a cyanine group (mitochondria-targeting), which made the whole molecular a large Stokes shift (210 nm) and the two well-resolved emission peaks separated by 140 nm. As a result, it is considered as a good candidate for high resolution hypochlorous acid imaging in live cells. The ratiometric fluorescent probe exhibited outstanding features of high sensitivity, high selectivity, rapid response time (within 50 s), and excellent mitochondria-targeting ability. Moreover, the probe can also be successfully applied to imaging endogenously hypochlorous acid in the mitochondria of living cells with low cytotoxicity, and high resolution.

  19. pH-Sensitive Polymeric Micelle-based pH Probe for Detecting and Imaging Acidic Biological Environments

    PubMed Central

    Lee, Young Ju; Kang, Han Chang; Hu, Jun; Nichols, Joseph W.; Jeon, Yong Sun; Bae, You Han

    2012-01-01

    To overcome the limitations of monomeric pH probes for acidic tumor environments, this study designed a mixed micelle pH probe composed of polyethylene glycol (PEG)-b- poly(L-histidine) (PHis) and PEG-b-poly(L-lactic acid) (PLLA), which is well-known as an effective antitumor drug carrier. Unlike monomeric histidine and PHis derivatives, the mixed micelles can be structurally destabilized by changes in pH, leading to a better pH sensing system in nuclear magnetic resonance (NMR) techniques. The acidic pH-induced transformation of the mixed micelles allowed pH detection and pH mapping of 0.2–0.3 pH unit differences by pH-induced “on/off”-like sensing of NMR and magnetic resonance spectroscopy (MRS). The micellar pH probes sensed pH differences in non-biological phosphate buffer and biological buffers such as cell culture medium and rat whole blood. In addition, the pH-sensing ability of the mixed micelles was not compromised by loaded doxorubicin. In conclusion, PHis-based micelles could have potential as a tool to simultaneously treat and map the pH of solid tumors in vivo. PMID:22861824

  20. Detection of Sialic Acid-Utilising Bacteria in a Caecal Community Batch Culture Using RNA-Based Stable Isotope Probing

    PubMed Central

    Young, Wayne; Egert, Markus; Bassett, Shalome A.; Bibiloni, Rodrigo

    2015-01-01

    Sialic acids are monosaccharides typically found on cell surfaces and attached to soluble proteins, or as essential components of ganglioside structures that play a critical role in brain development and neural transmission. Human milk also contains sialic acid conjugated to oligosaccharides, glycolipids, and glycoproteins. These nutrients can reach the large bowel where they may be metabolised by the microbiota. However, little is known about the members of the microbiota involved in this function. To identify intestinal bacteria that utilise sialic acid within a complex intestinal community, we cultured the caecal microbiota from piglets in the presence of 13C-labelled sialic acid. Using RNA-based stable isotope probing, we identified bacteria that consumed 13C-sialic acid by fractionating total RNA in isopycnic buoyant density gradients followed by 16S rRNA gene analysis. Addition of sialic acid caused significant microbial community changes. A relative rise in Prevotella and Lactobacillus species was accompanied by a corresponding reduction in the genera Escherichia/Shigella, Ruminococcus and Eubacterium. Inspection of isotopically labelled RNA sequences suggests that the labelled sialic acid was consumed by a wide range of bacteria. However, species affiliated with the genus Prevotella were clearly identified as the most prolific users, as solely their RNA showed significantly higher relative shares among the most labelled RNA species. Given the relevance of sialic acid in nutrition, this study contributes to a better understanding of their microbial transformation in the intestinal tract with potential implications for human health. PMID:25816158

  1. Detection of sialic acid-utilising bacteria in a caecal community batch culture using RNA-based stable isotope probing.

    PubMed

    Young, Wayne; Egert, Markus; Bassett, Shalome A; Bibiloni, Rodrigo

    2015-03-25

    Sialic acids are monosaccharides typically found on cell surfaces and attached to soluble proteins, or as essential components of ganglioside structures that play a critical role in brain development and neural transmission. Human milk also contains sialic acid conjugated to oligosaccharides, glycolipids, and glycoproteins. These nutrients can reach the large bowel where they may be metabolised by the microbiota. However, little is known about the members of the microbiota involved in this function. To identify intestinal bacteria that utilise sialic acid within a complex intestinal community, we cultured the caecal microbiota from piglets in the presence of 13C-labelled sialic acid. Using RNA-based stable isotope probing, we identified bacteria that consumed 13C-sialic acid by fractionating total RNA in isopycnic buoyant density gradients followed by 16S rRNA gene analysis. Addition of sialic acid caused significant microbial community changes. A relative rise in Prevotella and Lactobacillus species was accompanied by a corresponding reduction in the genera Escherichia/Shigella, Ruminococcus and Eubacterium. Inspection of isotopically labelled RNA sequences suggests that the labelled sialic acid was consumed by a wide range of bacteria. However, species affiliated with the genus Prevotella were clearly identified as the most prolific users, as solely their RNA showed significantly higher relative shares among the most labelled RNA species. Given the relevance of sialic acid in nutrition, this study contributes to a better understanding of their microbial transformation in the intestinal tract with potential implications for human health.

  2. Detection of Chromosomal Inversions Using Non-Repetitive Nucleic Acid Probes

    NASA Technical Reports Server (NTRS)

    Bailey, Susan M. (Inventor); Ray, F. Andrew (Inventor); Goodwin, Edwin H. (Inventor); Bedford, Joel S. (Inventor); Cornforth, Michael N. (Inventor)

    2014-01-01

    A method and a kit for the identification of chromosomal inversions are described. Single-stranded sister chromatids are generated, for example by CO-FISH. A plurality of non-repetitive, labeled probes of relatively small size are hybridized to portions of only one of a pair of single-stranded sister chromatids. If no inversion exists, all of the probes will hybridize to a first chromatid. If an inversion has occurred, these marker probes will be detected on the sister chromatid at the same location as the inversion on the first chromatid.

  3. Detection of chromosomal inversions using non-repetitive nucleic acid probes

    NASA Technical Reports Server (NTRS)

    Bailey, Susan M. (Inventor); Ray, F. Andrew (Inventor); Goodwin, Edwin H. (Inventor); Bedford, Joel S. (Inventor); Cornforth, Michael N. (Inventor)

    2012-01-01

    A method for the identification of chromosomal inversions is described. Single-stranded sister chromatids are generated, for example by CO-FISH. A plurality of non-repetitive, labeled probes of relatively small size are hybridized to portions of only one of a pair of single-stranded sister chromatids. If no inversion exists, all of the probes will hybridize to a first chromatid. If an inversion has occurred, these marker probes will be detected on the sister chromatid at the same location as the inversion on the first chromatid.

  4. Colorimetric and fluorescence detection of G-quadruplex nucleic acids with a coumarin-benzothiazole probe.

    PubMed

    Yan, Jin-wu; Tian, Yi-guang; Tan, Jia-heng; Huang, Zhi-shu

    2015-11-01

    A colorimetric and red-emitting fluorescent dual probe for G-quadruplexes was devised with a conjugated coumarin-benzothiazole scaffold. Its significant and distinct changes in both color and fluorescence enable the label-free and visual detection of G-quadruplex structures. In addition, this probe gives a distinct strong emission response to the nucleoli in fixed cells imaging, which might be attributed to the interaction between the probe and rDNA G-quadruplex based on the chromatin immunoprecipitation assay. All these results suggest its promising application prospects in the G-quadruplex research field.

  5. Human papillomavirus 35 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorincz, A.T.

    1989-07-18

    This patent describes an HPV 35 hybridization probe comprising a member selected from the group consisting of (i) HPV 35 DNA or fragments thereof labelled with a marker and (ii) HPV 35 RNA or fragments thereof labelled with a marker.

  6. Human papillomavirus 43 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorincz, A.T.

    1989-07-18

    This patent describes an HPV 43 hybridization probe comprising a member selected from the group consisting of (i) HPV 43 DNA or fragments thereof labelled with a marker and (ii) HPV 43 RNA or fragments thereof labelled with a marker.

  7. Human papillomavirus 44 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorincz, A.T.

    1989-07-18

    This patent describes an HPV 44 hybridization probe comprising a member selected from the group consisting of (1) HPV 44 DNA or fragments thereof labelled with a marker and (ii) HPV 44 RNA or fragments thereof labelled with a marker.

  8. Human papillomavirus 56 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorinez, A.T.

    1990-03-13

    This patent describes an HPV 56 hybridization probe. It comprises: a member selected from the group consisting of HPV 56 DNA or fragments thereof labelled with a marker and HPV 56 RNA or fragments thereof labelled with a marker.

  9. Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

    DOEpatents

    Gray, J.W.; Weier, H.U.

    1998-11-24

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences. 9 figs.

  10. Y chromosome specific nucleic acid probe and method for identifying the Y chromosome in SITU

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich

    1999-01-01

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

  11. Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich

    1998-01-01

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

  12. Y chromosome specific nucleic acid probe and method for identifying the Y chromosome in SITU

    DOEpatents

    Gray, J.W.; Weier, H.U.

    1999-03-30

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences. 9 figs.

  13. Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich

    2001-01-01

    A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

  14. Sensitivity and specificity of nucleic acid probes for potato leafroll luteovirus detection.

    PubMed

    Robinson, D J; Romero, J

    1991-01-01

    Complementary DNA (cDNA) probes, prepared by nick-translation or by oligolabelling of a 520 bp fragment representing residues 4741 to 5261 in the potato leafroll luteovirus (PLRV) sequence, were equally sensitive, with a detection limit equivalent to sap from about 600 micrograms of infected potato or Nicotiana clevelandii leaf tissue and to RNA from about 120 micrograms tissue. Increasing the concentration of oligolabelled probe gave similar results with shorter autoradiographic exposures, but also resulted in positive signals with sap extracts from healthy plants. In contrast, a complementary RNA (cRNA) probe made by in vitro transcription of the cDNA insert could be used at higher concentration without giving rise to reactions with healthy plant extracts, and had a detection limit equivalent to 5 micrograms tissue/spot. Five oligolabelled probes representing different regions of the PLRV genome detected PLRV equally well. A probe that represented a portion of the particle protein gene also detected beet western yellows luteovirus (BWYV), with which it has 69% nucleotide sequence homology, and an English strain of the RPV form of barley yellow dwarf luteovirus, and reacted weakly with extracts from plants infected with groundnut rosette assistor luteovirus or carrot red leaf luteovirus. Probes for regions on either side of the particle protein gene also detected RPV, but not any of the other luteoviruses tested, in agreement with earlier suggestions that RPV is more closely related to PLRV than are BWYV or the other luteoviruses tested. An attempt to improve the detection of weak heterologous reactions by using a cRNA probe was unsuccessful, perhaps because tests using cRNA are more affected by mismatching than tests using cDNA probes. PMID:1804851

  15. A Far-Red Emitting Probe for Unambiguous Detection of Mobile Zinc in Acidic Vesicles and Deep Tissue†

    PubMed Central

    Rivera-Fuentes, Pablo; Wrobel, Alexandra T.; Zastrow, Melissa L.; Khan, Mustafa; Georgiou, John; Luyben, Thomas T.; Roder, John C.; Okamoto, Kenichi

    2015-01-01

    Imaging mobile zinc in acidic environments remains challenging because most small-molecule optical probes display pH-dependent fluorescence. Here we report a reaction-based sensor that detects mobile zinc unambiguously at low pH. The sensor responds reversibly and with a large dynamic range to exogenously applied Zn2+ in lysosomes of HeLa cells, endogenous Zn2+ in insulin granules of MIN6 cells, and zinc-rich mossy fiber boutons in hippocampal tissue from mice. This long-wavelength probe is compatible with the green-fluorescent protein, enabling multicolor imaging, and facilitates visualization of mossy fiber boutons at depths of >100 µm, as demonstrated by studies in live tissue employing two-photon microscopy. PMID:25815162

  16. Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

    SciTech Connect

    Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

    2008-07-28

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg{sup 2+} as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 {angstrom} (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

  17. Proteomic-based stable isotope probing reveals taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

    DOE PAGES

    Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; Robert L. Hettich; Mayali, Xavier; Pan, Chongle; Mueller, Ryan S.

    2016-04-26

    Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual global primary production, yet defining how substrate utilization preferences and resource partitioning structure these microbial communities remains a challenge. In this study, we utilized proteomics-based stable isotope probing (proteomic SIP) to characterize the assimilation of amino acids by coastal marine bacterioplankton populations. We incubated microcosms of seawater collected from Newport, OR and Monterey Bay, CA with 1 M 13C-amino acids for 15 and 32 hours. Subsequent analysis of 13C incorporation into protein biomass quantified the frequency and extent of isotope enrichment for identifiedmore » proteins. Using these metrics we tested whether amino acid assimilation patterns were different for specific bacterioplankton populations. Proteins associated with Rhodobacterales and Alteromonadales tended to have a significantly high number of tandem mass spectra from 13C-enriched peptides, while Flavobacteriales and SAR11 proteins generally had significantly low numbers of 13C-enriched spectra. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time-point 2, while Alteromonadales ribosomal proteins were 13C- enriched across time-points. Overall, proteomic SIP facilitated quantitative comparisons of dissolved free amino acids assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population-level metabolic responses to resource acquisition in complex microbial communities.« less

  18. Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester.

    PubMed

    Brandt, Gabriel S; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J; Yep, Alejandra; Kenyon, George L; Petsko, Gregory A; Jordan, Frank; Ringe, Dagmar

    2008-07-22

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

  19. Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens.

    PubMed

    Hiroyasu, Makoto; Akatsuka, Shinya; Shirase, Tomoyuki; Toda, Yoshinobu; Hiai, Hiroshi; Toyokuni, Shinya

    2004-04-01

    Although the human genome project has been completed, the functions of many genes remain undetermined. In situ hybridization (ISH) is a key method for identifying cells in which a given messenger RNA is transcribed. Paraffin-embedded specimens remain precious materials for research, but preservation of high-quality RNA in these specimens is not expected unless ample caution was taken during fixation. Peptide nucleic acid (PNA) is a recently developed hybrid molecule with genetic information that has high stability and high affinity to the complementary DNA or RNA. We applied a PNA probe to mRNA ISH of liver specimens obtained by autopsy and embedded in paraffin 28-48 years ago. An 18-mer PNA probe for glyceraldehyde 3-phosphate dehydrogenase was used. Staining was then analyzed in association with morphology by hematoxylin and eosin staining, and with the time between death of the patient and tissue fixation. Notably, specimens fixed with formalin and embedded in paraffin 48 years ago yielded excellent results if the time before fixation was short enough (<8 h). There was a significant inverse correlation between the intensity of ISH staining and the time before fixation. Oligonucleotide PNA probe, albeit at high cost, would increase the value of paraffin-embedded specimens in storage for use in human medical research.

  20. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring. PMID:27154709

  1. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring.

  2. Mercaptopropionic acid-capped CdTe quantum dots as fluorescence probe for the determination of salicylic acid in pharmaceutical products.

    PubMed

    Bunkoed, Opas; Kanatharana, Proespichaya

    2015-11-01

    Mercaptopropionic acid (MPA)-capped cadmium telluride (CdTe) quantum dot (QDs) fluorescent probes were synthesized in aqueous solution and used for the determination of salicylic acid. The interaction between the MPA-capped CdTe QDs and salicylic acid was studied using fluorescence spectroscopy and some parameters that could modify the fluorescence were investigated to optimize the measurements. Under optimum conditions, the quenched fluorescence intensity of MPA-capped CdTe QDs was linearly proportional to the concentration of salicylic acid in the range of 0.5-40 µg mL(-1) with a coefficient of determination of 0.998, and the limit of detection was 0.15 µg mL(-1). The method was successfully applied to the determination of salicylic acid in pharmaceutical products, and satisfactory results were obtained that were in agreement with both the high pressure liquid chromatography (HPLC) method and the claimed values. The recovery of the method was in the range 99 ± 3% to 105 ± 9%. The proposed method is simple, rapid, cost effective, highly sensitivity and eminently suitable for the quality control of pharmaceutical preparation. The possible mechanisms for the observed quenching reaction was also discussed.

  3. Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

    PubMed Central

    2013-01-01

    Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. PMID:23586331

  4. Double-stem Hairpin Probe and Ultrasensitive Colorimetric Detection of Cancer-related Nucleic Acids.

    PubMed

    Xu, Jianguo; Li, Hongling; Wu, Zai-Sheng; Qian, Jun; Xue, Chang; Jia, Lee

    2016-01-01

    The development of a versatile biosensing platform to screen specific DNA sequences is still an essential issue of molecular biology research and clinic diagnosis of genetic disease. In this work, we for the first time reported a double-stem hairpin probe (DHP) that was simultaneously engineered to incorporate a DNAzyme, DNAzyme's complementary fragment and nicking enzyme recognition site. The important aspect of this hairpin probe is that, although it is designed to have a long ds DNA fragment, no intermolecular interaction occurs, circumventing the sticky-end pairing-determined disadvantages encountered by classic molecular beacon. For the DHP-based colorimetric sensing system, as a model analyte, cancer-related DNA sequence can trigger a cascade polymerization/nicking cycle on only one oligonucleotide probe. This led to the dramatic accumulation of G-quadruplexes directly responsible for colorimetric signal conversion without any loss. As a result, the target DNA is capable of being detected to 1 fM (six to eight orders of magnitude lower than that of catalytic molecular beacons) and point mutations are distinguished by the naked eye. The described DHP as a-proof-of-concept would not only promote the design of colorimetric biosensors but also open a good way to promote the diagnosis and treatment of genetic diseases. PMID:26909108

  5. Double-stem Hairpin Probe and Ultrasensitive Colorimetric Detection of Cancer-related Nucleic Acids

    PubMed Central

    Xu, Jianguo; Li, Hongling; Wu, Zai-Sheng; Qian, Jun; Xue, Chang; Jia, Lee

    2016-01-01

    The development of a versatile biosensing platform to screen specific DNA sequences is still an essential issue of molecular biology research and clinic diagnosis of genetic disease. In this work, we for the first time reported a double-stem hairpin probe (DHP) that was simultaneously engineered to incorporate a DNAzyme, DNAzyme's complementary fragment and nicking enzyme recognition site. The important aspect of this hairpin probe is that, although it is designed to have a long ds DNA fragment, no intermolecular interaction occurs, circumventing the sticky-end pairing-determined disadvantages encountered by classic molecular beacon. For the DHP-based colorimetric sensing system, as a model analyte, cancer-related DNA sequence can trigger a cascade polymerization/nicking cycle on only one oligonucleotide probe. This led to the dramatic accumulation of G-quadruplexes directly responsible for colorimetric signal conversion without any loss. As a result, the target DNA is capable of being detected to 1 fM (six to eight orders of magnitude lower than that of catalytic molecular beacons) and point mutations are distinguished by the naked eye. The described DHP as a-proof-of-concept would not only promote the design of colorimetric biosensors but also open a good way to promote the diagnosis and treatment of genetic diseases. PMID:26909108

  6. Double-stem Hairpin Probe and Ultrasensitive Colorimetric Detection of Cancer-related Nucleic Acids.

    PubMed

    Xu, Jianguo; Li, Hongling; Wu, Zai-Sheng; Qian, Jun; Xue, Chang; Jia, Lee

    2016-01-01

    The development of a versatile biosensing platform to screen specific DNA sequences is still an essential issue of molecular biology research and clinic diagnosis of genetic disease. In this work, we for the first time reported a double-stem hairpin probe (DHP) that was simultaneously engineered to incorporate a DNAzyme, DNAzyme's complementary fragment and nicking enzyme recognition site. The important aspect of this hairpin probe is that, although it is designed to have a long ds DNA fragment, no intermolecular interaction occurs, circumventing the sticky-end pairing-determined disadvantages encountered by classic molecular beacon. For the DHP-based colorimetric sensing system, as a model analyte, cancer-related DNA sequence can trigger a cascade polymerization/nicking cycle on only one oligonucleotide probe. This led to the dramatic accumulation of G-quadruplexes directly responsible for colorimetric signal conversion without any loss. As a result, the target DNA is capable of being detected to 1 fM (six to eight orders of magnitude lower than that of catalytic molecular beacons) and point mutations are distinguished by the naked eye. The described DHP as a-proof-of-concept would not only promote the design of colorimetric biosensors but also open a good way to promote the diagnosis and treatment of genetic diseases.

  7. Carboxylic Acid Ionophores as Probes of the Role of Calcium in Biological Systems

    NASA Technical Reports Server (NTRS)

    Reed, P. W.

    1983-01-01

    The biological effects of calcium ionophores are described, focusing on arachidonic acid oxygenation, and the formation of a number of oxygenated metabolites of arachidonic acid. These metabolites are involved in a number of bodily functions, and their production may be regulated by calcium.

  8. A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue.

    PubMed

    Vaughan, Roger A; Garrison, Richard L; Stamatikos, Alexis D; Kang, Minsung; Cooper, Jamie A; Paton, Chad M

    2015-11-01

    Recently, the pro-inflammatory effects of linoleic acid (LNA) have been re-examined. It is now becoming clear that relatively few studies have adequately assessed the effects of LNA, independent of obesity. The purpose of this work was to compare the effects of several fat-enriched but non-obesigenic diets on inflammation to provide a more accurate assessment of LNA's ability to induce inflammation. Specifically, 8-week-old male C57Bl/6 mice were fed either saturated (SFA), monounsaturated (MUFA), LNA, or alpha-linolenic acid enriched diets (50 % Kcal from fat, 22 % wt/wt) for 4 weeks. Chow and high-fat, hyper-caloric diets were used as negative and positive controls, respectively. Expression of pro-inflammatory and pro-coagulant markers from epididymal fat, liver, and plasma were measured along with food intake and body weights. Mice fed the high SFA, MUFA, and high-fat diets exhibited increased pro-inflammatory markers in liver and adipose tissue; however, mice fed LNA for four weeks did not display significant changes in pro-inflammatory or pro-coagulant markers in epididymal fat, liver, or plasma. The present study demonstrates that LNA alone is insufficient to induce inflammation. Instead, it is more likely that hyper-caloric diets are responsible for diet-induced inflammation possibly due to adipose tissue remodeling.

  9. Probing acid-amide intermolecular hydrogen bonding by NMR spectroscopy and DFT calculations

    NASA Astrophysics Data System (ADS)

    Chaudhari, Sachin Rama; Suryaprakash, N.

    2012-05-01

    Benzene carboxylic acids and benzamide act as their self-complement in molecular recognition to form inter-molecular hydrogen bonded dimers between amide and carboxylic acid groups, which have been investigated by 1H, 13C and 15N NMR spectroscopy. Extensive NMR studies using diffusion ordered spectroscopy (DOSY), variable temperature 1D, 2D NMR, established the formation of heterodimers of benzamide with benzoic acid, salicylic acid and phenyl acetic acid in deuterated chloroform solution. Association constants for the complex formation in the solution state have been determined. The results are ascertained by X-ray diffraction in the solid state. Intermolecular interactions in solution and in solid state were found to be similar. The structural parameters obtained by X-ray diffraction studies are compared with those obtained by DFT calculations.

  10. Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

    SciTech Connect

    Malgoyre, A.; Banzet, S.; Mouret, C.; Bigard, A.X.; Peinnequin, A. . E-mail: andrepeinnequin@crssa.net

    2007-03-02

    Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.

  11. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

    PubMed Central

    Reichau, Sebastian; Blackmore, Nicola J.; Jiao, Wanting; Parker, Emily J.

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal. PMID:27128682

  12. Molecular recognition of α-cyclodextrin (CD) to choral amino acids based on methyl orange as a molecular probe

    NASA Astrophysics Data System (ADS)

    Yuexian, Fan; Yu, Yang; Shaomin, Shuang; Chuan, Dong

    2005-03-01

    The molecular recognition interaction of α-CD to chiral amino acids was investigated by using spectrophotometry based on methyl orange as a molecular probe. The molecular recognition ability depended on the inclusion formation constants. The molecular recognition of α-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine ≈ DL-β-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L- iso-leucine > L-leucine ≈ L-methionine ≈ DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, Δ G, Δ H, Δ S, were determined. The experimental results indicated that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative or minor positive entropic contribution. The inclusion interaction between α-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K.

  13. ω-Azido fatty acids as probes to detect fatty acid biosynthesis, degradation, and modification[S

    PubMed Central

    Pérez, Alexander J.; Bode, Helge B.

    2014-01-01

    FAs play a central role in the metabolism of almost all known cellular life forms. Although GC-MS is regarded as a standard method for FA analysis, other methods, such as HPLC/MS, are nowadays widespread but are rarely applied to FA analysis. Here we present azido-FAs as probes that can be used to study FA biosynthesis (elongation, desaturation) or degradation (β-oxidation) upon their uptake, activation, and metabolic conversion. These azido-FAs are readily accessible by chemical synthesis and their metabolic products can be easily detected after click-chemistry based derivatization with high sensitivity by HPLC/MS, contributing a powerful tool to FA analysis, and hence, lipid analysis in general. PMID:25013232

  14. Probing the interaction of individual amino acids with inorganic surfaces using atomic force spectroscopy.

    PubMed

    Razvag, Yair; Gutkin, Vitaly; Reches, Meital

    2013-08-13

    This article describes single-molecule force spectroscopy measurements of the interaction between individual amino acid residues and inorganic surfaces in an aqueous solution. In each measurement, there is an amino acid residue, lysine, glutamate, phenylalanine, leucine, or glutamine, and each represents a class of amino acids (positively or negatively charged, aromatic, nonpolar, and polar). Force-distance curves measured the interaction of the individual amino acid bound to a silicon atomic force microscope (AFM) tip with a silcon substrate, cut from a single-crystal wafer, or mica. Using this method, we were able to measure low adhesion forces (below 300 pN) and could clearly determine the strength of interactions between the individual amino acid residues and the inorganic substrate. In addition, we observed how changes in the pH and ionic strength of the solution affected the adsorption of the residues to the substrates. Our results pinpoint the important role of hydrophobic interactions among the amino acids and the substrate, where hydrophobic phenylalanine exhibited the strongest adhesion to a silicon substrate. Additionally, electrostatic interactions also contributed to the adsorption of amino acid residues to inorganic substrates. A change in the pH or ionic strength values of the buffer altered the strength of interactions among the amino acids and the substrate. We concluded that the interplay between the hydrophobic forces and electrostatic interactions will determine the strength of adsorption among the amino acids and the surface. Overall, these results contribute to our understanding of the interaction at the organic-inorganic interface. These results may have implications for our perception of the specificity of peptide binding to inorganic surfaces. Consequently, it would possibly lead to a better design of composite materials and devices.

  15. DESIGN OF 2.4 GHZ CMOS DIRECT CONVERSION LNA AND MIXER COMBINATION FOR WIRLESS DATA LINK TRANSCEIVER.

    SciTech Connect

    ZHAO, D.; OCONNOR, P.

    2002-04-10

    Three LNA and mixer combinations in 0.6{micro}m and 0.4{micro}m standard CMOS processes for direct-conversion receiver of 2.4GHz ISM band short-range wireless data-link applications are described in this paper. Taking low power dissipation as first consideration, these designs, employing differential common-source LNA and double balanced mixer architectures, achieve total conversion gain as high as 42.4dB, DSB noise figure as low as 9.5dB, output-referred IP3 as high as of 21.3dBm at about 4mA DC current consumption. This proves it is possible to apply standard CMOS process to implement receiver front-end with low power dissipation for this kind of application, but gain changeable LNA is needed to combat the dominant flicker noise of the mixer in order to achieve acceptable sensitivity and dynamic range at the same time.

  16. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  17. Probing Phosphorus Efficient Low Phytic Acid Content Soybean Genotypes with Phosphorus Starvation in Hydroponics Growth System.

    PubMed

    Kumar, Varun; Singh, Tiratha Raj; Hada, Alkesh; Jolly, Monica; Ganapathi, Andy; Sachdev, Archana

    2015-10-01

    Phosphorus is an essential nutrient required for soybean growth but is bound in phytic acid which causes negative effects on both the environment as well as the animal nutrition. Lowering of phytic acid levels is associated with reduced agronomic characteristics, and relatively little information is available on the response of soybean plants to phosphorus (P) starvation. In this study, we evaluated the effects of different P starvation concentrations on the phytic acid content, growth, and yield of seven mutant genotypes along with the unirradiated control, JS-335, in a hydroponics growth system. The low phytic acid containing mutant genotypes, IR-JS-101, IR-DS-118, and IR-V-101, showed a relatively high growth rate in low P concentration containing nutrient solution (2 μM), whereas the high P concentration (50 μM) favored the growth of IR-DS-111 and IR-DS-115 mutant genotypes containing moderate phytate levels. The mutant genotypes with high phytic acid content, IR-DS-122, IR-DS-114, and JS-335, responded well under P starvation and did not have any significant effect on the growth and yield of plants. Moreover, the reduction of P concentration in nutrient solution from 50 to 2 μM also reduced the phytic acid content in the seeds of all the soybean genotypes under study. The desirable agronomic performance of low phytic acid containing mutant genotype IR-DS-118 reported in this study suggested it to be a P-efficient genotype which could be considered for agricultural practices under P limiting soils. PMID:26239443

  18. Probing Phosphorus Efficient Low Phytic Acid Content Soybean Genotypes with Phosphorus Starvation in Hydroponics Growth System.

    PubMed

    Kumar, Varun; Singh, Tiratha Raj; Hada, Alkesh; Jolly, Monica; Ganapathi, Andy; Sachdev, Archana

    2015-10-01

    Phosphorus is an essential nutrient required for soybean growth but is bound in phytic acid which causes negative effects on both the environment as well as the animal nutrition. Lowering of phytic acid levels is associated with reduced agronomic characteristics, and relatively little information is available on the response of soybean plants to phosphorus (P) starvation. In this study, we evaluated the effects of different P starvation concentrations on the phytic acid content, growth, and yield of seven mutant genotypes along with the unirradiated control, JS-335, in a hydroponics growth system. The low phytic acid containing mutant genotypes, IR-JS-101, IR-DS-118, and IR-V-101, showed a relatively high growth rate in low P concentration containing nutrient solution (2 μM), whereas the high P concentration (50 μM) favored the growth of IR-DS-111 and IR-DS-115 mutant genotypes containing moderate phytate levels. The mutant genotypes with high phytic acid content, IR-DS-122, IR-DS-114, and JS-335, responded well under P starvation and did not have any significant effect on the growth and yield of plants. Moreover, the reduction of P concentration in nutrient solution from 50 to 2 μM also reduced the phytic acid content in the seeds of all the soybean genotypes under study. The desirable agronomic performance of low phytic acid containing mutant genotype IR-DS-118 reported in this study suggested it to be a P-efficient genotype which could be considered for agricultural practices under P limiting soils.

  19. Array of nucleic acid probes on biological chips for diagnosis of HIV and methods of using the same

    DOEpatents

    Chee, Mark; Gingeras, Thomas R.; Fodor, Stephen P. A.; Hubble, Earl A.; Morris, MacDonald S.

    1999-01-19

    The invention provides an array of oligonucleotide probes immobilized on a solid support for analysis of a target sequence from a human immunodeficiency virus. The array comprises at least four sets of oligonucleotide probes 9 to 21 nucleotides in length. A first probe set has a probe corresponding to each nucleotide in a reference sequence from a human immunodeficiency virus. A probe is related to its corresponding nucleotide by being exactly complementary to a subsequence of the reference sequence that includes the corresponding nucleotide. Thus, each probe has a position, designated an interrogation position, that is occupied by a complementary nucleotide to the corresponding nucleotide. The three additional probe sets each have a corresponding probe for each probe in the first probe set. Thus, for each nucleotide in the reference sequence, there are four corresponding probes, one from each of the probe sets. The three corresponding probes in the three additional probe sets are identical to the corresponding probe from the first probe or a subsequence thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes.

  20. Functionalized ZnS quantum dots as luminescent probes for detection of amino acids.

    PubMed

    Mobarraz, Mahsa; Ganjali, Mohammad Reza; Chaichi, Mohammad Javad; Norouzi, Parviz

    2012-10-01

    In this work, L-cysteine capped-ZnS quantum dots were synthesized in aqueous medium, and their interaction with some of the amino acids was studied with fluorescence spectra. The results demonstrated that histidine could effectively quench the Quantum dots emission more than other amino acids. Electron transfer process between the capping ligands and histidine was mainly responsible for the remarkable quenching effect of histidine, because according to the structure of histidine, it is the strongest acceptor among essential amino acids. Under optimum conditions, the quenched fluorescence intensity increased linearly with the concentration of histidine ranging from 1.33×10(-6) to 1.49×10(-4) mol L(-1). The limit of detection for histidine was 0.05×10(-7) mol L(-1). PMID:22925905

  1. Facilitating unambiguous NMR assignments and enabling higher probe density through selective labeling of all methyl containing amino acids.

    PubMed

    Proudfoot, Andrew; Frank, Andreas O; Ruggiu, Fiorella; Mamo, Mulugeta; Lingel, Andreas

    2016-05-01

    The deuteration of proteins and selective labeling of side chain methyl groups has greatly enhanced the molecular weight range of proteins and protein complexes which can be studied using solution NMR spectroscopy. Protocols for the selective labeling of all six methyl group containing amino acids individually are available, however to date, only a maximum of five amino acids have been labeled simultaneously. Here, we describe a new methodology for the simultaneous, selective labeling of all six methyl containing amino acids using the 115 kDa homohexameric enzyme CoaD from E. coli as a model system. The utility of the labeling protocol is demonstrated by efficiently and unambiguously assigning all methyl groups in the enzymatic active site using a single 4D (13)C-resolved HMQC-NOESY-HMQC experiment, in conjunction with a crystal structure. Furthermore, the six fold labeled protein was employed to characterize the interaction between the substrate analogue (R)-pantetheine and CoaD by chemical shift perturbations, demonstrating the benefit of the increased probe density. PMID:27130242

  2. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  3. Allele specific locked nucleic acid quantitative PCR (ASLNAqPCR): an accurate and cost-effective assay to diagnose and quantify KRAS and BRAF mutation.

    PubMed

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.

  4. Adaptation and Validation of E-Probe Diagnostic Nucleic Acid Analysis for Detection of Escherichia coli O157:H7 in Metagenomic Data from Complex Food Matrices.

    PubMed

    Blagden, Trenna; Schneider, William; Melcher, Ulrich; Daniels, Jon; Fletcher, Jacqueline

    2016-04-01

    The Centers for Disease Control and Prevention recently emphasized the need for enhanced technologies to use in investigations of outbreaks of foodborne illnesses. To address this need, e-probe diagnostic nucleic acid analysis (EDNA) was adapted and validated as a tool for the rapid, effective identification and characterization of multiple pathogens in a food matrix. In EDNA, unassembled next generation sequencing data sets from food sample metagenomes are queried using pathogen-specific sequences known as electronic probes (e-probes). In this study, the query of mock sequence databases demonstrated the potential of EDNA for the detection of foodborne pathogens. The method was then validated using next generation sequencing data sets created by sequencing the metagenome of alfalfa sprouts inoculated with Escherichia coli O157:H7. Nonspecific hits in the negative control sample indicated the need for additional filtration of the e-probes to enhance specificity. There was no significant difference in the ability of an e-probe to detect the target pathogen based upon the length of the probe set oligonucleotides. The results from the queries of the sample database using E. coli e-probe sets were significantly different from those obtained using random decoy probe sets and exhibited 100% precision. The results support the use of EDNA as a rapid response methodology in foodborne outbreaks and investigations for establishing comprehensive microbial profiles of complex food samples. PMID:27052861

  5. Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan-MGB probes.

    PubMed

    Torija, M J; Mateo, E; Guillamón, J M; Mas, A

    2010-04-01

    A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10(2)-10(3) cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S-23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA. The RT-PCR assay used in this study is a reliable, specific and fast method for quantifying these five AAB species in wine and vinegar.

  6. Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.

    PubMed

    Michikawa, Yuichi; Fujimoto, Kentaro; Kinoshita, Kenji; Kawai, Seiko; Sugahara, Keisuke; Suga, Tomo; Otsuka, Yoshimi; Fujiwara, Kazuhiko; Iwakawa, Mayumi; Imai, Takashi

    2006-12-01

    In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3'-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

  7. Inhibition of Hsp27 Radiosensitizes Head-and-Neck Cancer by Modulating Deoxyribonucleic Acid Repair

    SciTech Connect

    Guttmann, David M.; Hart, Lori; Du, Kevin; Seletsky, Andrew; Koumenis, Constantinos

    2013-09-01

    Purpose: To present a novel method of tumor radiosensitization through Hsp27 knockdown using locked nucleic acid (LNA) and to investigate the role of Hsp27 in DNA double strand break (DSB) repair. Methods and Materials: Clonogenic survival assays, immunoblotting, the proximity ligation assay, and γH2AX foci analysis were conducted in SQ20B and FaDu human head-and-neck cancer cell lines treated with Hsp27 LNA and Hsp27 short hairpin RNA (shRNA). Additionally, nude mice with FaDu flank tumors were treated with fractionated radiation therapy after pretreatment with Hsp27 LNA and monitored for tumor growth. Results: Hsp27 LNA and Hsp27 shRNA radiosensitized head-and-neck cancer cell lines in an Hsp27-dependent manner. Ataxia-Telangectasia Mutated-mediated DNA repair signaling was impaired in irradiated cells with Hsp27 knockdown. ATM kinase inhibition abrogated the radiosensitizing effect of Hsp27. Furthermore, Hsp27 LNA and shRNA both attenuated DNA repair kinetics after radiation, and Hsp27 was found to colocalize with ATM in both untreated and irradiated cells. Last, combined radiation and Hsp27 LNA treatment in tumor xenografts in nude mice suppressed tumor growth compared with either treatment alone. Conclusions: These results support a radiosensitizing property of Hsp27 LNA in vitro and in vivo, implicate Hsp27 in double strand break repair, and suggest that Hsp27 LNA might eventually serve as an effective clinical agent in the radiotherapy of head-and-neck cancer.

  8. Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy.

    PubMed

    Juen, Michael Andreas; Wunderlich, Christoph Hermann; Nußbaumer, Felix; Tollinger, Martin; Kontaxis, Georg; Konrat, Robert; Hansen, D Flemming; Kreutz, Christoph

    2016-09-19

    In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear (13) C and (1) H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site-specific (2) H and (13) C labeling, spin topologies are introduced into DNA and RNA that make (1) H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A-site RNA was previously shown to undergo a two site exchange process in the micro- to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV-1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for (1) H RD experiments of nucleic acids.

  9. Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy.

    PubMed

    Juen, Michael Andreas; Wunderlich, Christoph Hermann; Nußbaumer, Felix; Tollinger, Martin; Kontaxis, Georg; Konrat, Robert; Hansen, D Flemming; Kreutz, Christoph

    2016-09-19

    In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear (13) C and (1) H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site-specific (2) H and (13) C labeling, spin topologies are introduced into DNA and RNA that make (1) H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A-site RNA was previously shown to undergo a two site exchange process in the micro- to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV-1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for (1) H RD experiments of nucleic acids. PMID:27533469

  10. Water uptake of internally mixed ammonium sulfate and dicarboxylic acid particles probed by infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Miñambres, Lorena; Méndez, Estíbaliz; Sánchez, María N.; Castaño, Fernando; Basterretxea, Francisco J.

    2013-05-01

    Tropospheric aerosols are usually mixtures of inorganic and organic compounds in variable proportions, and the relative amount of organic fraction can influence the hygroscopic properties of the particles. Infrared spectra of submicrometer internally mixed dry particles of ammonium sulfate (AS) with various dicarboxylic acids (oxalic, malonic, maleic, glutaric and pimelic) have been measured in an aerosol flow tube at several solute mass ratios. The spectra show a notable broadening in the bandwidth of sulfate ion ν3 vibrational band near 1115 cm-1 with respect to pure AS. We attribute these perturbations, that are biggest at AS/organic acid mass ratio near unity, to intermolecular interactions between inorganic ions and organic acid molecules in the internally mixed solids. The water uptake behavior of internally mixed particles has been measured by recording the infrared integrated absorbance of liquid water as a function of relative humidity (RH). The amount of water present in the particles prior to deliquescence correlates partially with the water solubilities of the dicarboxylic acids, and also with the relative magnitudes of intermolecular interactions in the internally mixed dry solids. Phase change of ammonium sulfate in the internally mixed particles with RH has been spectrally monitored, and it is shown that water uptaken before full deliquescence produces structural changes in the particles that are revealed by their vibrational spectra.

  11. A high-fat, high-oleic diet, but not a high-fat, saturated diet, reduces hepatic alpha-linolenic acid and eicosapentaenoic acid content in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Considerable research centers upon the role of linoleic acid (LNA; 18:2n6) as a competitive inhibitor of a-linolenic (ALA; 18:3n3) metabolism; however, little data exist as to the impact of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) on ALA metabolism. We tested the hypothesi...

  12. Probing inclusion complexes of cyclodextrins with amino acids by physicochemical approach.

    PubMed

    Roy, Mahendra Nath; Roy, Aditi; Saha, Subhadeep

    2016-10-20

    Formations of host-guest inclusion complexes of two natural amino acids, viz., l-Leucine and l-Isoleucine as guests with α and β-cyclodextrins have been investigated which include diverse applications in modern science such as controlled delivery in the field of pharmaceuticals, food processing etc. Surface tension and conductivity studies establish the formation of inclusion complexes with 1:1 stoichiometry. The interactions of cyclodextrins with amino acids have been supported by density, viscosity, refractive index, hydration and solvation number measurements indicating higher degree of inclusion in case of α-cyclodextrin. l-Leucine interacts more with the hydrophobic cavity of cyclodextrin than its isomer. With the help of stability constant by NMR titration, hydrophobic effect, H-bonds and structural effects the formations of inclusion complexes have been explained. PMID:27474589

  13. Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

    PubMed Central

    Öjemalm, Karin; Higuchi, Takashi; Lara, Patricia; Lindahl, Erik; Suga, Hiroaki

    2016-01-01

    Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of “snorkeling” of charged amino acids toward the lipid–water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp. These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells. PMID:27601675

  14. Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids.

    PubMed

    Öjemalm, Karin; Higuchi, Takashi; Lara, Patricia; Lindahl, Erik; Suga, Hiroaki; von Heijne, Gunnar

    2016-09-20

    Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells. PMID:27601675

  15. Using modern tools to probe the structure-function relationship of fatty acid synthases

    PubMed Central

    Burkart, Michael D.

    2015-01-01

    Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory. PMID:25676190

  16. Re-engineering of CYP2C9 to probe acid-base substrate selectivity.

    PubMed

    Tai, Guoying; Dickmann, Leslie J; Matovic, Nicholas; DeVoss, James J; Gillam, Elizabeth M J; Rettie, Allan E

    2008-10-01

    A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites.

  17. Mitochondrial, acidic, and cytosolic pHs determination by ³¹P NMR spectroscopy: design of new sensitive targeted pH probes.

    PubMed

    Culcasi, Marcel; Thétiot-Laurent, Sophie; Atteia, Ariane; Pietri, Sylvia

    2015-01-01

    (31)P nuclear magnetic resonance (NMR) is a unique technique to monitor noninvasively the energetics of living systems at real time through the detection of a variety of phosphorylated metabolites. Using adequately designed α-aminophosphonates as external probes, we have shown earlier that (31)P NMR can also give access simultaneously to the accurate pH of cytosolic and acidic compartments in normal and stressed cultured cells or isolated perfused organs, a feature that was not possible using endogenous inorganic phosphate as the probe. More recently, we obtained a series of derivatives of these new pH probes that incorporate a triphenylphosphonium cation as a specific vector to the mitochondrion. Here, we describe the synthesis, (31)P NMR pH titrating properties in buffers, and application in cultures of the green alga Chlamydomonas reinhardtii of two of these mitochondria-targeted pH probes in comparison with one nonvectorized, yet still informative α-aminophosphonate.

  18. Mitochondrial, acidic, and cytosolic pHs determination by ³¹P NMR spectroscopy: design of new sensitive targeted pH probes.

    PubMed

    Culcasi, Marcel; Thétiot-Laurent, Sophie; Atteia, Ariane; Pietri, Sylvia

    2015-01-01

    (31)P nuclear magnetic resonance (NMR) is a unique technique to monitor noninvasively the energetics of living systems at real time through the detection of a variety of phosphorylated metabolites. Using adequately designed α-aminophosphonates as external probes, we have shown earlier that (31)P NMR can also give access simultaneously to the accurate pH of cytosolic and acidic compartments in normal and stressed cultured cells or isolated perfused organs, a feature that was not possible using endogenous inorganic phosphate as the probe. More recently, we obtained a series of derivatives of these new pH probes that incorporate a triphenylphosphonium cation as a specific vector to the mitochondrion. Here, we describe the synthesis, (31)P NMR pH titrating properties in buffers, and application in cultures of the green alga Chlamydomonas reinhardtii of two of these mitochondria-targeted pH probes in comparison with one nonvectorized, yet still informative α-aminophosphonate. PMID:25634273

  19. Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

    PubMed

    Claisse, O; Lonvaud-Funel, A

    2001-06-01

    Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider. PMID:11403134

  20. Molecular beacons: a novel DNA probe for nucleic acid and protein studies.

    PubMed

    Tan, W; Fang, X; Li, J; Liu, X

    2000-04-01

    A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.

  1. On the use of 16-iodohexadecanoic acid (IHDA) as a probe for myocardial beta-oxidation of fatty acids

    SciTech Connect

    De Grado, T.R.; Gatley, S.J.; Ng, C.K.; Holden, J.E.; Bernstein, D.R.

    1984-01-01

    The rate of myocardial clearance of label IHDA was compared with the beta-oxidation rate by coinjection of 125-IHDA and 3-H-Palmitic Acid (THDA) into isolated rat hearts. I-125 residue curves and efflux I-125 and H-3 curves were obtained. Three groups of hearts were studied, control, ischemic and those from rats previously treated with the beta-oxidation inhibitor Ethyl 2(5(4-chlorophenyl)pentyl)-oxiran-2-carboxylate (POCA). Both labels were found to exit ischemic and POCA treated hearts more slowly than controls. In all cases, the clearance of label from IHDA was significantly slower than that of the labelled water from beta-oxidation of THDA, consistent with previous work indicating the efflux of iodide from the mitochondria to be rate-limiting. Assay of heart homogenates showed 60% of the extracted label to be iodide at 1 minute. Thus, iodide is pooled within the mitochondria and diffuses out with a characteristic rate governed by electrochemical and lemmal properties. By assuming parallel utilization of IHDA and THDA and transient clearance of both labelled metabolites from the cytosol, the H-3 curves were interpreted ad equivalent with the production of free iodide in the mitochondria. A rate constant describing the flux of iodide across the mitochondrial membrane was calculated by fitting the I-125 curve (I/sup -/ output) to the H-3 curve (I/sup -/ input) convolved with a single exponential. The rate constant (k/sub I/=2.25 +- .35 x 10/sup -3/ s/sup -1/ for normals) was similar to the largest rate constant from fitted residue curves (k=2.08 +- .05 x 10/sup -3/ s/sup -1/).

  2. Probing determinants of cyclopiazonic acid sensitivity of bacterial Ca2+-ATPases.

    PubMed

    Kotšubei, Aljona; Gorgel, Manuela; Morth, Jens P; Nissen, Poul; Andersen, Jacob L

    2013-11-01

    Cyclopiazonic acid (CPA) is a specific and potent inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a). Despite high sequence similarity to SERCA1a, Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1) is not inhibited by CPA. To test whether a CPA binding site could be created while maintaining the functionality of the ATPase we targeted four amino acid positions in LMCA1 for mutational studies based on a multiple sequence alignment of SERCA-like Ca(2+)-ATPases and structural analysis of the CPA site. The identification of CPA-sensitive gain-of-function mutants pinpointed key determinants of the CPA binding site. The importance of these determinants was further underscored by the characterization of the CPA sensitivity of two additional bacterial Ca(2+)-ATPases from Lactococcus lactis and Bacillus cereus. The CPA sensitivity was predicted from their sequence compared with the LMCA1 results, and this was experimentally confirmed. Interestingly, a cluster of Lactococcus bacteria applied in the production of fermented cheese display Ca(2+)-ATPases that are predictably CPA insensitive and may originate from their coexistence with CPA-producing Penicillum and Aspergillus fungi in the cheese. The differences between bacterial and mammalian binding pockets encompassing the CPA site suggest that CPA derivatives that are specific for bacteria or other pathogens can be developed. PMID:23621633

  3. New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

    PubMed Central

    Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre

    2006-01-01

    A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys. PMID:16569894

  4. ESTIMATION OF BACTERIAL CELL NUMBERS IN HUMIC ACID-RICH SALT MARSH SEDIMENTS WITH PROBES DIRECTED TO 16S RIBOSOMAL DNA

    EPA Science Inventory

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membr...

  5. Highly water-soluble monoboronic acid probes that show optical sensitivity to glucose based on 4-sulfo-1,8-naphthalic anhydride.

    PubMed

    Cao, Zhi; Nandhikonda, Premchendar; Heagy, Michael D

    2009-05-01

    Two highly water-soluble monoboronic acid probes that display the more desirable off-on fluorescence response were synthesized based on 4-sulfo-1,8-naphthalic anhydride and a remarkable sensitivity for glucose rather than fructose and galactose was also observed.

  6. Adaptation and validation of E-probe diagnostic nucleic acid analysis for detection of Escherichia coli O157:H7 in metagenomic data of complex food matrices

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foodborne pathogens are an increasing problem threatening the US food supply. The need for rapid sensitive diagnostic tools that can address multiple types and taxonomic classes of foodbourne pathogens is growing. This paper describes the adaptation of E-probe Diagnostic Nucleic acid Analysis (EDNA)...

  7. ESA Mission ROSETTA Will Probe for Chirality of Cometary Amino Acids

    NASA Astrophysics Data System (ADS)

    Thiemann, Wolfram H.-P.; Meierhenrich, Uwe

    2001-02-01

    New crucial theoretical investigations on the origin of biomolecular chirality are reviewed briefly. With the goal to investigate these theories our team is going to perform the `chirality-experiment' in the near future with cometary matter. In 2012 the robotical lander RoLand will detach from the orbiter of the ROSETTA spacecraft and set down on the surface of comet 46P/Wirtanen in order to separate and identify cometary organic compounds via GC-MS in situ. Chiral organics will be separated into their enantiomers by application of 3 capillary columns coated with different kinds of stationary phases. Non-volatile compounds like amino acids will be derivatized in especially developed gas phase alkylation steps avoiding reactions in the liquid phase. The results of these preliminary gas phase reactions are presented in this article.

  8. Label-free and selective sensing of uric acid with gold nanoclusters as optical probe.

    PubMed

    Wang, Jian; Chang, Yong; Wu, Wen Bi; Zhang, Pu; Lie, Shao Qing; Huang, Cheng Zhi

    2016-05-15

    Clinically, the amount of uric acid (UA) in biological fluids is closely related to some diseases such as hyperuricemia and gout, thus it is of great significance to sense UA in clinical samples. In this work, red gold nanoclusters (AuNCs) with relatively high fluorescence quantum yield and strong fluorescence emission were facilely available using bovine serum albumin (BSA) as template. The fluorescence of BSA-protected AuNCs can be sensitively quenched by H2O2, which is further capable of sensing UA through the specific catalytic oxidation with uricase, since it generates stoichiometric quantity of H2O2 by-product. The proposed assay allows for the selective detection of UA in the range of 10-800 μM with a detection limit of 6.6 μM, which is applicable to sense UA in clinical samples with satisfactory results, suggesting its great potential for diagnostic purposes. PMID:26992526

  9. Styrene oligomerization as a molecular probe reaction for Brønsted acidity at the nanoscale.

    PubMed

    Aramburo, Luis R; Wirick, Sue; Miedema, Piter S; Buurmans, Inge L C; de Groot, Frank M F; Weckhuysen, Bert M

    2012-05-21

    The Brønsted acid-catalyzed oligomerization of 4-fluorostyrene has been studied on a series of H-ZSM-5 zeolite powders, steamed under different conditions, with a combination of UV-Vis micro-spectroscopy and Scanning Transmission X-ray Microscopy (STXM). UV-Vis micro-spectroscopy and STXM have been used to monitor the relative formation of cyclic and linear dimeric carbocations as a function of the steaming post-treatment (i.e., parent vs. steaming at 600, 700 and 800 °C). It was found that the UV-Vis band intensity ratios of linear to cyclic dimeric species increase from 0.79 (parent H-ZSM-5) over 1.41 (H-ZSM-5 steamed at 600 °C) and 1.88 (H-ZSM-5 steamed at 700 °C) to 2.33 (H-ZSM-5 steamed at 800 °C). STXM confirms this trend in reaction product selectivity, as the relative intensities of the transitions attributed to the presence of the cyclic dimer in the carbon K-edge spectra decrease with increasing severity of the steaming post-treatment. Furthermore, STXM reveals spatial heterogeneities in reaction product formation within the H-ZSM-5 zeolite powders at the nanoscale. More specifically, a shrinking carbon core-shell distribution was detected within the zeolite aggregates, in which the relative amount of cyclic dimeric species is higher in the core relative to the shell of the zeolite aggregate and the relative amount of cyclic dimeric species in the zeolite core gradually decreases with increasing severity of the steaming post-treatment. These differences are rationalized in terms of spatial differences in Brønsted acidity within H-ZSM-5 zeolite powders as well as by changes in the formation process of linear and dimeric carbocations within H-ZSM-5 micro- and mesopores.

  10. Reversible two-photon fluorescent probe for imaging of hypochlorous acid in live cells and in vivo.

    PubMed

    Zhang, Wei; Liu, Wei; Li, Ping; kang, Junqing; Wang, Jiaoyang; Wang, Hui; Tang, Bo

    2015-06-25

    Herein, we have developed a novel reversible two-photon fluorescent probe that is well suited for monitoring HOCl levels selectively and instantaneously. Results showed the reversible and instantaneous responses of the probe towards intracellular HOCl. Moreover, the probe was successfully applied to the imaging of the HOCl levels in zebrafish and mice via two-photon imaging.

  11. Probing the interaction of the amino acid alanine with the surface of ZnO(1010).

    PubMed

    Gao, Y K; Traeger, F; Shekhah, O; Idriss, H; Wöll, C

    2009-10-01

    The adsorption modes and stability of the amino acid alanine (NH(2)-CH(CH(3))-COOH) have been studied on the nonpolar single crystal surface of zinc oxide, ZnO(1010), experimentally by X-ray photoelectron spectroscopy (XPS) and computationally using density functional theory (DFT). Deposition at 200 K was found to lead to the formation of multilayers identified by an XPS N1s peak at 401.7 eV assigned to the NH(3)(+) group, a fingerprint of the zwitterionic structure of alanine in the solid state. Heating to 300 K resulted in the removal of most of the multilayers with the remaining surface coverage estimated to 0.4 with respect to Zn cations. At this temperature most of the alanine molecules are found to be deprotonated (dissociated), yielding a carboxylate species (NH(2)-CH(CH(3))-COO(-) (a) + OH (s); where O is surface oxygen, (a) for adsorbed and (s) for surface species). Further heating of the surface resulted in a gradual decrease of the surface coverage and by 500 K a large fraction of adsorbed alanine molecules have desorbed from the surface. Total energy DFT computations of different adsorbate species identified two stable dissociative adsorption modes: bidentate and monodentate. The bidentate species with adsorption energy of 1.75 eV was found to be more stable than the monodentate species by about 0.7 eV.

  12. Probing the affinity of polyanions for acidic fibroblast growth factor by unfolding kinetics.

    PubMed

    Mach, H; Middaugh, C R

    1994-02-15

    The relationship between ligand-protein affinity and the extent of protein stabilization induced by such interactions has been investigated using the binding of polyanions to acidic fibroblast growth factor (aFGF) as a model system. It was found that the experimentally observed unfolding rate constant of aFGF consists of two components: one equal to the unfolding rate constant of the aFGF-ligand complex and the other the product of the unfolding rate constant of free aFGF, the aFGF-ligand dissociation constant (Kd), and the reciprocal of the molar ligand concentration. This reflects the presence of two possible unfolding pathways: at high ligand excess dissociation is suppressed and slow unfolding of the aFGF-ligand complex itself prevails. When lower concentrations of ligand allows equilibrium-driven appearance of free aFGF, a more rapid unfolding of dissociated protein predominates. Existence of a steady state of dissociated aFGF undergoing unfolding was demonstrated by computer simulation of the elementary events, using experimentally determined rate constants. The potential applications of such simulations are outlined. An equation allowing estimation of dissociation constants from equilibrium denaturation curves obtained in the presence of a varying amount of ligand is also proposed. In addition, determination of initial unfolding rates in the presence of excess protein permits the the stoichiometry of the interaction to be determined.

  13. Probing Gαi1 Protein Activation at Single Amino Acid Resolution

    PubMed Central

    Sun, Dawei; Maeda, Shoji; Matkovic, Milos; Mendieta, Sandro; Mayer, Daniel; Dawson, Roger; Schertler, Gebhard F.X.; Madan Babu, M.; Veprintsev, Dmitry B.

    2016-01-01

    We present comprehensive single amino acid resolution maps of the residues stabilising the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and of the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1-3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4-6. Key residues in this cluster are Y320, crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the inter-domain interface and release of GDP. PMID:26258638

  14. DNAzyme molecular beacon probes for target-induced signal-amplifying colorimetric detection of nucleic acids.

    PubMed

    Fu, Rongzhan; Li, Taihua; Lee, Soo Suk; Park, Hyun Gyu

    2011-01-15

    A novel DNAzyme molecular beacon (DNAzymeMB) strategy was developed for target-induced signal-amplifying colorimetric detection of target nucleic acids. The DNAzymeMB, which exhibits peroxidase activity in its free hairpin structure, was engineered to form a catalytically inactive hybrid through hybridization with a blocker DNA. The presence of target DNA leads to dissociation of the DNAzymeMB from the inactive hybrid through hybridization with the blocker DNA. This process results in recovery of the catalytically active DNAzymeMB, which can catalyze a colorimetric reaction that signals the presence of the target DNA. In addition, a primer was rationally designed to anneal to the blocker DNA of the blocker/target DNA duplex and displace the bound target DNA during the extension reaction. The released target DNA triggers the next cycle involving hybridization with blocker DNA, DNAzymeMB dissociation, primer extension, and target displacement. This unique amplifying strategy leads to the generation of multiple numbers of active DNAzymeMB molecules from a single target molecule and gives a detection limit down to 1 pM, a value that is nearly 3 or 5 orders of magnitude lower than those of previously reported DNAzyme molecular beacon-based DNA detection methods.

  15. Probing the interaction of the amino acid alanine with the surface of ZnO(1010).

    PubMed

    Gao, Y K; Traeger, F; Shekhah, O; Idriss, H; Wöll, C

    2009-10-01

    The adsorption modes and stability of the amino acid alanine (NH(2)-CH(CH(3))-COOH) have been studied on the nonpolar single crystal surface of zinc oxide, ZnO(1010), experimentally by X-ray photoelectron spectroscopy (XPS) and computationally using density functional theory (DFT). Deposition at 200 K was found to lead to the formation of multilayers identified by an XPS N1s peak at 401.7 eV assigned to the NH(3)(+) group, a fingerprint of the zwitterionic structure of alanine in the solid state. Heating to 300 K resulted in the removal of most of the multilayers with the remaining surface coverage estimated to 0.4 with respect to Zn cations. At this temperature most of the alanine molecules are found to be deprotonated (dissociated), yielding a carboxylate species (NH(2)-CH(CH(3))-COO(-) (a) + OH (s); where O is surface oxygen, (a) for adsorbed and (s) for surface species). Further heating of the surface resulted in a gradual decrease of the surface coverage and by 500 K a large fraction of adsorbed alanine molecules have desorbed from the surface. Total energy DFT computations of different adsorbate species identified two stable dissociative adsorption modes: bidentate and monodentate. The bidentate species with adsorption energy of 1.75 eV was found to be more stable than the monodentate species by about 0.7 eV. PMID:19596338

  16. Design and Performance of rRNA Targeted Oligonucleotide Probes for in Situ Detection and Phylogenetic Identification of Microorganisms Inhabiting Acid Mine Drainage Environments.

    PubMed

    Bond, P.L.; Banfield, J.F.

    2001-02-01

    At Iron Mountain, CA, there is an extreme occurrence of acid mine drainage (AMD). This is a result of past mining activity that has exposed a sulfide ore body to weathering and microbial activity. This study presents seven new oligonucleotide probes for the detection of microorganisms at this AMD site by fluorescent in situ hybridization. In the design of these probes we have accounted for a large body of 16S rRNA sequence data recently compiled by us. This was obtained by PCR and cloning directly from environmental DNA and was mostly represented by novel sequences. The probes were developed to include detection of novel and uncultivated organisms. This includes detection for the Thermoplasmales group, a new group of Leptospirillum, the genus Sulfobacillus, the Acidiphilium genus, Acidimicrobium and relatives, and for organisms within the delta Proteobacteria. These probes have been used to examine the abundance and distribution of organisms, including novel and uncultivated taxa, and to clarify their potential contributions to AMD production at the site. We anticipate that these probes will be useful tools for exploration of the microbiology of other natural acidic environments and bioleaching systems. PMID:12032620

  17. Adsorption of 4-picoline and piperidine to the hydrated SiO2 surface: probing the surface acidity with vibrational sum frequency generation spectroscopy.

    PubMed

    Liu, Dingfang; Ma, Gang; Allen, Heather C

    2005-04-01

    Vapor adsorption is an important process influencing the migration and the fate of many organic pollutants in the environment. In this study, vibrational sum frequency generation (SFG) spectroscopy was used to study the adsorption of two surface acidity probe molecules, 4-picoline (pKa = 5.94) and piperidine (pKa = 11.24), onto the amorphous SiO2 surface. The adsorption of 4-picoline onto the silica surface occurs by forming weak hydrogen bonds between the nitrogen atoms of 4-picoline molecules and the hydrogen atoms of surface silanol OH groups. Piperidine molecules are strongly chemisorbed onto the SiO2 surface through the protonation of piperidine molecules by surface silanol OH groups. The SFG results indicate that the surface acidity constant of silanol OH groups (pKa-(HOSi triple bond)) is in the range of 5.94-11.24 at the air/solid interface. Although this range of surface acidity constants is quite wide, it is possible to narrow it by choosing probe molecules with a smaller pKa range. Together with theoretical prediction methods, adsorption studies using vibrational SFG spectroscopy are capable of quantifying the surface acidity of mineral oxides by carefully choosing the acidity probe molecules.

  18. A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes.

    PubMed

    Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule

    2012-05-01

    Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications.

  19. pH at the micellar interface: synthesis of pH probes derived from salicylic acid, acid-base dissociation in sodium dodecyl sulfate micelles, and Poisson-Boltzmann simulation.

    PubMed

    Souza, T P; Zanette, D; Kawanami, A E; de Rezende, L; Ishiki, H M; do Amaral, A T; Chaimovich, H; Agostinho-Neto, A; Cuccovia, I M

    2006-05-01

    The study of the H+ concentration at the micellar interface is a convenient system for modeling the distribution of H+ at interfaces. We have synthesized salicylic acid derivatives to analyze the proton dissociation of both the carboxylic and phenol groups of the probes, determining spectrophotometrically the apparent pK(a)'s (pK(ap)) in sodium dodecyl sulfate, SDS, micelles with and without added salt. The synthesized probes were 2-hydroxy-5-(2-trimethylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumhexanoyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumundecanoyl)benzoate; 2-hydroxy-5-acetylbenzoic acid; and 2-hydroxy-5-dodecanoylbenzoic acid. Upon incorporation into SDS micelles the pK(ap)'s of both carboxylic and phenol groups increased by ca. 3 pH units and NaCl addition caused a decrease in the probe-incorporated pK(ap). The experimental results were fitted with a cell model Poisson-Boltzmann (P-B) equation taking in consideration the effect of salt on the aggregation number of SDS and using the distance of the dissociating group as a parameter. The conformations of the probes were analyzed theoretically using two dielectric constants, e.g., 2 and 78. Both the P-B analysis and conformation calculations can be interpreted by assuming that the acid groups dissociate very close to, or at, the interface. Our results are consistent with the assumption that the intrinsic pK(a)'s of both carboxylic and phenol groups of the salicylic acid probes used here can be taken as those in water. Using this assumption the micellar and salt effects on the pK(ap)'s of the (trialkylammonium)benzoate probes were described accurately using a cell model P-B analysis.

  20. 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

  1. Association of fluorescent probes 1-anilinonaphthalene-8-sulfonate and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid with T7 RNA polymerase.

    PubMed

    Ghosh, Utpal; Das, Mili; Dasgupta, Dipak

    2003-01-01

    T7 RNA polymerase is an enzyme that carries out transcription using DNA as the template and ribonucleotides as the substrates. Here we report the association of the polymerase with 1-anilinonaphthalene-8-sulfonate (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS), which are two fluorescent hydrophobic probes that are frequently used to study structural perturbations in proteins and intermediate states of proteins during folding and unfolding. Our results from the fluorescence titration data show that these two molecules bind to the enzyme with dissociation constants on the micromolar order. The results from the tryptic digestion of the enzyme in the absence and presence of the probes show that they inhibit the rate of tryptic digestion. Circular dichroism spectroscopic studies of the protein in the near UV region indicate that both probes induce tertiary structural changes in the polymerase. There is also a probe (ANS or bis-ANS) induced inhibition of the enzymatic activity. All these results are attributed to association of the probes with the enzyme, leading to an alteration in the conformation of T7 RNA polymerase. This limits the use of these extrinisic probes to the study of the folding properties of the enzyme.

  2. 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.

  3. Synthesis of fluorescent D-amino acids (FDAAs) and their use for probing peptidoglycan synthesis and bacterial growth in situ

    PubMed Central

    Kuru, Erkin; Tekkam, Srinivas; Hall, Edward

    2015-01-01

    Fluorescent D-amino acids (FDAAs) are efficiently incorporated into the peptidoglycan of diverse bacterial species at the sites of active peptidoglycan biosynthesis, allowing specific and covalent probing of bacterial growth with minimal perturbation. Here, we provide a protocol for the synthesis of four FDAAs emitting light in blue, green or red and for their use in peptidoglycan labeling of live bacteria. Our modular synthesis protocol gives easy access to a library of different FDAAs made with commercially available fluorophores. FDAAs can be synthesized in a typical chemistry laboratory in 2–3 days. The simple labeling procedure involves addition of the FDAAs to the bacterial sample for the desired labeling duration and stopping further label incorporation by fixation or by washing away excess dye. We discuss several scenarios for the use of these labels including short or long labeling durations, and the combination of different labels in pure culture or complex environmental samples. Depending on the experiment, FDAA labeling can take as little as 30 s for a rapidly growing species such as Escherichia coli. PMID:25474031

  4. Denaturant effects on HbGp hemoglobin as monitored by 8-anilino-1-naphtalene-sulfonic acid (ANS) probe.

    PubMed

    Barros, Ana E B; Carvalho, Francisco A O; Alves, Fernanda R; Carvalho, José W P; Tabak, Marcel

    2015-03-01

    Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB-HbGp-ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.

  5. A unique "turn-on" fluorescence signalling strategy for highly specific detection of ascorbic acid using carbon dots as sensing probe.

    PubMed

    Fong, Jessica Fung Yee; Chin, Suk Fun; Ng, Sing Muk

    2016-11-15

    Carbon dots (CDs) that showed strong blue fluorescence were successfully synthesised from sodium alginate via furnace pyrolysis. The single step pyrolytic synthesis was simple to perform while yielded CDs with high photostability, good water solubility and minimum by-products. In order to design the probe with "turn-on" sensing capability, the CDs were screened against a series of metal cations to first "turn-off" the fluorescence. It was found that ferric ions (Fe(3+)) were most responsive and effective in quenching the fluorescence of CDs. Based on this observation, the conditioning of the probe was performed to ensure the fluorescence was completely quenched, while not overloading the system with Fe(3+). At the optimised condition, the CDs-Fe(3+) mixture served as a highly specific detection probe for ascorbic acid (AA). The analytical potential of the probe was evaluated and showed a good linear range of response for AA concentration of 24-40μg/mL. The selectivity study against other possible co-existing species was carried out and proved that our unique "turn-on" fluorescence signalling strategy was highly effective and selective towards AA as the target analyte. The probe was demonstrated for quantification of AA in real samples, which was the commercially available vitamin C supplement. The result showed good accuracy with minimum deviation from standard method adopted for validation purpose. PMID:27290666

  6. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

    PubMed Central

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575

  7. Cytotoxicity of food preservatives in cultured rat hepatocytes loaded with linolenic acid.

    PubMed

    Sugihara, N; Shimomichi, K; Furuno, K

    1997-06-01

    We investigated the ability of eight food preservatives to induce lipid peroxidation in normal and alpha-linolenic acid (LNA)-loaded cultured rat hepatocytes. On the addition of sodium dehydroacetate (DHA-Na), potassium sorbate (SA-K) or thiabendazole (TBZ) to the cell culture, lipid peroxidation, assessed in terms of the production of malondialdehyde (MDA), was induced in LNA-loaded cells, but not in normal cells. At the low concentrations, induction of lipid peroxidation in LNA-loaded cells was highest with TBZ, whereas at high concentrations DHA-Na greatly induced lipid peroxidation. The occurrence of lipid peroxidation in LNA-loaded cells was accompanied by a decrease in cellular GSH levels with the three preservatives and by a decrease in cellular protein-SH levels with DHA-Na and TBZ. Furthermore, cell injury, measured by the release of LDH, was produced in LNA-loaded cells exposed to DHA-Na and SA-K. The addition of TBZ caused substantial cell injury in normal cells, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD) almost completely prevented DHA-Na- and SA-K-induced cell injury, and reduced TBZ-induced cell injury. The addition of diphenyl (DP), o-phenylphenol (OPP) or butyl p-hydroxybenzoate (BHB) caused severe cell injury, in association with a marked decrease in cellular levels of both of GSH and protein-SH in both groups of cells. However, lipid peroxidation was not detectable in either group of cells exposed to these preservatives. Sodium propionate (PA-Na) and sodium benzoate (BA-Na) had little effect on any cytotoxic parameter in either group of cells.

  8. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

  9. Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

    PubMed Central

    Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

    2014-01-01

    Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

  10. Top-down characterization of nucleic acids modified by structural probes using high-resolution tandem mass spectrometry and automated data interpretation.

    PubMed

    Kellersberger, Katherine A; Yu, Eizadora; Kruppa, Gary H; Young, Malin M; Fabris, Daniele

    2004-05-01

    A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base

  11. Targeting Acidity in Pancreatic Adenocarcinoma: Multispectral Optoacoustic Tomography Detects pH-low Insertion Peptide Probes in vivo

    PubMed Central

    Kimbrough, Charles W; Khanal, Anil; Zeiderman, Matthew; Khanal, Bigya R.; Burton, Neal C; McMasters, Kelly M; Vickers, Selwyn; Grizzle, William E; McNally, Lacey R

    2015-01-01

    Background pH-low Insertion Peptides (pHLIPs) can serve as a targeting moiety that enables pH-sensitive probes to detect solid tumors. Using these probes in conjunction with multispectral optoacoustic tomography (MSOT) is a promising approach to improve imaging for pancreatic cancer. Methods A pH-sensitive pHLIP (V7) was conjugated to 750 NIR fluorescent dye and evaluated as a targeted probe for pancreatic adenocarcinoma. The pH-insensitive K7 pHLIP served as an untargeted control. Probe binding was assessed in vitro at pH 7.4, 6.8, and 6.6 using human pancreatic cell lines S2VP10 and S2013. Using MSOT, semi-quantitative probe accumulation was then assessed in vivo with a murine orthotopic pancreatic adenocarcinoma model. Results In vitro, the V7–750 probe demonstrated significantly higher fluorescence at pH 6.6 compared to pH 7.4 (S2VP10, p=0.0119; S2013, p=0.0160), while no difference was observed with the K7–750 control (S2VP10, p=0.8783; S2013, p=0.921). In the in vivo S2VP10 model, V7–750 probe resulted in 782.5 MSOT a.u. signal compared to 5.3 MSOT a.u. in K7–750 control in tumor (p= 0.0001). Similarly, V7–750 probe signal was 578.3 MSOT a.u. in the S2013 model compared to K7–750 signal at 5.1 MSOT a.u. (p=0.0005). There was minimal off-target accumulation of the V7–750 probe within the liver or kidney, and probe distribution was confirmed with ex vivo imaging. Conclusion Compared to pH-insensitive controls, V7–750 pH-sensitive probe specifically targets pancreatic adenocarcinoma, and has minimal off-target accumulation. The non-invasive detection of pH-targeted probes by means of MSOT represents a promising modality to improve the detection and monitoring of pancreatic cancer. PMID:26124201

  12. A rapid microwave synthesis of nitrogen-sulfur co-doped carbon nanodots as highly sensitive and selective fluorescence probes for ascorbic acid.

    PubMed

    Duan, Junxia; Yu, Jie; Feng, Suling; Su, Li

    2016-06-01

    A ultrafast one-step microwave-assisted method was developed for the synthesis of nitrogen-sulfur co-doped carbon nanodots (N,S-CDs) by using ethylenediamine as the carbon source and sulfamic acid as the surface passivation reagent. The morphology and the properties of N,S-CDs were explored by a series of techniques, such as high-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, UV-vis absorption and fluorescence spectroscopy. The prepared N,S-CDs exhibit bright blue photoluminescence with a high fluorescence quantum yield (FLQY) up to 28%, and high stability and excellent water solubility. A N,S-CDs-based fluorescent probe was developed for sensitive detection ascorbic acid (AA) in the presence of Cu(2+), based on the mechanism that AA reduces Cu(2+) to Cu(+), then Cu(+) quenches the fluorescence of N,S-CDs through electron or energy transfer due to the interaction between Cu(+) and thiol ligand on the N,S-CDs surface. The observed linear response concentration range was from 0.057 to 4.0μM to AA with a detection limit as low as 18nM. The probe exhibited a highly selective response toward AA even in the presence of possible interfering substances, such as uric acid and citric acid. Moreover, these promising features made the sensing system used for the analysis of human serum and urine samples. PMID:27130124

  13. Locked nucleic acid based beacons for surface interaction studies and biosensor development.

    PubMed

    Martinez, Karen; Estevez, M-Carmen; Wu, Yanrong; Phillips, Joseph A; Medley, Colin D; Tan, Weihong

    2009-05-01

    DNA sensors and microarrays permit fast, simple, and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective, and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon's poor stability because of the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics, and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity, and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization.

  14. Locked nucleic acid based beacons for surface interaction studies and biosensor development

    PubMed Central

    Martinez, Karen; Estevez, M.-Carmen; Wu, Yanrong; Phillips, Joseph A.; Medley, Colin D.; Tan, Weihong

    2011-01-01

    DNA sensors and microarrays permit fast, simple and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon’s poor stability due to the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization. PMID:19351140

  15. Linolenic acid grafted hyaluronan: Process development, structural characterization, biological assessing, and stability studies.

    PubMed

    Huerta-Angeles, Gloria; Brandejsová, Martina; Kulhánek, Jaromír; Pavlík, Vojtěch; Šmejkalová, Daniela; Vágnerová, Hana; Velebný, Vladimír

    2016-11-01

    In this study, hyaluronan (HA) was grafted with alpha-linolenic acidLNA) by benzoyl mixed anhydrides methodology, which allowed the derivatization of HA under mild reaction conditions. The reaction was optimized and transferred from laboratory to semi-scale production. The derivative revealed an unexpected cytotoxicity after oven drying and storage at 40°C. For this reason, the storage conditions of sodium linolenyl hyaluronate (αLNA-HA) were optimized in order to preserve the beneficial effect of the derivative. Oven, spray dried and lyophilized samples were prepared and stored at -20°C, 4°C and 25°C up to 6 months. A comprehensive material characterization including stability study of the derivative, as well as evaluation of possible changes on chemical structure and presence of peroxidation products were studied by Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), thermogravimetric analysis (TGA) and complemented with assessment of in vitro viability on mouse fibroblasts NIH-3T3. The most stable αLNA-HA derivative was obtained after spray drying and storage at ambient temperature under inert atmosphere. The choice of inert atmosphere is recommended to suppress oxidation of αLNA supporting the positive influence of the derivative on cell viability. The encapsulation of hydrophobic drugs of αLNA-HA were also demonstrated. PMID:27516333

  16. A peptide nucleic acid-functionalized carbon nitride nanosheet as a probe for in situ monitoring of intracellular microRNA.

    PubMed

    Liao, Xianjiu; Wang, Quanbo; Ju, Huangxian

    2015-06-21

    A novel probe for recognition of both cancer cells and intracellular microRNA (miRNA) is designed by functionalizing a carbon nitride nanosheet (f-CNNS) with a Cy5-labeled peptide nucleic acid (Cy5-PNA) and folate. The interaction between Cy5-PNA and CNNS quenches the fluorescence of Cy5, and the presence of folate endows the probe with good specificity to folate acceptor overexpressed cells. The probe can be specifically taken up by cancer cells with an incubation step. Upon the recognition of the PNA to complementary miRNA, the hybridization product is released from the CNNS surface, which leads to the fluorescence recovery and provides a specific method for sensing of miRNA. Thus, this probe can be used for cell-specific intracellular miRNA sensing with a confocal microscope. Using miRNA-18a as a target model, the dynamic changes of its expression level inside living cells can be monitored with the proposed method. This method possesses promising applications in the study of miRNA related bioprocesses and biomedicine.

  17. Simultaneous brain and blood microdialysis study with a new removable venous probe. Serotonin and 5-hydroxyindolacetic acid changes after D-norfenfluramine or fluoxetine.

    PubMed

    Páez, X; Hernández, L

    1996-01-01

    A removable intravenous microdialysis probe was developed and simultaneously used with a removable microdialysis probe placed in the lateral hypothalamus (LH). Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) changes in blood and brain dialysates were measured by HPLC-EC after an i.p. injection of 5 mg/kg d-norfenfluramine (dNF) or 10 mg/kg fluoxetine (FLU) in freely moving rats. 5-HT in the LH significantly increased after both drugs, but the rise was larger and faster with dNF [F(7,28)=4.0 p<0.05] than with FLU [F(5,20)=5.0 p<0.01]. By contrast, in venous blood 5-HT increased after FLU [F(5,20)=2.96 p<0.05] but not after dNF. 5-HIAA after both drugs continued decreasing significantly in the LH [dNF F(7,28)=11.4 p<0.01; FLU F(5,20)=22.8 p<0.01], but it did not change in blood. Simultaneous dialysis in brain and blood allowed evaluation of the differential effects of dNF and FLU on 5-HT and 5-HIAA in the two places. Removable venous probes prevented the inflammatory reaction that may occur around permanently implanted probes, and the dialysis could be more efficient and with less risk of clogging.

  18. A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid.

    PubMed

    Yang, Wen; Tian, Jianniao; Ma, Yefei; Wang, Lijun; Zhao, Yanchun; Zhao, Shulin

    2015-11-01

    A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3'-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully. PMID:26572843

  19. A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid.

    PubMed

    Yang, Wen; Tian, Jianniao; Ma, Yefei; Wang, Lijun; Zhao, Yanchun; Zhao, Shulin

    2015-11-01

    A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3'-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully.

  20. Assessing the acidity of high silica chabazite H-SSZ-13 by FTIR using CO as molecular probe: Comparison with H-SAPO-34.

    PubMed

    Bordiga, Silvia; Regli, Laura; Cocina, Donato; Lamberti, Carlo; Bjørgen, Morten; Lillerud, Karl Petter

    2005-02-24

    Zeolitic materials based on the chabazite topology, such as H-SAPO-34, possess unique shape-selectivity properties for converting methanol into light olefins. In addition to the topology, zeolite acidity is inherently linked to catalyst activity and selectivity. The acidic properties of high silica chabazite (H-SSZ-13) have attracted much attention in the past decade because the material represents an idealized model system having one acidic site per cage. Conclusions drawn so far have essentially been founded on quantum chemical methods. An experimentally based benchmark of the acidity of H-SSZ-13 has hitherto not been available. In this work, transmission FTIR spectroscopy provides a description of the different acidic sites of H-SSZ-13 by using CO as molecular probe at 70 K. The results demonstrate that H-SSZ-13 is a strongly Brønsted acidic material, essentially having two distinct families of acidic sites. In contrast to numerous preceding reports, we find it fundamental to consider proton distributions among all four possible sites, and do not delimit the interpretations to only two sites. The present data consistently suggest the most abundant family of protons to have three members being located on different crystalline positions on the eight-membered-ring window giving access to the chabazite cage. Consequently, these protons are exposed to two neighboring cages. The second, and less abundant family, is constituted by only one site that is situated on the six-membered ring defining the top/bottom of the barrel-shaped chabazite cage. This proton is therefore only exposed to one cage and requires a higher CO pressure to form adducts. Toward CO, both families of sites possess the same acidity. Parallel experiments were also carried out for the isostructural and commercially important H-SAPO-34 having an equal density of acidic sites. This is the first attempt to directly compare, on an experimental basis, the acidity of these two materials.

  1. Assessing the acidity of high silica chabazite H-SSZ-13 by FTIR using CO as molecular probe: Comparison with H-SAPO-34.

    PubMed

    Bordiga, Silvia; Regli, Laura; Cocina, Donato; Lamberti, Carlo; Bjørgen, Morten; Lillerud, Karl Petter

    2005-02-24

    Zeolitic materials based on the chabazite topology, such as H-SAPO-34, possess unique shape-selectivity properties for converting methanol into light olefins. In addition to the topology, zeolite acidity is inherently linked to catalyst activity and selectivity. The acidic properties of high silica chabazite (H-SSZ-13) have attracted much attention in the past decade because the material represents an idealized model system having one acidic site per cage. Conclusions drawn so far have essentially been founded on quantum chemical methods. An experimentally based benchmark of the acidity of H-SSZ-13 has hitherto not been available. In this work, transmission FTIR spectroscopy provides a description of the different acidic sites of H-SSZ-13 by using CO as molecular probe at 70 K. The results demonstrate that H-SSZ-13 is a strongly Brønsted acidic material, essentially having two distinct families of acidic sites. In contrast to numerous preceding reports, we find it fundamental to consider proton distributions among all four possible sites, and do not delimit the interpretations to only two sites. The present data consistently suggest the most abundant family of protons to have three members being located on different crystalline positions on the eight-membered-ring window giving access to the chabazite cage. Consequently, these protons are exposed to two neighboring cages. The second, and less abundant family, is constituted by only one site that is situated on the six-membered ring defining the top/bottom of the barrel-shaped chabazite cage. This proton is therefore only exposed to one cage and requires a higher CO pressure to form adducts. Toward CO, both families of sites possess the same acidity. Parallel experiments were also carried out for the isostructural and commercially important H-SAPO-34 having an equal density of acidic sites. This is the first attempt to directly compare, on an experimental basis, the acidity of these two materials. PMID:16851287

  2. Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis.

    PubMed

    Rossi, Stefano; Lesignoli, Francesca; Germini, Andrea; Faccini, Andrea; Sforza, Stefano; Corradini, Roberto; Marchelli, Rosangela

    2007-04-01

    PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.

  3. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid) complex as a carrier.

    PubMed

    Geng, Yao; Lin, Dajie; Shao, Lijia; Yan, Feng; Ju, Huangxian

    2013-01-01

    A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.

  4. Cox-2 inhibitory effects of naturally occurring and modified fatty acids.

    PubMed

    Ringbom, T; Huss, U; Stenholm, A; Flock, S; Skattebøl, L; Perera, P; Bohlin, L

    2001-06-01

    In the search for new cyclooxygenase-2 (COX-2) selective inhibitors, the inhibitory effects of naturally occurring fatty acids and some of their structural derivatives on COX-2-catalyzed prostaglandin biosynthesis were investigated. Among these fatty acids, linoleic acid (LA), alpha-linolenic acid (alpha-LNA), myristic acid, and palmitic acid were isolated from a CH(2)Cl(2) extract of the plant Plantago major by bioassay-guided fractionation. Inhibitory effects of other natural, structurally related fatty acids were also investigated: stearic acid, oleic acid, pentadecanoic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Further, the inhibitory effects of these compounds on COX-2- and COX-1-catalyzed prostaglandin biosynthesis was compared with the inhibition of some synthesized analogues of EPA and DHA with ether or thioether functions. The most potent COX-2-catalyzed prostaglandin biosynthesis inhibitor was all-(Z)-5-thia-8,11,14,17-eicosatetraenoic acid (2), followed by EPA, DHA, alpha-LNA, LA, (7E,11Z,14Z,17Z)-5-thiaeicosa-7,11,14,17-tetraenoic acid, all-(Z)-3-thia-6,9,12,15-octadecatetraenoic acid, and (5E,9Z,12Z,15Z,18Z)-3-oxaheneicosa-5,9,12,15,18-pentaenoic acid, with IC(50) values ranging from 3.9 to180 microM. The modified compound 2 and alpha-LNA were most selective toward COX-2, with COX-2/COX-1 ratios of 0.2 and 0.1, respectively. This study shows that several of the natural fatty acids as well as all of the semisynthetic thioether-containing fatty acids inhibited COX-2-catalyzed prostaglandin biosynthesis, where alpha-LNA and compound 2 showed selectivity toward COX-2. PMID:11421736

  5. A naphthalene-based two-photon fluorescent probe for selective and sensitive detection of endogenous hypochlorous acid.

    PubMed

    Zhou, Xiao-Hong; Jiang, Yu-Ren; Zhao, Xiong-Jie; Guo, Dong

    2016-11-01

    An efficient naphthalene-based two-photon fluorescent probe for endogenous HClO has been reported in the present study, which consists of a 6-(2-benzothiazolyl)-2-naphthalenol fluorophore connected with a 4-aminophenol (the fluorescence quenching and response group). This probe exhibits a high selectivity and excellent sensitivity with a detection limit of 7.6nM over other reactive oxygen species and analyte species, and the fluorescence intensity enhanced 103-fold when responsed. Furthermore, it was successfully used for two-photon imaging of endogenous HClO in live cells with high-resolution. PMID:27591640

  6. A naphthalene-based two-photon fluorescent probe for selective and sensitive detection of endogenous hypochlorous acid.

    PubMed

    Zhou, Xiao-Hong; Jiang, Yu-Ren; Zhao, Xiong-Jie; Guo, Dong

    2016-11-01

    An efficient naphthalene-based two-photon fluorescent probe for endogenous HClO has been reported in the present study, which consists of a 6-(2-benzothiazolyl)-2-naphthalenol fluorophore connected with a 4-aminophenol (the fluorescence quenching and response group). This probe exhibits a high selectivity and excellent sensitivity with a detection limit of 7.6nM over other reactive oxygen species and analyte species, and the fluorescence intensity enhanced 103-fold when responsed. Furthermore, it was successfully used for two-photon imaging of endogenous HClO in live cells with high-resolution.

  7. Sensing of a nucleic acid binding protein via a label-free perylene probe fluorescence recovery assay.

    PubMed

    Liao, Dongli; Li, Wenying; Chen, Jian; Jiao, Huping; Zhou, Huipeng; Wang, Bin; Yu, Cong

    2013-10-01

    A novel label-free fluorescence recovery assay for the sensing of a DNA binding protein has been developed. A transcription factor c-Jun protein, and a 21 base pair duplex DNA containing the c-Jun protein binding site (J-DNA) were selected. J-DNA was mixed with a cationic fluorescent perylene probe (compound 1), and induced aggregation of the probe. Quenching of the probe's fluorescence was observed. However, when c-Jun protein was mixed with the J-DNA, c-Jun bound to the duplex DNA, which reduced the degree of the induced perylene probe aggregation, and a turn on fluorescence signal was observed. The recovered fluorescence intensity was directly related to the amount of c-Jun added. The method is highly selective, six non-DNA binding proteins and one randomly selected 21 base pair duplex DNA (con-1) were tested. No noticeable compound 1 fluorescence recovery was observed. Mutations were also introduced to the c-Jun recognition sequence and much reduced fluorescence recovery was observed. Our assay is label-free, convenient, inexpensive, and fast. It can be used in biomedical research such as high throughput screening of drugs targeted at DNA-binding proteins.

  8. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

    PubMed Central

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L.

    2014-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating 13C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% 13C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation. PMID:25217022

  9. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for Applications in Stable Isotope Probing.

    PubMed

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L; Mohn, William W

    2014-12-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating (13)C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography-tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% (13)C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation.

  10. Molecular cloning and functional characterization of arachidonate 5-lipoxygenase (Alox5), and its expression in response to the ratio of linolenic acid to linoleic acid in diets of large yellow croaker (Larmichthys crocea).

    PubMed

    Wang, Tianjiao; Zuo, Rantao; Mai, Kangsen; Xu, Wei; Ai, Qinghui

    2016-11-01

    This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA. PMID:27378407

  11. Molecular cloning and functional characterization of arachidonate 5-lipoxygenase (Alox5), and its expression in response to the ratio of linolenic acid to linoleic acid in diets of large yellow croaker (Larmichthys crocea).

    PubMed

    Wang, Tianjiao; Zuo, Rantao; Mai, Kangsen; Xu, Wei; Ai, Qinghui

    2016-11-01

    This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA.

  12. Mapping the nicking efficiencies of nickase R.BbvCI for side-specific LNA-substituted substrates using rolling circle amplification

    PubMed Central

    Wei, Hua; Zhao, Guojie; Hu, Tianyu; Tang, Suming; Jiang, Jiquan; Hu, Bo; Guan, Yifu

    2016-01-01

    We used a novel asymmetric cleavage analysis method based on rolling circle amplification (RCA) to determine the effects of LNA modification of substrate on the two subunits of R.BbvCI cleavage. We designed two sets of cleavage circular substrates by using two different ligation strategies and analyzed the single strand cleavage efficiency affected by different modification positions both from the cleaved strands and the uncleaved strands. Results showed that the effects of LNA on cleavage rates of modified strands and unmodified strands were both site-dependent. The Nb.BbvCI and Nt.BbvCI were affected by LNA modification in different way. Most of the modification positions showed strong inhibition of both of these two nickases cleavage. However, the modification in T3 position of bottom strand hardly affected both of the two nickases activities. The results suggested an intimated interaction between the two subunits of R.BbvCI, and the T3 position in bottom strand might be a less tight position which was hard to be disturbed. PMID:27582033

  13. Detection of hydrofluoric acid by a SiO2 sol-gel coating fiber-optic probe based on reflection-based localized surface plasmon resonance.

    PubMed

    Chen, I-Cherng; Lin, Shiu-Shiung; Lin, Tsao-Jen; Du, Je-Kang

    2011-01-01

    A novel fiber-optic probe based on reflection-based localized surface plasmon resonance (LSPR) was developed to quantify the concentration of hydrofluoric acid (HF) in aqueous solutions. The LSPR sensor was constructed with a gold nanoparticle-modified PMMA fiber, integrated with a SiO(2) sol-gel coating. This fiber-sensor was utilized to assess the relationship between HF concentration and SiO(2) sol-gel layer etching reduction. The results demonstrated the LSPR sensor was capable of detecting HF-related erosion of hydrofluoric acid solutions of concentrations ranging from 1% to 5% using Relative RI Change Rates. The development of the LSPR sensor constitutes the basis of a detector with significant sensitivity for practical use in monitoring HF solution concentrations.

  14. Detection of Hydrofluoric Acid by a SiO2 Sol-Gel Coating Fiber-Optic Probe Based on Reflection-Based Localized Surface Plasmon Resonance

    PubMed Central

    Chen, I-Cherng; Lin, Shiu-Shiung; Lin, Tsao-Jen; Du, Je-Kang

    2011-01-01

    A novel fiber-optic probe based on reflection-based localized surface plasmon resonance (LSPR) was developed to quantify the concentration of hydrofluoric acid (HF) in aqueous solutions. The LSPR sensor was constructed with a gold nanoparticle-modified PMMA fiber, integrated with a SiO2 sol-gel coating. This fiber-sensor was utilized to assess the relationship between HF concentration and SiO2 sol-gel layer etching reduction. The results demonstrated the LSPR sensor was capable of detecting HF-related erosion of hydrofluoric acid solutions of concentrations ranging from 1% to 5% using Relative RI Change Rates. The development of the LSPR sensor constitutes the basis of a detector with significant sensitivity for practical use in monitoring HF solution concentrations. PMID:22319388

  15. Assay of the human liver citric acid cycle probe phenylacetylglutamine and of phenylacetate in plasma by gas chromatography-mass spectrometry.

    PubMed

    Yang, D; Beylot, M; Agarwal, K C; Soloviev, M V; Brunengraber, H

    1993-07-01

    Phenylacetate, derived from phenylalanine, is converted in human and primate liver to phenylacetylglutamine. The latter has been used to assess the labeling pattern of liver citric acid cycle intermediates. We present gas chromatographic-mass spectrometric assays of phenylacetylglutamine, phenylacetate, and phenylalanine in biological fluids. The compounds are derivatized with dimethylformamide dimethyl acetal. Limits of detection are 0.1 nmol for phenylacetylglutamine and phenylacetate and 2 nmol for phenylalanine. Baseline plasma concentrations of phenylacetate and phenylacetylglutamine and 1 and 3 microM, respectively. The 24-h urinary excretions of phenylacetate and phenylacetylglutamine are about 4 mumol and 1 mmol, respectively. Ingestion of phenylalanine (in the form of aspartame) by a human is followed by sequential increases in phenylacetate and phenylacetylglutamine concentrations in plasma and urine. This assay opens the way to noninvasive probing of the 13C-labeling pattern of liver citric acid cycle intermediates in humans.

  16. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

    PubMed

    Jaye, M; de la Salle, H; Schamber, F; Balland, A; Kohli, V; Findeli, A; Tolstoshev, P; Lecocq, J P

    1983-04-25

    A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

  17. Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry.

    PubMed

    Zhu, Quan-Fei; Hao, Yan-Hong; Liu, Ming-Zhou; Yue, Jiang; Ni, Jian; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-09-01

    Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes

  18. Incorporation of extra amino acids in peptide recognition probe to improve specificity and selectivity of an electrochemical peptide-based sensor.

    PubMed

    Zaitouna, Anita J; Maben, Alex J; Lai, Rebecca Y

    2015-07-30

    We investigated the effect of incorporating extra amino acids (AA) at the n-terminus of the thiolated and methylene blue-modified peptide probe on both specificity and selectivity of an electrochemical peptide-based (E-PB) HIV sensor. The addition of a flexible (SG)3 hexapeptide is, in particular, useful in improving sensor selectivity, whereas the addition of a highly hydrophilic (EK)3 hexapeptide has shown to be effective in enhancing sensor specificity. Overall, both E-PB sensors fabricated using peptide probes with the added AA (SG-EAA and EK-EAA) showed better specificity and selectivity, especially when compared to the sensor fabricated using a peptide probe without the extra AA (EAA). For example, the selectivity factor recorded in the 50% saliva was ∼2.5 for the EAA sensor, whereas the selectivity factor was 7.8 for both the SG-EAA and EK-EAA sensors. Other sensor properties such as the limit of detection and dynamic range were minimally affected by the addition of the six AA sequence. The limit of detection was 0.5 nM for the EAA sensor and 1 nM for both SG-EAA and EK-EAA sensors. The saturation target concentration was ∼200 nM for all three sensors. Unlike previously reported E-PB HIV sensors, the peptide probe functions as both the recognition element and antifouling passivating agent; this modification eliminates the need to include an additional antifouling diluent, which simplifies the sensor design and fabrication protocol.

  19. Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with α-linolenic acid and the transport mechanism of the conjugates.

    PubMed

    Xiao, Yuliang; Li, Pingli; Cheng, Yanna; Zhang, Xinke; Sheng, Juzheng; Wang, Decai; Li, Juan; Zhang, Qian; Zhong, Chuanqing; Cao, Rui; Wang, Fengshan

    2014-04-25

    The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides-based self-assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. α-Linolenic acid-low molecular weight chondroitin sulfate polymers(α-LNA-LMCS) were successfully synthesized, and characterized by FTIR, (1)HNMR, TGA/DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half-life (t₁/₂) extension of LNA-LMCS2 in rats were evidenced by intragastric administration in comparison with CS and LMCS. Caco-2 transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2 was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, α-LNA-LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with α-LNA-LMCS micelles, which further certified the capacity of α-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, LNA-LMCS2 with low cytotoxicity and high bioavailability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc.

  20. Fluorescence sensing of phosdrin pesticide by the luminescent Eu(III)- and Tb(III)-bis(coumarin-3-carboxylic acid) probes

    NASA Astrophysics Data System (ADS)

    Hussein, Belal H. M.; Khairy, Gasser M.; Kamel, Rasha M.

    2016-04-01

    Luminescence quenching of the Eu(III)- and Tb(III)-bis (coumarin-3-carboxylic acid) (Ln(III)-(CCA)2) probes has been studied in the presence of organophosphorus or organochlorine pesticides; Phosdrin (P1), Malathion (P2), Profenofos (P3), Formothion (P4), Heptachlor (P5), and Endosulfan (P6). The luminescence intensity of lanthanide complex probes Ln(III)-(CCA)2 decreases as the concentration of the Phosdrin pesticide increases, while the other investigated pesticides have no significant influence on the lanthanide fluorescent intensities. It is observed that the quenching of Eu(III) and Tb(III)-coumarin-3-carboxylic acid by Phosdrin proceeds via static quenching processes according to Stern-Volmer plot. The binding constants (K) and the thermodynamic parameters of the interaction of Ln(III)-(CCA)2 with Phosdrin have been determined. A direct method for the determination of the Phosdrin in ethanol has been developed based on the luminescence changes of the Ln(III)-(CCA)2-phosdrin ternary complexes. The detection limits of P1 were 6.28 and 1.07 μM in case of Eu(III) and Tb(III)-complex, respectively. The influence of various interfering species on the detection of P1 has been investigated to assess the analytical applicability of the method. The new method was applied to determine the Phosdrin pesticide in different types of water samples.

  1. Fluorescence sensing of phosdrin pesticide by the luminescent Eu(III)- and Tb(III)-bis(coumarin-3-carboxylic acid) probes.

    PubMed

    Hussein, Belal H M; Khairy, Gasser M; Kamel, Rasha M

    2016-04-01

    Luminescence quenching of the Eu(III)- and Tb(III)-bis (coumarin-3-carboxylic acid) (Ln(III)-(CCA)2) probes has been studied in the presence of organophosphorus or organochlorine pesticides; Phosdrin (P1), Malathion (P2), Profenofos (P3), Formothion (P4), Heptachlor (P5), and Endosulfan (P6). The luminescence intensity of lanthanide complex probes Ln(III)-(CCA)2 decreases as the concentration of the Phosdrin pesticide increases, while the other investigated pesticides have no significant influence on the lanthanide fluorescent intensities. It is observed that the quenching of Eu(III) and Tb(III)-coumarin-3-carboxylic acid by Phosdrin proceeds via static quenching processes according to Stern-Volmer plot. The binding constants (K) and the thermodynamic parameters of the interaction of Ln(III)-(CCA)2 with Phosdrin have been determined. A direct method for the determination of the Phosdrin in ethanol has been developed based on the luminescence changes of the Ln(III)-(CCA)2-phosdrin ternary complexes. The detection limits of P1 were 6.28 and 1.07 μM in case of Eu(III) and Tb(III)-complex, respectively. The influence of various interfering species on the detection of P1 has been investigated to assess the analytical applicability of the method. The new method was applied to determine the Phosdrin pesticide in different types of water samples.

  2. A novel adenosine-based molecular beacon probe for room temperature nucleic acid rapid detection in cotton thread device.

    PubMed

    Du, Ting-E; Wang, Yiyun; Zhang, Yi; Zhang, Tian; Mao, Xun

    2015-02-25

    We used cotton thread as substrate to develop a novel room temperature DNA detection device for low-cost, sensitive and rapid detection of a human genetic disease, hereditary tyrosinemia type I related DNA sequences. A novel adenosine based molecular beacon (ABMB) probe modified on gold nanoparticle was used as reporter probe. In the presence of coralyne, a small molecule which can react with adenosines, the ABMB would form a hairpin structure just like traditional molecular beacon used extensively. In the presence of target DNA sequences, the hairpin structure of ABMB modified on gold nanoparticles will be opened and the biotin group modified at one end of the DNA probes will be released and react with the streptavidin immobilized on the test zone of the cotton thread. The response of the thread based DNA test device is linear over the range of 2.5-100 nM complementary DNA. The ability of our developed device for discriminating the single base mismatched DNA related to a human genetic disease, hereditary tyrosinemia type I, was improved comparing with previous report. It is worth mentioning that the whole assay procedure for DNA test is performed under room temperature which simplified the assay procedures greatly.

  3. Probing the general time scale question of boronic acid binding with sugars in aqueous solution at physiological pH.

    PubMed

    Ni, Nanting; Laughlin, Sarah; Wang, Yingji; Feng, You; Zheng, Yujun; Wang, Binghe

    2012-05-01

    The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pairs examined, reactions were complete within seconds. The k(on) values with various sugars follow the order of D-fructose>D-tagatose>D-mannose>D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the 'on' rate is the key factor determining the binding constant.

  4. Acid-base equilibrium dynamics in methanol and dimethyl sulfoxide probed by two-dimensional infrared spectroscopy.

    PubMed

    Lee, Chiho; Son, Hyewon; Park, Sungnam

    2015-07-21

    Two-dimensional infrared (2DIR) spectroscopy, which has been proven to be an excellent experimental method for studying thermally-driven chemical processes, was successfully used to investigate the acid dissociation equilibrium of HN3 in methanol (CH3OH) and dimethyl sulfoxide (DMSO) for the first time. Our 2DIR experimental results indicate that the acid-base equilibrium occurs on picosecond timescales in CH3OH but that it occurs on much longer timescales in DMSO. Our results imply that the different timescales of the acid-base equilibrium originate from different proton transfer mechanisms between the acidic (HN3) and basic (N3(-)) species in CH3OH and DMSO. In CH3OH, the acid-base equilibrium is assisted by the surrounding CH3OH molecules which can directly donate H(+) to N3(-) and accept H(+) from HN3 and the proton migrates through the hydrogen-bonded chain of CH3OH. On the other hand, the acid-base equilibrium in DMSO occurs through the mutual diffusion of HN3 and N3(-) or direct proton transfer. Our 2DIR experimental results corroborate different proton transfer mechanisms in the acid-base equilibrium in protic (CH3OH) and aprotic (DMSO) solvents.

  5. Field-Based Stable Isotope Probing Reveals the Identities of Benzoic Acid-Metabolizing Microorganisms and Their In Situ Growth in Agricultural Soil▿

    PubMed Central

    Pumphrey, Graham M.; Madsen, Eugene L.

    2008-01-01

    We used a combination of stable isotope probing (SIP), gas chromatography-mass spectrometry-based respiration, isolation/cultivation, and quantitative PCR procedures to discover the identity and in situ growth of soil microorganisms that metabolize benzoic acid. We added [13C]benzoic acid or [12C]benzoic acid (100 μg) once, four times, or five times at 2-day intervals to agricultural field plots. After monitoring 13CO2 evolution from the benzoic acid-dosed soil, field soils were harvested and used for nucleic acid extraction and for cultivation of benzoate-degrading bacteria. Exposure of soil to benzoate increased the number of culturable benzoate degraders compared to unamended soil, and exposure to benzoate shifted the dominant culturable benzoate degraders from Pseudomonas species to Burkholderia species. Isopycnic separation of heavy [13C]DNA from the unlabeled fraction allowed terminal restriction fragment length polymorphism (T-RFLP) analyses to confirm that distinct 16S rRNA genes were localized in the heavy fraction. Phylogenetic analysis of sequenced 16S rRNA genes revealed a predominance (15 of 58 clones) of Burkholderia species in the heavy fraction. Burkholderia sp. strain EBA09 shared 99.5% 16S rRNA sequence similarity with a group of clones representing the dominant RFLP pattern, and the T-RFLP fragment for strain EBA09 and a clone from that cluster matched the fragment enriched in the [13C]DNA fraction. Growth of the population represented by EBA09 during the field-dosing experiment was demonstrated by using most-probable-number-PCR and primers targeting EBA09 and the closely related species Burkholderia hospita. Thus, the target population identified by SIP not only actively metabolized benzoic acid but reproduced in the field upon the addition of the substrate. PMID:18469130

  6. Field-based stable isotope probing reveals the identities of benzoic acid-metabolizing microorganisms and their in situ growth in agricultural soil.

    PubMed

    Pumphrey, Graham M; Madsen, Eugene L

    2008-07-01

    We used a combination of stable isotope probing (SIP), gas chromatography-mass spectrometry-based respiration, isolation/cultivation, and quantitative PCR procedures to discover the identity and in situ growth of soil microorganisms that metabolize benzoic acid. We added [(13)C]benzoic acid or [(12)C]benzoic acid (100 microg) once, four times, or five times at 2-day intervals to agricultural field plots. After monitoring (13)CO(2) evolution from the benzoic acid-dosed soil, field soils were harvested and used for nucleic acid extraction and for cultivation of benzoate-degrading bacteria. Exposure of soil to benzoate increased the number of culturable benzoate degraders compared to unamended soil, and exposure to benzoate shifted the dominant culturable benzoate degraders from Pseudomonas species to Burkholderia species. Isopycnic separation of heavy [(13)C]DNA from the unlabeled fraction allowed terminal restriction fragment length polymorphism (T-RFLP) analyses to confirm that distinct 16S rRNA genes were localized in the heavy fraction. Phylogenetic analysis of sequenced 16S rRNA genes revealed a predominance (15 of 58 clones) of Burkholderia species in the heavy fraction. Burkholderia sp. strain EBA09 shared 99.5% 16S rRNA sequence similarity with a group of clones representing the dominant RFLP pattern, and the T-RFLP fragment for strain EBA09 and a clone from that cluster matched the fragment enriched in the [(13)C]DNA fraction. Growth of the population represented by EBA09 during the field-dosing experiment was demonstrated by using most-probable-number-PCR and primers targeting EBA09 and the closely related species Burkholderia hospita. Thus, the target population identified by SIP not only actively metabolized benzoic acid but reproduced in the field upon the addition of the substrate.

  7. A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases*

    PubMed Central

    Kallemeijn, Wouter W.; Witte, Martin D.; Voorn-Brouwer, Tineke M.; Walvoort, Marthe T. C.; Li, Kah-Yee; Codée, Jeroen D. C.; van der Marel, Gijsbert A.; Boot, Rolf G.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.

    2014-01-01

    Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes. PMID:25344605

  8. Development of a Peptide Nucleic Acid Probe to Trichosporon Species and Identification of Trichosporonosis by Use of In Situ Hybridization in Formalin-Fixed and Paraffin-Embedded (FFPE) Sections

    PubMed Central

    Shinozaki, Minoru; Okubo, Yoichiro; Sasai, Daisuke; Nakayama, Haruo; Murayama, Somay Yamagata; Ide, Tadashi; Wakayama, Megumi; Ishiwatari, Takao; Tochigi, Naobumi; Nemoto, Tetsuo

    2013-01-01

    In order to identify Trichosporon species in formalin-fixed and paraffin-embedded sections from which visual discrimination of non-glabrata Candida species is mostly ineffective but critical for the choice of antifungals, we tested the usefulness of a newly designed peptide nucleic acid probe (PNA) for in situ hybridization (ISH). Results confirmed the usefulness of ISH with our PNA probe in identifying Trichosporon species from Candida albicans. PMID:23100341

  9. Thio-ketosides of sialic acid containing aryl azides: potential photo-affinity probes for analysis of neuraminidases and sialic acid binding proteins

    SciTech Connect

    Warner, T.G.; Lee, L.A.

    1986-05-01

    To date, only a single report describing the synthesis of thio-ketosides of sialic acid has appeared. In this procedure, the pseudo thiourea of acetoneuraminic acid methyl ester (NTU) was used to prepare the sodium thiolate salt. However, in their hands, the preparation of NTU was not straight-forward, and in subsequent reactions thio glycosides were not obtained. Therefore, they have developed an alternate route for introduction of the sulfhydryl group and have prepared novel thio-ketosides with aryl azides. The thio linkage is advantageous since it is not easily cleaved by neuraminidases and it allows incorporation of /sup 35/S as a convenient radioactive label. 2-deoxy-2-S-acetyl-4,7,8,9,- tetra-0-acetyl-N-acetyl neuraminic acid methyl ester was prepared (70% yield) from 2-chloro aceto- neuraminic acid methyl ester and potassium thioacetate in acetone at room temperature (RT) for 90 min. Selective hydrolysis of the thio acetate group was accomplished with equimolar sodium methoxide in DMF. After 10 min at RT, 4-fluoro-3-nitrophenyl azide was added and reaction continued for 60 min. Silicic acid purification, base hydrolysis, and gel filtration chromatography, gave 2'-deoxy-2'-(2-nitro-4-azido-thiophenyl)-..cap alpha..-D-N-acetyl neuraminic acid (35% yield). Other thio-arylazido ketosides were prepared similarly.

  10. Di- and triarylmethylium ions as probes for the ambident reactivities of carbanions derived from 5-benzylated Meldrum's acid.

    PubMed

    Chen, Xi; Tan, Yue; Berionni, Guillaume; Ofial, Armin R; Mayr, Herbert

    2014-08-25

    The kinetics of the reactions of carbocations with carbanions 1 derived from 5-benzyl-substituted Meldrum's acids 1-H (Meldrum's acid = 2,2-dimethyl-1,3-dioxane-4,6-dione) were investigated by UV/Vis spectroscopic methods. Benzhydryl cations Ar2CH(+) added exclusively to C-5 of the Meldrum's acid moiety. As the second-order rate constants (kC) of these reactions in DMSO followed the linear free-energy relationship lg k = sN (N+E), the nucleophile-specific reactivity parameters N and sN for the carbanions 1 could be determined. In contrast, trityl cations Ar3C(+) reacted differently. While tritylium ions of low electrophilicity (E<-2) reacted with 1 through rate-determining β-hydride abstraction, more Lewis acidic tritylium ions initially reacted at the carbonyl oxygen of 1 to form trityl enolates, which subsequently reionized and eventually yielded triarylmethanes and 5-benzylidene Meldrum's acids by hydride transfer. PMID:25099696

  11. Real-time polymerase chain reaction (PCR) quantitative detection of Brassica napus using a locked nucleic acid TaqMan probe.

    PubMed

    Schmidt, Anna-Mary; Rott, Michael E

    2006-02-22

    Several countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms. Real-time quantitative Polymerase Chain Reaction (PCR) has quickly become the method of choice in support of these regulations and requires the development of separate PCR assays targeting the transgenic sequence as well as a specific endogenous gene sequence. To develop a Brassica napus-specific PCR assay, partial sequences of the acetyl-CoA carboxylase BnACCg8 gene from B. napus and the closely related Brassica rapa were determined and compared, and a region of unique nucleotide sequence was identified. Universal amplification primers were designed to either side of this region, and a locked nucleic acid TaqMan probe was designed to the B. napus-specific sequence. Evaluation of this primer/probe combination indicated a high level of specificity to B. napus: no amplification signal was observed with any other species tested, including five closely related Brassica species. The method was assayed with 14 different B. napus cultivars, and comparable amplification curves were consistently obtained for all. The assay was highly sensitive, with a limit of detection between 1 and 10 haploid copies. Practically, the method was demonstrated to be effective for the detection of processed food samples and for the quantification of Roundup Ready canola content in mixed samples.

  12. A potential fluorescent probe: Maillard reaction product from glutathione and ascorbic acid for rapid and label-free dual detection of Hg(2+) and biothiols.

    PubMed

    Dong, Jiang Xue; Song, Xiao Fang; Shi, Yan; Gao, Zhong Feng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

    2016-07-15

    Maillard reactions and their fluorescent products have drawn much attention in the fields of food and life science, however, the application of fluorescent products separated from the reaction as an indicator for detection of certain substances in sensor field has not been mentioned. In this article, we report on an easy-to-synthesize and water-soluble fluorescent probe separated from the typical Maillard reaction products of glutathione and ascorbic acid, with excellent stability and high quantum yield (18.2%). The further application of the probe has been explored for dual detection of Hg(2+) and biothiols including cysteine, homocysteine, and glutathione, which is based on Hg(2+)-induced fluorescence quenching of the Maillard reaction fluorescent products (MRFPs) and the fluorescence recovery as the introduction of biothiols. This sensing system exhibits a good selectivity and sensitivity, and the linear ranges for Hg(2+), cysteine, homocysteine, and glutathione are 0.05-12, 0.5-10, 0.3-20, and 0.3-20μM, respectively. The detection limits for Hg(2+), cysteine, homocysteine, and glutathione are 22, 47, 96, and 30nM at a signal-to-noise ratio of 3, respectively. Furthermore, the practical applications of this sensor for Hg(2+) and biothiols determination in water samples and human plasma sample have been demonstrated with satisfactory results. PMID:27015151

  13. A potential fluorescent probe: Maillard reaction product from glutathione and ascorbic acid for rapid and label-free dual detection of Hg(2+) and biothiols.

    PubMed

    Dong, Jiang Xue; Song, Xiao Fang; Shi, Yan; Gao, Zhong Feng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

    2016-07-15

    Maillard reactions and their fluorescent products have drawn much attention in the fields of food and life science, however, the application of fluorescent products separated from the reaction as an indicator for detection of certain substances in sensor field has not been mentioned. In this article, we report on an easy-to-synthesize and water-soluble fluorescent probe separated from the typical Maillard reaction products of glutathione and ascorbic acid, with excellent stability and high quantum yield (18.2%). The further application of the probe has been explored for dual detection of Hg(2+) and biothiols including cysteine, homocysteine, and glutathione, which is based on Hg(2+)-induced fluorescence quenching of the Maillard reaction fluorescent products (MRFPs) and the fluorescence recovery as the introduction of biothiols. This sensing system exhibits a good selectivity and sensitivity, and the linear ranges for Hg(2+), cysteine, homocysteine, and glutathione are 0.05-12, 0.5-10, 0.3-20, and 0.3-20μM, respectively. The detection limits for Hg(2+), cysteine, homocysteine, and glutathione are 22, 47, 96, and 30nM at a signal-to-noise ratio of 3, respectively. Furthermore, the practical applications of this sensor for Hg(2+) and biothiols determination in water samples and human plasma sample have been demonstrated with satisfactory results.

  14. Probing thermal stability of the β-lactoglobulin-oleic acid complex by fluorescence spectroscopy and molecular modeling

    NASA Astrophysics Data System (ADS)

    Simion (Ciuciu), Ana-Maria; Aprodu, Iuliana; Dumitrașcu, Loredana; Bahrim, Gabriela Elena; Alexe, Petru; Stănciuc, Nicoleta

    2015-09-01

    Bovine β-lactoglobulin is able to interact with different bioactive compounds, thus being an important candidate in the development of delivery systems with improved functionality. The heat induced changes in the β-lactoglobulin-oleic acid complex were examined by means of fluorescence spectroscopy and molecular modeling techniques. Fluorescence spectroscopy results indicated a rigid protein structure in the temperature range 25-70 °C, whereas at temperatures over 75 °C, the rearrangements of the polypeptide chains led to higher exposure of hydrophobic residues. The most significant increase of the accessible surface area with temperature increase was identified in case of Tyr99 and Tyr102. The phase diagram method indicated an all or none transition between two conformations. Due to conformational changes, no contact between Ile56 or Lys60 and the fatty acid could be identified at 85 °C, but new non-bonding interaction were established with Ile12 and Val15. The results obtained in this study provide important details about thermal induced changes in the conformation of β-lactoglobulin-oleic acid complex. Significant conformational changes were registered above 75 °C, suggesting the possibility of obtaining highly functional complexes between whey proteins and natural unsaturated fatty acids.

  15. Probing High School Students' Cognitive Structures and Key Areas of Learning Difficulties on Ethanoic Acid Using the Flow Map Method

    ERIC Educational Resources Information Center

    Zhou, Qing; Wang, Tingting; Zheng, Qi

    2015-01-01

    The purpose of this study was primarily to explore high school students' cognitive structures and to identify their learning difficulties on ethanoic acid through the flow map method. The subjects of this study were 30 grade 1 students from Dong Yuan Road Senior High School in Xi'an, China. The interviews were conducted a week after the students…

  16. Probing metal ion complexation with salicylic acid and its derivatives with excited state proton transfer and luminescence anisotropy

    SciTech Connect

    Wang, Z.; Friedrich, D.M.; Ainsworth, C.C.

    1996-10-01

    Salicylic acid and its derivatives in which the phenolic proton is preserved show a characteristic dual fluorescence: one band in the UV, due to a {open_quotes}normal{close_quotes} excited state emission, and the other in the visible range, is assigned to excited state intramolecular proton transfer (ESIPT). The transition energy, quantum yield and fluorescence lifetime as well as fluorescence anisotropy are sensitive to the solvent environment, temperature and properties of the substituents (complexation) at the phenolic and carboxylic oxygens. The ESIPT band disappears in molecules in which the intramolecular hydrogen bond between phenolic hydrogen and the carbonyl oxygen is prohibited. In this work, the complexation of Na(I), Ca(II), Al(III) and La(III) with salicylic acid, 3-hydroxybenzoic acid, methylsalicylate and anisic acid in both aqueous and non-aqueous solvents has been studied by absorption and steady state luminescence spectroscopy, picosecond to nanosecond luminescence lifetimes and luminescence anisotropy measurements in a range of solvent and in ethanol at 77 K. Speciation in these complex systems, binding characteristics between the metal ion and the ligand, and ligand-centered energetics are discussed in terms of the spectroscopic properties, luminescence and anisotropy decay kinetics.

  17. QUANTITATIVE CONTRIBUTIONS OF DIET AND LIVER SYNTHESIS TO DOCOSAHEXAENOIC ACID HOMEOSTASIS

    PubMed Central

    Rapoport, Stanley I.; Igarashi, Miki; Gao, Fei

    2010-01-01

    Dietary requirements for maintaining brain and heart docosahexaenoic acid (DHA, 22:6n-3) homeostasis are not agreed on, in part because rates of liver DHA synthesis from circulating α-linolenic acid (α-LNA, 18:2n-3) have not been quantified. These rates can be estimated in vivo using intravenous radiotracer- or heavy isotope-labeled α-LNA infusion. In adult unanesthetized male rats, such infusion shows that liver synthesis-secretion rates of DHA from α-LNA markedly exceed brain and heart DHA synthesis rates and brain DHA consumption rate, and that liver but not heart or brain synthesis is upregulated as dietary n-3 PUFA content is reduced. These differences in rate reflect much higher expression of DHA-synthesizing enzymes in liver, and upregulation of liver but not heart or brain enzyme expression by reduced dietary n-3 PUFA content. A noninvasive intravenous [U-13C]α-LNA infusion method that produces steady-state liver tracer metabolism gives exact liver DHA synthesis-secretion rates and could be extended for human studies. PMID:20226642

  18. Topographic probes of angiotensin and receptor: potent angiotensin II agonist containing diphenylalanine and long-acting antagonists containing biphenylalanine and 2-indan amino acid in position 8.

    PubMed

    Hsieh, K H; LaHann, T R; Speth, R C

    1989-04-01

    A series of phenylalanine-mimicking amino acids with increasing conformational restraint were prepared and incorporated into angiotensin II, in order to develop topographic probes of angiotensin useful for probing receptor boundaries by molecular graphics analysis and for conformational analysis of the ligand by NMR. In binding studies, all analogues displayed high affinity for rat uterus (Ki of 0.74-6.08 nM) and brain (0.46-1.82 nM) receptors. In smooth muscle (rat uterus) contraction assay, the diphenylalanine-containing [Sar1,Dip8]AII and [Sar1,D-Dip8]AII were potent agonists with respectively 284% and 48% activity of [Asn1]AII. In contrast, the biphenylalanine-containing [Sar1,Bip8]AII, [Sar1,D-Bip8]AII, and the 2-indan amino acid containing [Sar1,2-Ind8]AII were potent inhibitors, approximately 9, 2, and 1.4 times more effective than a standard antagonist, [Sar1,Leu8]AII. Their respective pA10 values in rat uterus assay were 8.87, 8.70, and 8.82. By comparison, the pA10 value for [Sar1,Leu8]AII was 8.35. In rats, a single dose of 10 micrograms of [Sar1,2-Ind8]AII or [Sar1,Bip8]AII produced prolonged blockade of the pressor response toward angiotensin II for over 90 min. The very different pharmacological profiles of these rigid aromatic analogues suggest that the angiotensin receptor activation site consists of a relatively wide and elongated pocket with a narrow opening.

  19. Hexagonal cobalt oxyhydroxide-carbon dots hybridized surface: high sensitive fluorescence turn-on probe for monitoring of ascorbic acid in rat brain following brain ischemia.

    PubMed

    Li, Linbo; Wang, Chao; Liu, Kangyu; Wang, Yuhan; Liu, Kun; Lin, Yuqing

    2015-03-17

    In this study, we report a novel and efficient fluorescence probe synthesized by Tris(hydroxymethyl)aminomethane-derived carbon dots (CDs)-modified hexagonal cobalt oxyhydroxide(CoOOH) nanoflakes (Tris-derived CDs-CoOOH) for monitoring of cerebral ascorbic acid (AA) in brain microdialysate. The as-prepared Tris-derived CDs with the fluorescence quantum yield of 7.3% are prepared by a one-step pyrolysis strategy of the sole precursor and used as the signal output. After being hybridized with CoOOH nanoflakes to form Tris-derived CDs-CoOOH, the luminescence of the Tris-derived CDs can be efficiently quenched by CoOOH via fluorescence resonance energy transfer (FRET). Due to the specific redox reaction between the enediol group of AA and hexagonal CoOOH nanoflakes, AA can reduce the hexagonal CoOOH nanoflakes in the Tris-derived CDs-CoOOH and lead to collapse of the hybrized structure, then the release of Tris-derived CDs, and thus finally the fluorescence recovery. Moreover, cobalt ions (II), generated by CoOOH nanoflakes oxidizing AA, almost have no obvious interference on the fluorescence probe, i.e., Tris-derived CDs, which could be ascribed to the surface of Tris-derived CDs containing a few strong chelation groups such as amino/carboxyl/thiol groups, instead of plenty of -OH groups with weak chelation with Co(2+). On the basis of this feature, the Tris-derived CDs-CoOOH fluorescent probe demonstrates a linear range from 100 nM to 20 μM with the detection limit of ∼50 nM, i.e., with an improved sensitivity toward AA detection. Compared with other turn-on fluorescent methods using convenient fluorophore-nitroxide fluorescent probes for detection of AA, the method demonstrated here possesses a facial synthesis route, lower limit of detection, and wider linear range, which validates sensing of AA in the cerebral systems during the calm/ischemia process. This study provides a fluorescence assay for the simple yet facial detection of AA in the cerebral systems and

  20. A new dual-channel optical signal probe for Cu2+ detection based on morin and boric acid.

    PubMed

    Wang, Peng; Yuan, Bin Fang; Li, Nian Bing; Luo, Hong Qun

    2014-01-01

    In this work we utilized the common analytical reagent morin to develop a new a dual-channel, cost-effective, and sensitive method for determination of Cu(2+). It is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid. Moreover, the fluorescence of complexes of morin with boric acid is quenched linearly by Cu(2+) in a certain concentration range. Under optimum conditions, the fluorescence quenching efficiency was linearly proportional to the concentration of cupric ions in the range of 0.5-25 μM with high sensitivity, and the detection limit for Cu(2+) was 0.38 μM. The linear range was 1-25 μM determined by spectrophotometry, and the detection limit for cupric ions was 0.8 μM. Furthermore, the mechanism of sensitive fluorescence quenching response of morin to Cu(2+) is discussed. PMID:25199115

  1. A new dual-channel optical signal probe for Cu2+ detection based on morin and boric acid.

    PubMed

    Wang, Peng; Yuan, Bin Fang; Li, Nian Bing; Luo, Hong Qun

    2014-01-01

    In this work we utilized the common analytical reagent morin to develop a new a dual-channel, cost-effective, and sensitive method for determination of Cu(2+). It is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid. Moreover, the fluorescence of complexes of morin with boric acid is quenched linearly by Cu(2+) in a certain concentration range. Under optimum conditions, the fluorescence quenching efficiency was linearly proportional to the concentration of cupric ions in the range of 0.5-25 μM with high sensitivity, and the detection limit for Cu(2+) was 0.38 μM. The linear range was 1-25 μM determined by spectrophotometry, and the detection limit for cupric ions was 0.8 μM. Furthermore, the mechanism of sensitive fluorescence quenching response of morin to Cu(2+) is discussed.

  2. Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent

    SciTech Connect

    Julin, D.A.; Wiesinger, H.; Toney, M.D.; Kirsch, J.F. )

    1989-05-02

    The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shown that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.

  3. Gold-catalysed cross-coupling between aryldiazonium salts and arylboronic acids: probing the usefulness of photoredox conditions.

    PubMed

    Cornilleau, Thomas; Hermange, Philippe; Fouquet, Eric

    2016-08-21

    The synthesis of biaryl compounds from aryldiazonium salts and arylboronic acids was achieved using PPh3AuCl as catalyst, CsF as base and acetonitrile as solvent. Combined to photosensitizers, irradiation by blue LEDs allowed accelerating the reaction and expanding its scope. Various functional groups were compatible including bromoaryls, iodoaryls, aldehydes and alcohols. A 2-iodobenzyl alcohol moiety was smoothly introduced by this method, enabling its consecutive isotopic labelling by a Pd-catalysed alkoxycarbonylation. PMID:27452177

  4. Spectrophotometric probe

    DOEpatents

    Prather, William S.; O'Rourke, Patrick E.

    1994-01-01

    A support structure bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe.

  5. Spectrophotometric probe

    DOEpatents

    Prather, W.S.; O'Rourke, P.E.

    1994-08-02

    A support structure is described bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe. 3 figs.

  6. Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.

    PubMed

    Dempsey, Daniel R; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J

    2015-11-01

    Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.

  7. Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

    PubMed

    Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

    2015-10-01

    Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

  8. Probing the active site of MIO-dependent 2,3-aminomutases, key catalysts in the biosynthesis of beta-amino acids incorporated in secondary metabolites

    PubMed Central

    Bruner, Steven D.; Cooke, Heather

    2012-01-01

    The tyrosine aminomutase SgTAM produces (S)-β-tyrosine from l-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form α,β-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been no reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the α,β-unsaturated intermediates to form β-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray co-crystal structure of the SgTAM mutant of the catalytic base with l-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis. PMID:20577998

  9. Probing the Active Site of MIO-dependent Aminomutases, Key Catalysts in the Biosynthesis of amino Acids Incorporated in Secondary Metabolites

    SciTech Connect

    Cooke, H.; Bruner, S

    2010-01-01

    The tyrosine aminomutase SgTAM produces (S)-{beta}-tyrosine from L-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form {alpha},{beta}-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been limited reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the {alpha},{beta}-unsaturated intermediates to form {beta}-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis.

  10. Investigation of Humidity Dependent Surface Morphology and Proton Conduction in Multi-Acid Side Chain Membranes by Conductive Probe Atomic Force Microscopy.

    PubMed

    Economou, Nicholas J; Barnes, Austin M; Wheat, Andrew J; Schaberg, Mark S; Hamrock, Steven J; Buratto, Steven K

    2015-11-01

    In this report, we employ phase-contrast tapping mode and conductive probe atomic force microscopy (cp-AFM) as tools to investigate the nanoscale morphology and proton conductance of a 3M perfluoro-imide acid (PFIA) membrane (625 EW) over a large range of relative humidity (3-95% RH). As a point of comparison, we also investigate 3M perfluorosulfonic acid (PFSA) (825 EW) and Nafion 212. With AFM, we assess the membrane's water retention and mechanical stability at low RH and high RH, respectively. Cp-AFM allows us to spatially resolve the hydrophilic and electrochemically active domains under a similar set of conditions and observe directly the ties between membrane morphology and proton conductance. From our data, we are able to correlate the improved water retention indicated by the size of the hydrophilic domains with the proton conductance in the PFIA membrane at elevated temperature and compare the result with that observed for the PFSA and Nafion. At high RH conditions, we see evidence of a nearly continuous hydrophilic phase, which indicates a high degree of swelling. PMID:26439098

  11. Use of Dimedone-Based Chemical Probes for Sulfenic Acid Detection: Methods to Visualize and Identify Labeled Proteins

    PubMed Central

    Nelson, Kimberly J.; Klomsiri, Chananat; Codreanu, Simona G.; Soito, Laura; Liebler, Daniel C.; Rogers, LeAnn C.; Daniel, Larry W.; Poole, Leslie B.

    2013-01-01

    Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines. PMID:20513473

  12. A novel magnetic field probing technique for determining state of health of sealed lead-acid batteries

    NASA Astrophysics Data System (ADS)

    Khare, Neeta; Singh, Pritpal; Vassiliou, John K.

    2012-11-01

    State of Health (SOH) is a critical index for a Sealed Lead-Acid (SLA) battery diagnostic which provides the information about battery replacement and aging effects. SOH is a complex function of chemical parameters of a battery such as stratification in electrolyte, electrode structure (sulfation and hard sulfation) in addition to electrical parameters of a battery. This paper describes a method of online determination of stratification, electrode structure, electrode polarization and current profile within the battery under the influence of a magnetic field. An AC magnetic field is used as a noninvasive tool during battery cycles. An induced emf in a secondary coil (SCV) is used as a measure of change in the magnetic field. The H+ proton density varies with change in sulfuric acid (electrolyte) concentration during battery cycles. The magnetic flux lines are affected by the density of H+ protons whose magnetic dipole moments try to align along the magnetic flux lines. The stratification is seen by a 12% decrease in magnetic flux linking from the top to the bottom of the electrolyte in a battery. Additional experimental results demonstrate the variation in magnetic flux linking which correlates with current profile across the electrode and electrode structure.

  13. Probing the adsorption of weak acids on graphite using amplitude modulation-frequency modulation atomic force microscopy.

    PubMed

    Moustafa, Ahmed M A; Huang, Jun; McPhedran, Kerry N; Zeng, Hongbo; El-Din, Mohamed Gamal

    2015-03-17

    Recent thermodynamics calculations and adsorption isotherms showed that the adsorption of a self-assembled layer (SAL) of ionized weak acids to carbon was attributed to the negatively charged hydrogen bonding (-CAHB), yet the direct visualization and characterization of this adsorption behavior have not been reported. Here, an amplitude modulation-frequency modulation atomic force microscopy (AM-FM AFM) technique was applied to discriminate the adsorption of decanoic acids (DA) on highly ordered pyrolytic graphite (HOPG). Thermodynamics calculations revealed that the adsorption of SAL was driven by the formation of -CAHB with negatively charged functional groups of HOPG. Multilayer adsorption could occur over the adsorbed ionized SAL, leading to the development of aggregates. AM-FM AFM imaging showed that the adsorption of the DA molecules forming aggregates occurred only for the HOPG-functionalized steps, while DA molecules were found to adsorb over the entire functionalized HOPG surface after water-plasma treatment, as evident from the frequency shifts identified in AFM images. PMID:25710305

  14. Probing the adsorption of weak acids on graphite using amplitude modulation-frequency modulation atomic force microscopy.

    PubMed

    Moustafa, Ahmed M A; Huang, Jun; McPhedran, Kerry N; Zeng, Hongbo; El-Din, Mohamed Gamal

    2015-03-17

    Recent thermodynamics calculations and adsorption isotherms showed that the adsorption of a self-assembled layer (SAL) of ionized weak acids to carbon was attributed to the negatively charged hydrogen bonding (-CAHB), yet the direct visualization and characterization of this adsorption behavior have not been reported. Here, an amplitude modulation-frequency modulation atomic force microscopy (AM-FM AFM) technique was applied to discriminate the adsorption of decanoic acids (DA) on highly ordered pyrolytic graphite (HOPG). Thermodynamics calculations revealed that the adsorption of SAL was driven by the formation of -CAHB with negatively charged functional groups of HOPG. Multilayer adsorption could occur over the adsorbed ionized SAL, leading to the development of aggregates. AM-FM AFM imaging showed that the adsorption of the DA molecules forming aggregates occurred only for the HOPG-functionalized steps, while DA molecules were found to adsorb over the entire functionalized HOPG surface after water-plasma treatment, as evident from the frequency shifts identified in AFM images.

  15. Mefenamic acid anti-inflammatory drug: probing its polymorphs by vibrational (IR and Raman) and solid-state NMR spectroscopies.

    PubMed

    Cunha, Vanessa R R; Izumi, Celly M S; Petersen, Philippe A D; Magalhães, Alviclér; Temperini, Marcia L A; Petrilli, Helena M; Constantino, Vera R L

    2014-04-24

    This work deals with the spectroscopic (supported by quantum chemistry calculations), structural, and morphological characterization of mefenamic acid (2-[(2,3-(dimethylphenyl)amino] benzoic acid) polymorphs, known as forms I and II. Polymorph I was obtained by recrystallization in ethanol, while form II was reached by heating form I up to 175 °C, to promote the solid phase transition. Experimental and theoretical vibrational band assignments were performed considering the presence of centrosymmetric dimers. Besides band shifts in the 3345-3310 cm(-1) range, important vibrational modes to distinguish the polymorphs are related to out-of-phase and in-phase N-H bending at 1582 (Raman)/1577 (IR) cm(-1) and 1575 (Raman)/1568 (IR) cm(-1) for forms I and II, respectively. In IR spectra, bands assigned to N-H bending out of plane are observed at 626 and 575 cm(-1) for polymorphs I and II, respectively. Solid-state (13)C NMR spectra pointed out distinct chemical shifts for the dimethylphenyl group: 135.8 to 127.6 ppm (carbon bonded to N) and 139.4 to 143.3 ppm (carbon bonded to methyl group) for forms I and II, respectively. PMID:24654805

  16. A high-fat, high-oleic diet, but not a high-fat, saturated diet, reduces hepatic n3 fatty acid content in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While considerable research has centered upon the role of linoleic acid (LNA; 18:2n6) as a competitive inhibitor of alpha-linolenic (ALA; 18:3n3) metabolism, a growing literature indicates that the amount of fat consumed can reduce the elongation and desaturation process. However, little data exist ...

  17. Using rumen probes to examine effects of conjugated linoleic acids and dietary concentrate proportion on rumen pH and rumen temperature of periparturient dairy cows.

    PubMed

    Petzold, M; Meyer, U; Spilke, J; Dänicke, S

    2014-08-01

    The study aimed to examine the influence of supplemented conjugated linoleic acids (CLA) to periparturient cows receiving different concentrate proportions antepartum on rumen pH (RpH) and rumen temperature (RT). Twenty pregnant German Holstein cows were equipped with rumen probes for continuous RpH and RT measurement in a frequency of 15 min to investigate effects of dietary concentrate and CLA around parturition and the impact of parturition itself on RpH and RT. Cows had ad libitum access to partial mixed rations, 3 weeks prior to calving until day 7 post-partum. Antepartum, cows received 100 g/day control fat (CON) or CLA supplement, either in low (20%; CON-20, CLA-20) or high concentrate diet (60%; CON-60, CLA-60). Post-partum, concentrate proportion was adjusted to 50% while fat supplementation continued. Compared with adapted feeding, high concentrate proportions antepartum tended to increase DMI and reduced RpH. Groups CON-60 and CLA-60 spent more than 4 h per day below RpH 5.6 during late pregnancy, indicating the presence of subacute rumen acidosis (SARA). The RT remained unaffected antepartum. Before calving, cows spent less time below RpH 5.6 and SARA could be detected in each group post-partum. Mean RpH increased slightly antepartum, whereas few hours before parturition a sharp decrease in RpH could be observed, accompanied with increased RT. Overall, it seems that CLA supplementation influences RpH and RT. Bearing in mind that rumen parameters fluctuate during day and herd level must be known, rumen probes for continuous RpH and RT measurement could be a useful management tool for animal health surveillance and may also help to predict parturition.

  18. Development of a fatty acid and RNA stable isotope probing-based method for tracking protist grazing on bacteria in wastewater.

    PubMed

    Kuppardt, Steffen; Chatzinotas, Antonis; Kästner, Matthias

    2010-12-01

    Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on (13)C-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate Uronema sp. and (13)C-labeled Escherichia coli as model prey. The transfer of bacterial (13)C into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP). Uronema sp. showed ingestion rates of ∼390 bacteria protist(-1) h(-1), and the temporal patterns of (13)C assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for Uronema sp. were found (20:0, i20:0, 22:0, 24:0, 20:1ω9c, 20:1ω9t, 22:1ω9c, 22:1ω9t, and 24:1). Four of these fatty acids (22:0, 20:1ω9t, 22:1ω9c, and 22:1ω9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1ω9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the E. coli-derived (13)C was incorporated into Uronema biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (y(x)(/)(s) ≈ 0.2), and the temporal pattern of (13)C labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (∼98 atom% (13)C) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of (13)C-labeled protist RNA demonstrated

  19. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins.

    PubMed

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  20. Probing roles of lipopolysaccharide, type 1 fimbria, and colanic acid in the attachment of Escherichia coli strains on inert surfaces.

    PubMed

    Chao, Yuanqing; Zhang, Tong

    2011-09-20

    The roles of bacterial surface polymers in reversible (phase I) and irreversible (phase II) attachment (i.e., lipopolysaccharides (LPS), type 1 fimbria, and capsular colanic acid (CA)) were investigated in situ by combining fluorescence microscopy and atomic force microscopy. Fluorescence microscopy was used to evaluate the phase I attachment by counting the total number of cells on the substrata, and AFM was applied to image the phase II cells and measure the lateral detachment force to characterize phase II attachment. Also, by comparing the number of cells in phases I and II, the transformation ratio was calculated and used as an index to evaluate the roles of different polymers in the attachment process. Escherichia coli K-12 and its six mutants, which had different surface polymers in terms of LPS structures, CA contents, and type 1 fimbriae, were used as the test strains. Six different materials were applied as substrata, including glass, two metals (aluminum and stainless steel), and three plastics (polyvinyl chloride, polycarbonate, and polyethylene). The results indicated that LPS significantly enhanced phases I and II attachment as well as the transformation ratio from phase I to II. Like LPS, type 1 fimbriae largely increased the phase I attachment and the transformation ratio; however, they did not significantly influence the adhesion strength in phase II. CA had a negative effect on attachment in phases I and II by decreasing the adhered number of cells and the lateral detachment force, respectively, but had no influence on the transformation ratio. PMID:21842859

  1. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins

    PubMed Central

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  2. Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level.

    PubMed

    Pérez Santero, Silvia; Favretto, Filippo; Zanzoni, Serena; Chignola, Roberto; Assfalg, Michael; D'Onofrio, Mariapina

    2016-09-15

    Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell. PMID:27457417

  3. ANTS-anchored Zn-Al-CO3-LDH particles as fluorescent probe for sensing of folic acid

    NASA Astrophysics Data System (ADS)

    Liu, Pengfei; Liu, Dan; Liu, Yanhuan; Li, Lei

    2016-09-01

    A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO3-layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn2+ ions of Zn-Al-CO3-LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO3-LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO3-LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO3 groups in ANTS-anchored on the surface of Zn-Al-CO3-LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO3-LDH particles can afford a very sensitive system for the sensing FA in aqueous solution.

  4. Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry.

    PubMed

    Ren, Xiao-Min; Cao, Lin-Ying; Zhang, Jing; Qin, Wei-Ping; Yang, Yu; Wan, Bin; Guo, Liang-Hong

    2016-04-01

    Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.

  5. Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

    PubMed

    Shin, Kyong-Oh; Kim, Kunpyo; Jeon, Sanghun; Seo, Cho-Hee; Lee, Yong-Moon; Cho, Yunhi

    2015-10-01

    Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1. PMID:26233818

  6. Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

    PubMed

    Shin, Kyong-Oh; Kim, Kunpyo; Jeon, Sanghun; Seo, Cho-Hee; Lee, Yong-Moon; Cho, Yunhi

    2015-10-01

    Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1.

  7. Effect of glucose on the fatty acid composition of Cupriavidus necator JMP134 during 2,4-dichlorophenoxyacetic acid degradation: implications for lipid-based stable isotope probing methods.

    PubMed

    Lerch, Thomas Z; Dignac, Marie-France; Barriuso, Enrique; Mariotti, André

    2011-10-01

    Combining lipid biomarker profiling with stable isotope probing (SIP) is a powerful technique for studying specific microbial populations responsible for the degradation of organic pollutants in various natural environments. However, the presence of other easily degradable substrates may induce significant physiological changes by altering both the rate of incorporation of the target compound into the biomass and the microbial lipid profiles. In order to test this hypothesis, Cupriavidus necator JMP134, a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium, was incubated with [(13)C]2,4-D, [(13)C]glucose, or mixtures of both substrates alternatively labeled with (13)C. C. necator JMP134 exhibited a preferential use of 2,4-D over glucose. The isotopic analysis showed that glucose had only a small effect on the incorporation of the acetic chain of 2,4-D into the biomass (at days 2 and 3) and no effect on that of the benzenic ring. The addition of glucose did change the fatty acid methyl ester (FAME) composition. However, the overall FAME isotopic signature reflected that of the entire biomass. Compound-specific individual isotopic analyses of FAME composition showed that the (13)C-enriched FAME profiles were slightly or not affected when tracing the 2,4-D acetic chain or 2,4-D benzenic ring, respectively. This batch study is a necessary step for validating the use of lipid-based SIP methods in complex environments.

  8. Probing the interaction induced conformation transitions in acid phosphatase with cobalt ferrite nanoparticles: Relation to inhibition and bio-activity of Chlorella vulgaris acid phosphatase.

    PubMed

    Ahmad, Farooq; Zhou, Xing; Yao, Hongzhou; Zhou, Ying; Xu, Chao

    2016-09-01

    The present study explored the interaction and kinetics of cobalt ferrite nanoparticles (NPs) with acid phosphatase (ACP) by utilizing diverse range of spectroscopic techniques. The results corroborate, the CoFe2O4 NPs cause fluorescence quenching in ACP by static quenching mechanism. The negative values of van't Hoff thermodynamic expressions (ΔH=-0.3293Jmol(-1)K(-1) and ΔG=-3.960kJmol(-1)K(-1)) corroborate the spontaneity and exothermic nature of static quenching. The positive value of ΔS (13.2893Jmol(-1)K(-1)) corroborate that major contributors of higher and stronger binding affinity among CoFe2O4 NPs with ACP were electrostatic. In addition, FTIR, UV-CD, UV-vis spectroscopy and three dimensional fluorescence (3D) techniques confirmed that CoFe2O4 NPs binding induces microenvironment perturbations leading to secondary and tertiary conformation changes in ACP to a great extent. Furthermore, synchronous fluorescence spectroscopy (SFS) affirmed the comparatively significant changes in microenvironment around tryptophan (Trp) residue by CoFe2O4 NPs. The effect of CoFe2O4 NPs on the activation kinetics of ACP was further examined in Chlorella vulgaris. Apparent Michaelis constant (Km) values of 0.57 and 26.5mM with activation energy values of 0.538 and 3.428kJmol(-1) were determined without and with 200μM CoFe2O4 NPs. Apparent Vmax value of -7Umml(-1) corroborate that enzyme active sites were completely captured by the NPs leaving no space for the substrate. The results confirmed that CoFe2O4 NPs ceased the activity by unfolding of ACP enzyme. This suggests CoFe2O4 NPs perturbed the enzyme activity by transitions in conformation and hence the metabolic activity of ACP. This study provides the pavement for novel and simple approach of using sensitive biomarkers for sensing NPs in environment. PMID:27209386

  9. A turn-on highly selective and ultrasensitive determination of copper (II) in an aqueous medium using folic acid capped gold nanoparticles as the probe

    NASA Astrophysics Data System (ADS)

    Vasimalai, N.; Prabhakarn, A.; John, S. Abraham

    2013-12-01

    This paper describes a ‘turn-on’ fluorescent determination of Cu(II) in an aqueous medium using folic acid capped gold nanoparticles (FA-AuNPs) as the probe. The FA-AuNPs were synthesized by the wet chemical method and were characterized by UV-visible, fluorescence, HR-TEM, XRD, zeta potential, and DLS techniques. The FA-AuNPs show an absorption maximum at 510 nm and an emission maximum at 780 nm (λex: 510 nm). On adding 10 μM Cu(II), the wine-red color of FA-AuNPs changed to purple and the absorbance at 510 nm decreased. The observed changes were ascribed to the aggregation of AuNPs. This was confirmed by DLS and HR-TEM studies. Interestingly, the emission intensity of FA-AuNPs was enhanced even in the presence of a picomolar concentration of Cu(II). Based on the enhancement of the emission intensity, the concentration of Cu(II) was determined. The FA-AuNPs showed an extreme selectivity towards the determination of 10 nM Cu(II) in the presence of 10 000-fold higher concentration of interferences except EDTA and the carboxylate anion. A good linearity was observed from 10 × 10-9 to 1 × 10-12 M Cu(II), and the detection limit was found to be 50 fM l-1 (S/N = 3). The proposed method was successfully applied to determine Cu(II) in real samples. The results obtained were validated with ICP-AES.

  10. Optical probe

    DOEpatents

    Hencken, Kenneth; Flower, William L.

    1999-01-01

    A compact optical probe is disclosed particularly useful for analysis of emissions in industrial environments. The instant invention provides a geometry for optically-based measurements that allows all optical components (source, detector, rely optics, etc.) to be located in proximity to one another. The geometry of the probe disclosed herein provides a means for making optical measurements in environments where it is difficult and/or expensive to gain access to the vicinity of a flow stream to be measured. Significantly, the lens geometry of the optical probe allows the analysis location within a flow stream being monitored to be moved while maintaining optical alignment of all components even when the optical probe is focused on a plurality of different analysis points within the flow stream.

  11. Solid-state (17)O NMR as a sensitive probe of keto and gem-diol forms of alpha-keto acid derivatives.

    PubMed

    Zhu, Jianfeng; Geris, Amanda J; Wu, Gang

    2009-08-28

    We have used solid-state (17)O NMR experiments to measure the (17)O quadrupole coupling (QC) and chemical shift (CS) tensors for two alpha-keto acids: sodium [2-(17)O]pyruvate and lithium [2,2'-(17)O(2)]pyruvate. In the solid state, sodium [2-(17)O]pyruvate is in the keto form (-C(double bond (17)O)-) whereas lithium [2,2'-(17)O(2)]pyruvate takes the gem-diol form (-C((17)OH)(2)-). This study represents the first time that a full set of (17)O NMR tensors are experimentally determined for alpha-keto acids in these two different tautomeric forms. We have found that the two forms exhibit drastically different (17)O QC and CS tensors: for the keto form, delta(iso) = 543 +/- 1 ppm, C(Q) = 10.8 +/- 0.2 MHz, eta(Q) = 0.48 +/- 0.05, delta(11) = 1020 +/- 10, delta(22) = 640 +/- 10, delta(33) = -40 +/- 10 ppm, alpha = 80 +/- 5 degrees, beta = 90 +/- 2 degrees, and gamma = 83 +/- 2 degrees; for the gem-diol form, delta(iso) = 62 +/- 1 ppm, C(Q) = 8.5 +/- 0.5 MHz, eta(Q) = 1.0 +/- 0.05, delta(11) = 140 +/- 5, delta(22) = 45 +/- 5, delta(33) = 0 +/- 5 ppm, alpha = 55 +/- 5 degrees, beta = 90 +/- 5 degrees, and gamma = 80 +/- 2 degrees. The (17)O chemical shift tensor observed for the gem-diol functional group also represents the first such measurement for any -ol functional group (e.g., alcohols, phenols, carbohydrates, etc.) Using these accurate experimental (17)O NMR tensors, we were able to evaluate the accuracy of quantum chemical calculations. Our results showed that quantum chemical calculations using the crystal lattice approach are in much better agreement with the experimental solid-state (17)O NMR data than those calculated using the molecular cluster approach. Quantum chemical calculations have also provided information about the sign of the (17)O quadrupolar coupling constants and about the (17)O NMR tensor orientations in the molecular frame of reference. Our findings suggest that solid-state (17)O NMR may be useful in probing the tautomeric form of the alpha

  12. Amino acid precursors from a simulated lower atmosphere of titan: experiments of cosmic ray energy source with ¹³C- and ¹⁸O-stable isotope probing mass spectrometry.

    PubMed

    Taniuchi, Toshinori; Takano, Yoshinori; Kobayashi, Kensei

    2013-01-01

    The organic haze of aerosols that shrouds the Saturnian moon Titan has previously been studied by both observations and laboratory simulation experiments. Here we report the abiotic formation of amino acid precursors in complex organic molecules during experimental simulation of the environment near Titan's surface with proton irradiation. Pyrolysis of the organic molecules formed in the simulated Titan atmosphere by proton irradiation at 600°C yielded compounds that contained HCN and NH₃ (m/z = 27 and 17). These experimental results are consistent with the molecular information obtained by pyrolysis gas chromatography/mass spectrometry (pyrolysis GC/MS) of samples collected by the Huygens probe to Titan. Scanning electron microscopy (SEM) and three-dimensional atomic force microscopy (AFM) images of the irradiation products reveal nanometer-scale filaments and globules in complex amorphous structures (approximately 1000 Da). Isotope probing experiments by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) show that oxygen atoms were incorporated into the racemic amino acids by hydrolysis of ¹⁸O-labeled water. We suggest that the amino acid precursors possibly formed after water hydrolysis, as suggested in a previous observational study (C. A. Griffith, T. Owen, T. R. Geballe, J. Rayner, and P. Rannou, Science, 2003, 300, 628). We propose that cosmic rays are a significant and effective energy source for producing complex organics and amino acid precursors in Titan's atmospheric haze.

  13. Conductivity Probe

    NASA Technical Reports Server (NTRS)

    2008-01-01

    The Thermal and Electrical Conductivity Probe (TECP) for NASA's Phoenix Mars Lander took measurements in Martian soil and in the air.

    The needles on the end of the instrument were inserted into the Martian soil, allowing TECP to measure the propagation of both thermal and electrical energy. TECP also measured the humidity in the surrounding air.

    The needles on the probe are 15 millimeters (0.6 inch) long.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  14. Design, synthesis, and biological evaluation of 4-(5-dimethylamino-naphthalene-1-sulfon-amido)-3-(4-iodophenyl)butanoic acid as a novel molecular probe for apoptosis imaging

    SciTech Connect

    Zeng, Wenbin; Miao, Weimin; Le Puil, Michael; Shi, Guangqing; Biggerstaff, John; Kabalka, George W.; Townsend, David

    2010-07-30

    Research highlights: {yields} Annexin V is the gold standard probe for imaging apoptosis. {yields} Unfavorable profiles of Annexin V make it difficult to apply in the clinic. {yields} A novel small-molecular probe DNSBA was designed as an alternative to Annexin V. {yields} DNSBA specifically and selectively detect apoptotic cancer cells at all stages. {yields} DNSBA is a potential SPECT and PET agent when labeled with radioiodine. -- Abstract: Apoptosis (programmed cell death) plays a crucial role in the pathogenesis of many disorders, thus the detection of apoptotic cells can provide the physician with important information to further therapeutic strategies and would substantially advance patient care. A small molecule, 4-(5-dimethylamino-naphthalene-1-sulfonamido)-3-(4-iodo-phenyl)butanoic acid (DNSBA), was designed as a novel probe for imaging apoptosis and synthesized with good yield. The biological characterization demonstrated that DNSBA can be used to specifically and selectively detect apoptotic cancer cells at all stages. DNSBA is also designed as a potential SPECT and PET probe when labeled with radioiodine (I-123, -124, and -131).

  15. Pollution Probe.

    ERIC Educational Resources Information Center

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  16. New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Forster resonance energy transfer probes in 5'-nuclease assays.

    PubMed

    Kutyavin, Igor V

    2010-03-01

    The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5'-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem-loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5'-nuclease activity of Taq. The stem-loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5'-flap sequence. It was found that at a certain length of these 5'-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology's real-time performance was compared to a number of conventional assays including Taqman, 3'-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay's cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection.

  17. LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging.

    PubMed

    Roy, Kislay; Kanwar, Rupinder K; Kanwar, Jagat R

    2015-12-01

    This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin

  18. Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids.

    PubMed

    Zened, A; Troegeler-Meynadier, A; Nicot, M C; Combes, S; Cauquil, L; Farizon, Y; Enjalbert, F

    2011-11-01

    Trans isomers of fatty acids exhibit different health properties. Among them, trans-10,cis-12 conjugated linoleic acid has negative effects on milk fat production and can affect human health. A shift from the trans-11 to the trans-10 pathway of biohydrogenation (BH) can occur in the rumen of dairy cows receiving high-concentrate diets, especially when the diet is supplemented with highly unsaturated fat sources. The differences of BH patterns between linoleic acid (LeA) and linolenic acid (LnA) in such ruminal conditions remain unknown; thus, the aim of this work was to investigate in vitro the effects of starch and sunflower oil in the diet of the donor cows and starch level in the incubates on the BH patterns and efficiencies of LeA and LnA. The design was a 4 × 4 Latin square design with 4 cows, 4 periods, and 4 diets with combinations of 21 or 34% starch and 0 or 5% sunflower oil. The rumen content of each cow during each period was incubated with 4 substrates, combining 2 starch levels and either LeA or LnA addition. Capillary electrophoresis single-strand conformation polymorphism of incubates showed that dietary starch decreased the diversity of the bacterial community and the high-starch plus oil diet modified its structure. High-starch diets poorly affected isomerization and first reduction of LeA and LnA, but decreased the efficiencies of trans-11,cis-15-C18:2 and trans C18:1 reduction. Dietary sunflower oil increased the efficiency of LeA isomerization but decreased the efficiency of trans C18:1 reduction. An interaction between dietary starch and dietary oil resulted in the highest trans-10 isomers production in incubates when the donor cow received the high-starch plus oil diet. The partition between trans-10 and trans-11 isomers was also affected by an interaction between starch level and the fatty acid added to the incubates, showing that the trans-10 shift only occurred with LeA, whereas LnA was mainly hydrogenated via the more usual trans-11

  19. Role of Omega-3 Polyunsaturated Fatty Acids in the Production of Prostaglandin E2 and Nitric Oxide during Experimental Murine Paracoccidioidomycosis

    PubMed Central

    Sargi, S. C.; Dalalio, M. M. O.; Moraes, A. G.; Visentainer, J. E. L.; Morais, D. R.; Visentainer, J. V.

    2013-01-01

    There has recently been increased interest in the potential health effects of omega-3 polyunsaturated fatty acids on the immune system. Paracoccidioidomycosis is the most important endemic mycosis in Latin America. Macrophages have a fundamental role and act as first line of organism defense. The purpose of this study was to analyze the effect of n-3 fatty acids on the production of PGE2 and NO by mice infected with Pb18 and fed a diet enriched with LNA for 8 weeks. To study the effect of omega-3 fatty acids on macrophage activity during experimental paracoccidioidomycosis, mice were infected with Pb18 and fed a diet supplemented with LNA. PGE2 in the serum of animals was analyzed and NO in the supernatants of macrophages cultured and challenged in vitro with Pb18 was measured. Omega-3 fatty acids seemed to decrease the production of PGE2 in vivo in the infected group fed an LNA-supplemented diet during the 4th and 8th weeks of the experiment. At the same time, we observed an increase in synthesis of NO by peritoneal macrophages in this group. Omega-3 fatty acids thus appear to have an immunomodulatory effect in paracoccidioidomycosis. PMID:24455741

  20. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    PubMed

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  1. Investigation of an unnatural amino acid for use as a resonance Raman probe: Detection limits, solvent and temperature dependence of the νC≡N band of 4-cyanophenylalanine.

    PubMed

    Weeks, Colin L; Polishchuk, Alexei; Getahun, Zelleka; Degrado, William F; Spiro, Thomas G

    2008-11-01

    The incorporation of unnatural amino acids into proteins that act as spectroscopic probes can be used to study protein structure and function. One such probe is 4-cyanophenylalanine (PheCN), the nitrile group of which has a stretching mode that occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins and the wavenumber is sensitive to the polarity of its environment. In this work we evaluate the potential of UV resonance Raman spectroscopy for monitoring the sensitivity of the νC≡N band of PheCN incorporated into proteins to the protein environment. Measurement of the Raman excitation profile of PheCN showed that considerable resonance enhancement of the Raman signal was obtained using UV excitation and the best signal-to-noise ratios were obtained with excitation wavelengths of 229 and 244 nm. The detection limit for PheCN in proteins was ~10 μM, approximately a hundred-fold lower than the concentrations used in IR studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νC≡N band was strongly dependent on the polarity of its environment, when the solvent was changed from H(2)O to THF it decreased by 8 cm(-1). The presence of liposomes caused a similar though smaller decrease in νC≡N for a peptide, mastoparan X, modified to contain PheCN. The selectivity and sensitivity of resonance Raman spectroscopy of PheCN mean that it can be a useful probe of intra- and intermolecular interactions in proteins and opens the door to its application in the study of protein dynamics using time-resolved resonance Raman spectroscopy.

  2. A 3-10 GHz ultra-wideband SiGe LNA with wideband LC matching network

    NASA Astrophysics Data System (ADS)

    del Pino, J.; Khemchandani, S. L.; García, H.; Pulido, R.; Goñi, A.; Hernández, A.

    2007-05-01

    A fully-integrated SiGe wide band amplifier implemented in a standard low cost 0.35 μm process up to 12 dB of gain and a bandwidth of 3-10 GHz is presented. This circuit is divided in 3 stages. The first one is the input matching where the use of an inductively degenerated amplifier is expanded by embedding the input network of the amplifying device in a multisection reactive network so that the overall input reactance is resonated over a wider bandwidth. The second stage is a cascode transistor to obtain a great power gain and a high isolation between input and output ports. In adition, by adjusting the area and the multiplicity of these transistors, we can reduce the noise figure of the circuit. Finally at the output a new technique is used to increase the bandwidth. This technique is based in the replacement of the load resistor by a shunt-peaking resistor composed by an inductor and a resistor. The addition of an inductance gives an output impedance that remains roughly constant over a broader frequency range. Chip dimensions are 0.665 × 0.665 mm2 and power dissipation is 39 mW, drawn from a 3.3V supply. The noise figure ranges from 3.5 to 7.5 in the band between 2 GHz and 8.5 GHz. Finally, the circuit core draws 5.3 mA from a 3.3-V supply. All this results were measured in a probe station.

  3. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    PubMed

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  4. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

    PubMed Central

    Kasuya, Takeshi; Hori, Shin-ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  5. Direct observation of the cyclic dimer in liquid acetic acid by probing the C=O vibration with ultrafast coherent Raman spectroscopy.

    PubMed

    Lütgens, Matthias; Friedriszik, Frank; Lochbrunner, Stefan

    2014-09-01

    We present a comparison of spontaneous Raman and ultrafast coherent anti-Stokes Raman scattering (CARS) spectra of the C=O vibration of liquid acetic acid. The former technique cannot clearly reveal the number of contributions in the spectrum. However, the additional time and spectrally resolved CARS experiment supports strictly the existence of four modes, which proves the coexistence of more than one H-bonded configuration in liquid acetic acid. A comparably slowly dephasing mode which is obscured by a broad band in the linear Raman spectrum is assigned to the cyclic dimer and can be observed freed from all other contributions by ultrafast CARS.

  6. A probe on the intermolecular forces in diisopropyl ether-n-butyric acid mixture by dielectric, FTIR studies and quantum chemical calculations.

    PubMed

    Arivazhagan, G; Shanmugam, R; Elangovan, A

    2013-03-15

    The results of FTIR spectral measurement on equimolar diisopropyl ether-butyric acid binary mixture and quantum chemical calculations on the complex molecule have been presented. Dielectric studies have been carried out on the binary mixture over the entire composition range and at four different temperatures 303 K, 308 K, 313 K and 318 K. n-Butyric acid seems to prefer less polar ether to interact with it. It appears that the usual interpretation of variation of static dielectric constant and positive deviation of excess permittivity from ideal mixture behavior needs to be relooked.

  7. Use of H2S to Probe the Active Sites in FeNC Catalysts for the Oxygen Reduction Reaction (ORR) in Acidic Media

    SciTech Connect

    Singh, Deepika; Mamtani, Kuldeep; Bruening, Christopher R.; Miller, Jeffrey T.; Ozkan, Umit S.

    2014-10-01

    H2S has been used as a probe molecule both in an “in situ” poisoning experiment and in intermediate-temperature heat-treatment steps during and after the preparation of FeNC catalysts in an attempt to analyze its effect on their ORR activity. The heat treatments were employed either on the ball-milled precursor of FeNC or after the Ar-NH3 high temperature heat treatments. ORR activity of the H2S-treated catalysts was seen to be significantly lower than the sulfur-free catalysts, whether the sulfur exposure was during a half-cell testing, or as an intermediate-temperature exposure to H2S. The incorporation of sulfur species and interaction of Fe with sulfur were confirmed by characterization using XPS, EXAFS, TPO, and TPD. This study provides crucial evidence regarding differences in active sites in FeNC versus nitrogen-containing carbon nanostructured (CNx) catalysts.

  8. A novel sandwich assay with molecular beacon as report probe for nucleic acids detection on one-dimensional microfluidic beads array.

    PubMed

    Zuo, Xinbing; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Wen, Jianhui

    2007-03-21

    A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of approximately 0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.

  9. Molecular beacon-based junction probes for efficient detection of nucleic acids via a true target-triggered enzymatic recycling amplification.

    PubMed

    Kong, Rong-Mei; Zhang, Xiao-Bing; Zhang, Liang-Liang; Huang, Yan; Lu, Dan-Qing; Tan, Weihong; Shen, Guo-Li; Yu, Ru-Qin

    2011-01-01

    This work reports the development of a new molecular beacon-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNA-cleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system.

  10. Constrained photophysics of partially and fully encapsulated charge transfer probe (E)-3-(4-Methylaminophenyl) acrylic acid methyl ester inside cyclodextrin nano-cavities: Evidence of cyclodextrins cavity dependent complex stoichiometry

    NASA Astrophysics Data System (ADS)

    Ghosh, Shalini; Jana, Sankar; Guchhait, Nikhil

    2011-12-01

    The polarity sensitive intra-molecular charge transfer (ICT) emission from (E)-3-(4-Methylaminophenyl) acrylic acid methyl ester (MAPAME) is found to show distinct changes once introduced into the nano-cavities of cyclodextrins in aqueous environment. Movement of the molecule from the more polar aqueous environment to the less polar, hydrophobic cyclodextrin interior is marked by the blue shift of the CT emission band with simultaneous fluorescence intensity enhancement. The emission spectral changes on complexation with the α- and β-CD show different stoichiometries as observed from the Benesi-Hildebrand plots. Fluorescence anisotropy and lifetime measurements were performed to probe the different behaviors of MAPAME in aqueous α- and β-CD solutions.

  11. THE APPLICATION OF PEPTIDE NUCLEIC ACID PROBES FOR RAPID DETECTION AND ENUMERATION OF EUBACTERIA, STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN RECREATIONAL BEACHES OF S. FLORIDA. (R828830)

    EPA Science Inventory

    A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, S...

  12. Electrospray ionization mass spectrometric investigations of [alpha]-dicarbonyl compounds--Probing intermediates formed in the course of the nonenzymatic browning reaction of l-ascorbic acid

    NASA Astrophysics Data System (ADS)

    Schulz, Anke; Trage, Claudia; Schwarz, Helmut; Kroh, Lothar W.

    2007-05-01

    A new method is presented which allows the simultaneous detection of various [alpha]-dicarbonyl compounds generated in the course of the nonenzymatic browning reaction initiated by thermal treatment of l-ascorbic acid, namely: glyoxal, methylglyoxal, diacetyl, 3-deoxy-l-pentosone, and l-threosoneE 3-Deoxy-l-threosone was successfully identified as a new C4-[alpha]-dicarbonyl structure for the first time in the degradation of Vitamin C by application of this non-chromatographic mass spectrometric approach. Moreover, a more detailed elucidation of the mechanistic scenario with respect to the oxidative and nonoxidative pathways is presented by using dehydro-l-ascorbic acid and 2,3-diketo-l-gulonic acid instead of l-ascorbic acid as a starting material. Furthermore, the postulated pathways are corroborated with the aid of 13C-isotopic labeling studies. The investigations were extended to baby food, and the successful detection of [alpha]-dicarbonyl compounds characteristic for Vitamin C degradation proved the matrix tolerance of the introduced method.

  13. Nine New Fluorescent Probes

    NASA Astrophysics Data System (ADS)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  14. In vivo incorporation of unnatural amino acids to probe structure, dynamics and ligand binding in a large protein by Nuclear Magnetic Resonance spectroscopy

    PubMed Central

    Cellitti, Susan E.; Jones, David H.; Lagpacan, Leanna; Hao, Xueshi; Zhang, Qiong; Hu, Huiyong; Brittain, Scott M.; Brinker, Achim; Caldwell, Jeremy; Bursulaya, Badry; Spraggon, Glen; Brock, Ansgar; Ryu, Youngha; Uno, Tetsuo; Schultz, Peter G.; Geierstanger, Bernhard H.

    2008-01-01

    In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by co-expressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy) phenyl) propanoic acid (OCF3Phe), 13C/15N-labeled p-methoxyphenylalanine (OMePhe), and 15N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of E. coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in 1H-15N, 1H-13C HSQC and 19F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF3Phe mutants of an active site tyrosine inhibited binding; incorporating 15N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with

  15. Acid-base interactions and secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and Ab initio model calculations.

    PubMed

    Dos, Alexandra; Schimming, Volkmar; Tosoni, Sergio; Limbach, Hans-Heinrich

    2008-12-11

    The interactions of the 15N-labeled amino groups of dry solid poly-L-lysine (PLL) with various halogen and oxygen acids HX and the relation to the secondary structure have been studied using solid-state 15N and 13C CPMAS NMR spectroscopy (CP = cross polarization and MAS = magic angle spinning). For comparison, 15N NMR spectra of an aqueous solution of PLL were measured as a function of pH. In order to understand the effects of protonation and hydration on the 15N chemical shifts of the amino groups, DFT and chemical shielding calculations were performed on isolated methylamine-acid complexes and on periodic halide clusters of the type (CH3NH3(+)X(-))n. The combined experimental and computational results reveal low-field shifts of the amino nitrogens upon interaction with the oxygen acids HX = HF, H2SO4, CH3COOH, (CH3)2POOH, H3PO4, HNO3, and internal carbamic acid formed by reaction of the amino groups with gaseous CO2. Evidence is obtained that only hydrogen-bonded species of the type (Lys-NH2***H-X)n are formed in the absence of water. 15N chemical shifts are maximum when H is located in the hydrogen bond center and then decrease again upon full protonation, as found for aqueous solution at low pH. By contrast, halogen acids interact in a different way. They form internal salts of the type (Lys-NH3(+)X(-))n via the interaction of many acid-base pairs. This salt formation is possible only in the beta-sheet conformation. By contrast, the formation of hydrogen-bonded complexes can occur both in beta-sheet domains as well as in alpha-helical domains. The 15N chemical shifts of the protonated ammonium groups increase when the size of the interacting halogen anions is increased from chloride to iodide and when the number of the interacting anions is increased. Thus, the observed high-field 15N shift of ammonium groups upon hydration is the consequence of replacing interacting halogen atoms by oxygen atoms.

  16. What Is a pH Probe Study?

    MedlinePlus

    What is a pH Probe Study ? What is pH a probe study? M easuring the pH in the esophagus helps determine whether or not acid is coming up from the stomach. A pH probe study is usually done in patients where ...

  17. Probing the Lewis acidity and catalytic activity of the metal-organic framework [Cu3(btc)2] (BTC=benzene-1,3,5-tricarboxylate).

    PubMed

    Alaerts, Luc; Séguin, Etienne; Poelman, Hilde; Thibault-Starzyk, Frédéric; Jacobs, Pierre A; De Vos, Dirk E

    2006-09-25

    An optimized procedure was designed for the preparation of the microporous metal-organic framework (MOF) [Cu3(btc)2] (BTC=benzene-1,3,5-tricarboxylate). The crystalline material was characterized by X-ray diffraction, optical microscopy, SEM, X-ray photoelectron spectroscopy, N2 sorption, thermogravimetry, and IR spectroscopy of adsorbed CO. CO adsorbs on a small number of Cu2O impurities, and particularly on the free CuII coordination sites in the framework. [Cu3(btc)2] is a highly selective Lewis acid catalyst for the isomerization of terpene derivatives, such as the rearrangement of alpha-pinene oxide to campholenic aldehyde and the cyclization of citronellal to isopulegol. By using the ethylene ketal of 2-bromopropiophenone as a test substrate, it was demonstrated that the active sites in [Cu3(btc)2] are hard Lewis acids. Catalyst stability, re-usability, and heterogeneity are critically assessed. PMID:16881030

  18. A DFT study of the acid-base properties of anatase TiO2 and tetragonal ZrO2 by adsorption of CO and CO2 probe molecules

    NASA Astrophysics Data System (ADS)

    Chen, Hsin-Yi Tiffany; Tosoni, Sergio; Pacchioni, Gianfranco

    2016-10-01

    We have performed a comparative study of the acid-base characteristics of the surfaces of anatase TiO2 and tetragonal ZrO2. To this end we performed DFT + U calculations on CO and CO2 probe molecules adsorbed both on terraces and steps of the two oxides. For titania, CO adsorption results in a moderate adsorption energy (about - 0.3 eV) and in a positive shift of the Csbnd O stretching frequency (about + 40 cm- 1), typical of Lewis acid sites, with no clear difference in the acidity between terraces or steps. For zirconia we found a similar CO binding energy as for titania, and a CO vibrational shift that depends on the location of the Zr cation: negligible on terraces, similar to TiO2 on steps. We conclude that the acidic properties are similar in the two oxide surfaces. Things are different for CO2 adsorption. On titania the interaction is weak and surface carbonates compete with physisorbed CO2, indicating a weak basic character. On the contrary, on zirconia three types of stable carbonates have been identified. Their vibrational frequencies are consistent with IR measurements reported in the literature. The most stable species forms on steps of the t-ZrO2 surface and consists of a CO32 - unit which lies flat on the surface with the O atoms pointing towards three Zr ions. The species forms spontaneously by extraction of a lattice oxygen by an incoming CO2 molecule. The different reactivity points towards a much more pronounced basic character of zirconia compared to titania, at least if measured by CO2 adsorption.

  19. A dinuclear ruthenium(II) complex as turn-on luminescent probe for hypochlorous acid and its application for in vivo imaging

    PubMed Central

    Liu, Zonglun; Gao, Kuo; Wang, Beng; Yan, Hui; Xing, Panfei; Zhong, Chongmin; Xu, Yongqian; Li, Hongjuan; Chen, Jianxin; Wang, Wei; Sun, Shiguo

    2016-01-01

    A dinuclear ruthenium(II) complex Ruazo was designed and synthesized, in which oxidative cyclization of the azo and o-amino group was employed for the detection of hypochlorous acid (HClO) in aqueous solution. The non-emissive Ruazo formed highly luminescent triazole-ruthenium(II) complex in presence of HClO and successfully imaged HClO in living cell and living mouse. PMID:27356618

  20. A dinuclear ruthenium(II) complex as turn-on luminescent probe for hypochlorous acid and its application for in vivo imaging

    NASA Astrophysics Data System (ADS)

    Liu, Zonglun; Gao, Kuo; Wang, Beng; Yan, Hui; Xing, Panfei; Zhong, Chongmin; Xu, Yongqian; Li, Hongjuan; Chen, Jianxin; Wang, Wei; Sun, Shiguo

    2016-06-01

    A dinuclear ruthenium(II) complex Ruazo was designed and synthesized, in which oxidative cyclization of the azo and o-amino group was employed for the detection of hypochlorous acid (HClO) in aqueous solution. The non-emissive Ruazo formed highly luminescent triazole-ruthenium(II) complex in presence of HClO and successfully imaged HClO in living cell and living mouse.

  1. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

    SciTech Connect

    Price, E.M.; Smith, P.L.; Klein, T.E.; Freisheim, J.H.

    1987-07-28

    N/sup ..cap alpha../-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-(/sup 125/I)iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the /sup 125/I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.

  2. A novel DAD type and folic acid conjugated fluorescent monomer as a targeting probe for imaging of folate receptor overexpressed cells.

    PubMed

    Ekiz Kanik, Fulya; Ag, Didem; Seleci, Muharrem; Barlas, Firat Baris; Kesik, Melis; Hizalan, Gonul; Akpinar, Hava; Timur, Suna; Toppare, Levent

    2014-01-01

    We describe a modification and post-functionalization technique for a donor-acceptor-donor type monomer; 6-(4,7-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-2H-benzo[d][1,2, 3]triazol-2-yl)hexan-1-amine. Folic acid was attached to the fluorescent structure. The conjugation was confirmed via NMR and Fourier transform infrared analyses. Cytotoxicity was investigated and the comparison of association of targeted monomeric structures in tumor cells was monitored via fluorescence microscopy.

  3. Probing the molecular and electronic structure of norhipposudoric and hipposudoric acids from the red sweat of Hippopotamus amphibius: a DFT investigation.

    PubMed

    Galasso, Vinicio; Pichierri, Fabio

    2009-03-19

    Molecular structure and tautomeric/conformational preferences of norhipposudoric and hipposudoric acids, the recently isolated pigments of the Hippopotamus amphibius' sweat, were investigated using the density functional theory (DFT) PBE0 formalism. Among a large variety of possible structures, two similar keto-enol tautomer/conformers are nearly isoenergetic and markedly more stable than the others both in the gas phase and aqueous solution. The bulk solvent effect was accounted for with the polarizable continuum model (PCM). A distinctive structural feature is the strong intramolecular hydrogen bonding in the keto-enol O-H...O bridge, as shown by analysis of the atoms-in-molecules topological properties of the electron density. To elucidate the claimed strong acidity of these pigments, the site-specific microscopic dissociation constants were also calculated using the cluster-continuum model, a hybrid approach based on inclusion of explicit solvent molecules and solvation of the cluster by the dielectric continuum. Notably, the first deprotonation should occur predominantly from the enolic group with a remarkably low pk(i) value. This factor could play an important role in the potent antibiotic activity of the pigments. The absorption spectra of the undissociated and dissociated compounds in aqueous solution were interpreted with time-dependent DFT/PCM calculations. The pi-pi* diquinoid excitations, mainly occurring in the fluorenoid nucleus, are the major contributors to the color and strong absorption bands in the UVA and UVB regions, which are closely related to the efficient sunscreen activity exerted by the pigments.

  4. Ethylene Diamine Tetraacetic Acid Etched Quantum Dots as a "Turn-On" Fluorescence Probe for Detection of Trace Zinc in Food.

    PubMed

    Liu, Wei; Wei, Fangdi; Xu, Guanhong; Wu, Yanzi; Hu, Chunting; Song, Quan; Yang, Jing; Hu, Qin

    2016-06-01

    In the present paper, a simple and rapid "turn-on" fluorescence sensor for Zn2+ based on ethylene diamine tetraacetic acid (EDTA) etched CdTe quantum dots (QDs) was developed. First, the initial bright fluorescence of mercaptopropionic acid (MPA) capped CdTe QDs was effectively quenched by EDTA, and then the presence of Zn2+ could "turn on" the weak fluorescence of QDs quenched by EDTA due to the formation of ZnS passivation shell. The increase of fluorescence intensity of EDTA etched QDs was found to be linear with the concentration of Zn2+ added. Under the optimum conditions, the calibration curve of this method showed good linearity in the concentration range of 9.1-1 09.1 μM of Zn2+ with the correlation coefficient R2 = 0.998. The limit of detection (3σ/K) was 2 μM. The developed QDs-based sensor was successfully applied to detect trace zinc in zinc fortified table salts and energy drinks with satisfactory results. PMID:27427745

  5. Hydrodynamic ultrasonic probe

    DOEpatents

    Day, Robert A.; Conti, Armond E.

    1980-01-01

    An improved probe for in-service ultrasonic inspection of long lengths of a workpiece, such as small diameter tubing from the interior. The improved probe utilizes a conventional transducer or transducers configured to inspect the tubing for flaws and/or wall thickness variations. The probe utilizes a hydraulic technique, in place of the conventional mechanical guides or bushings, which allows the probe to move rectilinearly or rotationally while preventing cocking thereof in the tube and provides damping vibration of the probe. The probe thus has lower friction and higher inspection speed than presently known probes.

  6. Stealth CD44-targeted hyaluronic acid supramolecular nanoassemblies for doxorubicin delivery: probing the effect of uncovalent pegylation degree on cellular uptake and blood long circulation.

    PubMed

    Han, Xiaopeng; Li, Zhenbao; Sun, Jin; Luo, Cong; Li, Lin; Liu, Yuhai; Du, Yuqian; Qiu, Shuhong; Ai, Xiaoyu; Wu, Chunnuan; Lian, He; He, Zhonggui

    2015-01-10

    Stealth active targeting nanoparticles (NPs) usually include two types of ligand sites: ligand anchored on distal ends of the polyethylene glycol (PEG) and ligand buried under pegylated layer. The latter typical case is hyaluronic acid (HA)-based NPs; however, there is little information available for the latter NPs about effect of the optimal density of surface PEG coating on the blood circulation time, cellular uptake and in vivo anticancer activity. Thus, in this study, in order to optimize the anticancer effects of HA-based NPs, we focus on how uncovalent pegylation degree modulates blood circulation time and cellular uptake of HA-based NPs. We firstly designed a new double-hydrophilic copolymer by conjugating HP-β-cyclodextrin with HA, and this carrier was further pegylated with adamantyl-peg (ADA-PEG) to form inclusion complex HA-HPCD/ADA-PEG, termed as HCPs. The supramolecular nanoassemblies were fabricated by host-guest and polar interactions between HCPs and doxorubicin (Dox), with vitamin E succinate (VES) being a nanobridge. Despite the active recognition between HA and CD44 receptor, the cellular uptake and targeting efficiency of HA-NPs decreased with the increasing peg density, demonstrating HA was partly buried by high density peg coating. However, the high density of peg coating was beneficial to long circulation time, tumor biodistribution and anticancer activity in vivo. NPs with 5% peg coating had the optimal cellular targeting efficiency in vitro and anticancer effects in vivo. The findings suggest that balancing long circulation property and cellular uptake is important to achieve the optimal antitumor efficacy for pegylated HA-based NPs, and that PEG coating densities cannot be extended beyond a certain density for shielding effect without compromising the efficacy of hyaluronic acid targeted delivery.

  7. Probing polymorphism and reactivity in the organic solid state using 13C NMR spectroscopy: Studies of p-Formyl- trans-cinnamic acid

    NASA Astrophysics Data System (ADS)

    Harris, Kenneth D. M.; Thomas, John M.

    1991-09-01

    p-Formyl- trans-cinnamic acid (p-FCA) is known to exist in two different crystal phases (denoted β and γ). When crystals of the β phase of p-FCA are exposed to UV radiation, a solid state dimerization reaction occurs to produce 4,4'-diformyl-β-truxinic acid. In contrast, crystals of the γ phase of p-FCA are photostable. It is shown in this paper that high resolution solid state 13C NMR spectroscopy is a sensitive technique for distinguishing the β and γ phases of p-FCA, and can be used to investigate, in detail, the chemical transformation that occurs upon UV irradiation of the β phase. Specifically, the 13C NMR spectra presented here were recorded using the TOSS (total suppression of sidebands) pulse sequence; this is based upon the standard 13C CPMAS (cross polarization/magic angle sample spinning/high power 1H decoupling) method, but has the additional feature that all orders of spinning sidebands are eliminated from the spectrum. The photoproduct obtained from UV irradiation of β-p-FCA contains a significant noncrystalline component (assessed via powder X-ray diffraction), and our NMR studies suggest that this noncrystalline component of the photoproduct contains some amount of the γ phase of the monomer p-FCA. A mechanism is proposed to explain the fact that UV irradiation of β-p-FCA can generate, in addition to the expected photodimer, an impurity amount of the γ phase of p-FCA.

  8. Probing the molecular and electronic structure of norhipposudoric and hipposudoric acids from the red sweat of Hippopotamus amphibius: a DFT investigation.

    PubMed

    Galasso, Vinicio; Pichierri, Fabio

    2009-03-19

    Molecular structure and tautomeric/conformational preferences of norhipposudoric and hipposudoric acids, the recently isolated pigments of the Hippopotamus amphibius' sweat, were investigated using the density functional theory (DFT) PBE0 formalism. Among a large variety of possible structures, two similar keto-enol tautomer/conformers are nearly isoenergetic and markedly more stable than the others both in the gas phase and aqueous solution. The bulk solvent effect was accounted for with the polarizable continuum model (PCM). A distinctive structural feature is the strong intramolecular hydrogen bonding in the keto-enol O-H...O bridge, as shown by analysis of the atoms-in-molecules topological properties of the electron density. To elucidate the claimed strong acidity of these pigments, the site-specific microscopic dissociation constants were also calculated using the cluster-continuum model, a hybrid approach based on inclusion of explicit solvent molecules and solvation of the cluster by the dielectric continuum. Notably, the first deprotonation should occur predominantly from the enolic group with a remarkably low pk(i) value. This factor could play an important role in the potent antibiotic activity of the pigments. The absorption spectra of the undissociated and dissociated compounds in aqueous solution were interpreted with time-dependent DFT/PCM calculations. The pi-pi* diquinoid excitations, mainly occurring in the fluorenoid nucleus, are the major contributors to the color and strong absorption bands in the UVA and UVB regions, which are closely related to the efficient sunscreen activity exerted by the pigments. PMID:19239216

  9. 5 prime -Azido-(3,6- sup 3 H sub 2 )-1-naphthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: Identification of a 23-kDa protein from maize coleoptile plasma membranes

    SciTech Connect

    Zettl, R.; Feldwisch, J.; Schell, J.; Palme, K. ); Boland, W. )

    1992-01-15

    1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, the authors have synthesized a tritiated, photolabile NPA analogue, 5{prime}-azido-(3,6-{sup 3}H{sub 2})NPA (({sup 3}H{sub 2})N{sub 3}NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of ({sup 3}H{sub 2})N{sub 3}NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with ({sup 3}H{sub 2})N{sub 3}NPA and exposure to UV light, they observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-(7-{sup 3}H)indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.

  10. Probing the Efficacy of Peptide-Based Inhibitors against Acid- and Zinc-Promoted Oligomerization of Amyloid-β Peptide via Single-Oligomer Spectroscopy

    PubMed Central

    Powell, Lyndsey R.; Dukes, Kyle D.; Lammi, Robin K.

    2011-01-01

    One avenue for prevention and treatment of Alzheimer's disease involves inhibiting the aggregation of amyloid-β peptide (Aβ). Given the deleterious effects reported for Aβ dimers and trimers, it is important to investigate inhibition of the earliest association steps. We have employed quantized photobleaching of dye-labeled Aβ peptides to characterize four peptide-based inhibitors of fibrillogenesis and/or cytotoxicity, assessing their ability to inhibit association in the smallest oligomers (n = 2–5). Inhibitors were tested at acidic pH and in the presence of zinc, conditions that may promote oligomerization in vivo. Distributions of peptide species were constructed by examining dozens of surface-tethered monomers and oligomers, one at a time. Results show that all four inhibitors shift the distribution of Aβ species toward monomers; however, efficacies vary for each compound and sample environment. Collectively, these studies highlight promising design strategies for future oligomerization inhibitors, affording insight into oligomer structures and inhibition mechanisms in two physiologically significant environments. PMID:21945664

  11. Thioglycolic Acid-Capped CdS Quantum Dots Conjugated to α-Amylase as a Fluorescence Probe for Determination of Starch at Low Concentration.

    PubMed

    Tayebi, Mahnoush; Tavakkoli Yaraki, Mohammad; Mogharei, Azadeh; Ahmadieh, Mahnaz; Tahriri, Mohammadreza; Vashaee, Daryoosh; Tayebi, Lobat

    2016-09-01

    In the present research, water soluble thioglycolic acid-capped CdS quantum dots (QDs) were synthesized by chemical precipitation method. The characteristics of prepared quantum dots were determined using X-Ray Diffraction (XRD) and Transmission Electron Microscopy (TEM). The obtained results revealed that CdS QDs have 5.60 nm crystallite size, hexagonal wurtzite structure and spherical morphology with less than 10 nm diameter. The photoluminescence (PL) spectroscopy was performed in order to study the effect of the presence of starch solutions. Blue emission peaks were positioned at 488 nm and its intensity quenched by increasing the concentration of starch solutions. The result of PL quenches in range of studied concentrations (0-100 ppm) was best described by Michaelis-Menten model. The amount of Michaelis constant (Km) for immobilized α-amylase in this system was about 68.08 ppm which showed a great tendency of enzyme to hydrolyze the starch as substrate. Finally, the limit of detection (LOD) was found to be about 2.24 ppm. PMID:27392974

  12. Triacontanol and jasmonic acid differentially modulate the lipid organization as evidenced by the fluorescent probe behavior and 31P nuclear magnetic resonance shifts in model membranes.

    PubMed

    Sivakumar Swamy, G; Swamy, Sivakumar G; Ramanarayan, K; Inamdar, Laxmi S; Inamdar, Sanjeev R

    2009-04-01

    Fluorescence resonance energy transfer (FRET), time-resolved fluorescence and anisotropy decays were determined in large unilamellar vesicles (LUVs) of egg phosphatidylcholine with the FRET pair N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine as donor and lissamine rhodamine B 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine as acceptor, using 2-ps pulses from a Ti:sapphire laser on LUVs with incorporated plant growth regulators: triacontanol (TRIA) and jasmonic acid (JA). FRET efficiency, energy transfer rate, rotation correlation time, microviscosity, and diffusion coefficient of lateral diffusion of lipids were calculated from these results. It was observed that TRIA and JA differentially modulated all parameters studied. The effect of JA in such modulations was always partially reversed by TRIA. Also, the generalized polarization of laurdan fluorescence indicated that JA enhances the degree of hydration in lipid bilayers to a larger extent than does TRIA. Solid-state (31)P magic-angle spinning nuclear magnetic resonance spectra of LUVs showed two chemical shifts, at 0.009 and -11.988 ppm, at low temperatures (20 degrees C), while at increasing temperatures (20-60 degrees C) only one (at -11.988 ppm) was prominent and the other (0.009 ppm) gradually became obscure. However, LUVs with TRIA exhibited only one of the shifts at 0.353 ppm even at lower temperatures and JA did not affect the chemical shifts.

  13. Probing the roles of Ca(2+) and Mg(2+) in humic acids-induced ultrafiltration membrane fouling using an integrated approach.

    PubMed

    Wang, Long-Fei; He, Dong-Qin; Chen, Wei; Yu, Han-Qing

    2015-09-15

    Membrane fouling induced by natural organic matter (NOM) negatively affects the performance of ultrafiltration (UF) technology in producing drinking water. Divalent cation is found to be an important factor that affects the NOM-induced membrane fouling process. In this work, attenuated total reflection-Fourier transformation infrared spectroscopy (ATR-FTIR) coupled with quartz crystal microbalance (QCM), assisted by isothermal titration calorimetry (ITC), is used to explore the contribution of Mg(2+) and Ca(2+), the two abundant divalent cations in natural water, to the UF membrane fouling caused by humic acid (HA) at a molecular level. The results show that Ca(2+) exhibited superior performance in accelerating fouling compared to Mg(2+). The hydrophobic polyethersulfone (PES) membrane exhibited greater complexation with HA in the presence of Mg(2+) and Ca(2+), compared to the hydrophilic cellulose membrane, as evidenced by the more intense polysaccharide C-O, aromatic C=C and carboxylic C=O bands in the FTIR spectra. The QCM and ITC measurements provide quantitative evidence to support that Ca(2+) was more effective than Mg(2+) in binding with HA and accumulating foulants on the membrane surfaces. The higher charge neutralization capacity and more favorable binding ability of Ca(2+) were found to be responsible for its greater contribution to the NOM-induced membrane fouling than Mg(2+). This work offers a new insight into the mechanism of cation-mediated NOM-induced membrane fouling process, and demonstrates that such an integrated ATR-FTIR/QCM/ITC approach could be a useful tool to explore other complicated interaction processes in natural and engineered environments. PMID:26094086

  14. Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

    PubMed Central

    Cerqueira, Laura; Fernandes, Ricardo M.; Ferreira, Rui M.; Oleastro, Mónica; Carneiro, Fátima; Brandão, Catarina; Pimentel-Nunes, Pedro; Dinis-Ribeiro, Mário; Figueiredo, Céu; Keevil, Charles W.; Vieira, Maria J.

    2013-01-01

    Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach. PMID:23596234

  15. Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein–Protein Interactions

    PubMed Central

    2016-01-01

    Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein–protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries. PMID:27417384

  16. The unique probe selector: a comprehensive web service for probe design and oligonucleotide arrays

    PubMed Central

    Chen, Shu-Hwa; Lo, Chen-Zen; Tsai, Ming-Chi; Hsiung, Chao A; Lin, Chung-Yen

    2008-01-01

    Background Nucleic acid hybridization, a fundamental technique in molecular biology, can be modified into very effective and sensitive methods for detecting particular targets mixed with millions of non-target sequences. Therefore, avoiding cross-hybridization is the most crucial issue for developing diagnostic methods based on hybridization. Results To develop a probe with a high discriminating power, this study constructed a web service, the Unique Probe Selector (UPS), for customized probe design. The UPS service integrates a probe design mechanism and a scoring system for evaluating the performance of probe annealing and the uniqueness of a probe in a user-defined genetic background. Starting from an intuitive web interface, the UPS accepts a query with single or multiple sequences in fasta format. The best probe(s) for each sequence can be downloaded from result pages in a fasta or .csv format with a summary of probe characteristics. The option "Unique probe within group" selects the most unique probe for each target sequence with low probability to hybridize to the other sequences in the same submitted query. The option "Unique probe in the specific organism" devises probes for each submitted sequence to identify its target among selected genetic backgrounds based on Unigene. Conclusion The UPS evaluates probe-to-target hybridization under a user-defined condition in silico to ensure high-performance hybridization and minimizes the possibility of non-specific reactions. UPS has been applied to design human arrays for gene expression studies and to develop several small arrays of gene families that were inferred as molecular signatures of cancer typing/staging or pathogen signatures. Notably, UPS is freely accessible at . PMID:18315861

  17. Development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes.

    PubMed

    Sidoti, Francesca; Bergallo, Massimiliano; Terlizzi, Maria Elena; Piasentin Alessio, Elsa; Astegiano, Sara; Gasparini, Giorgio; Cavallo, Rossana

    2012-03-01

    Evidence demonstrating that human rhinovirus (HRV) disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of HRV infections. In this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. We described a simple method to accurately quantify RNA target by computing the time to positivity (TTP) values for HRV RNA. Quantification capacity was assessed by plotting these TTP values against the starting number of target molecules. By using this simple method, we have significantly increased the diagnostic accuracy, precision, and trueness of real-time NASBA assay. Specificity of the method was verified in both in silico and experimental studies. Moreover, for assessment of clinical reactivity of the assay, NASBA has been validated on bronchoalveolar lavage (BAL) specimens. Our quantitative NASBA assay was found to be very specific, accurate, and precise with high repeatability and reproducibility.

  18. Transformation of 17β-estradiol in humic acid solution by ε-MnO2 nanorods as probed by high-resolution mass spectrometry combined with (13)C labeling.

    PubMed

    Sun, Kai; Liang, Shangtao; Kang, Fuxing; Gao, Yanzheng; Huang, Qingguo

    2016-07-01

    Steroidal estrogens (SEs), widespread in aquatic systems, have a potential to disrupt the endocrine system of wildlife species and humans. In our experiments, the performance of ε-MnO2 nanorods in transforming 17β-estradiol (E2) was investigated, and the effect of humic acid (HA) on the reaction behaviors was systematically characterized. Reconfiguration of humic molecules was also investigated by high-performance size exclusion chromatography (HPSEC). Results indicated that ε-MnO2 nanomaterials ensured efficient removal of E2 from the aqueous solution. The presence of HA hindered the transformation of E2, while enhanced the cross-coupling between E2 and humic molecules. In particular, we used a mixture of un-labeled E2 and (13)C3-labeled E2 at a 1: 1 set ratio (w/w) to probe the reaction products via high-resolution mass spectrometry (HRMS). The combination of HRMS and (13)C3-labeling revealed the intermediate products including estrone (E1), and hydroxylated, quinone-like, and ring-opened species, as well as E2 dimer and trimer. More importantly, possible cross-coupling products between E2 and HA were also identified. A reaction mechanism including two-electron oxidation and single-electron oxidation was proposed. The applied analytical approach using HRMS along with (13)C3-labeling for reaction-product identification is crucial to understanding the role of HA in the transformation of SEs.

  19. Transformation of 17β-estradiol in humic acid solution by ε-MnO2 nanorods as probed by high-resolution mass spectrometry combined with (13)C labeling.

    PubMed

    Sun, Kai; Liang, Shangtao; Kang, Fuxing; Gao, Yanzheng; Huang, Qingguo

    2016-07-01

    Steroidal estrogens (SEs), widespread in aquatic systems, have a potential to disrupt the endocrine system of wildlife species and humans. In our experiments, the performance of ε-MnO2 nanorods in transforming 17β-estradiol (E2) was investigated, and the effect of humic acid (HA) on the reaction behaviors was systematically characterized. Reconfiguration of humic molecules was also investigated by high-performance size exclusion chromatography (HPSEC). Results indicated that ε-MnO2 nanomaterials ensured efficient removal of E2 from the aqueous solution. The presence of HA hindered the transformation of E2, while enhanced the cross-coupling between E2 and humic molecules. In particular, we used a mixture of un-labeled E2 and (13)C3-labeled E2 at a 1: 1 set ratio (w/w) to probe the reaction products via high-resolution mass spectrometry (HRMS). The combination of HRMS and (13)C3-labeling revealed the intermediate products including estrone (E1), and hydroxylated, quinone-like, and ring-opened species, as well as E2 dimer and trimer. More importantly, possible cross-coupling products between E2 and HA were also identified. A reaction mechanism including two-electron oxidation and single-electron oxidation was proposed. The applied analytical approach using HRMS along with (13)C3-labeling for reaction-product identification is crucial to understanding the role of HA in the transformation of SEs. PMID:27086077

  20. Determination of essential fatty acid composition among mutant lines of Canola (Brassica napus), through high pressure liquid chromatography.

    PubMed

    Raza, Ghulam; Siddique, Aquil; Khan, Imtiaz Ahmad; Ashraf, Muhammed Yasin; Khatri, Abdullah

    2009-12-01

    The present study aimed to quantify the methyl esters of lenoleic acid (LA), gamma-lenolenic acid (LNA) and oleic acid (OL) in the oil of Brassica napus mutants. Five stable mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of B. napus cv. 'Rainbow' (P) and three mutants (W97-95/16, W97-0.75/11 and W97-.075/13) of B. napus cv. 'Westar' (P) at M6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield was observed in the mutant (ROO-100/6) followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parent plants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performance liquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, ranging from 39.1 to 66.3%; LA was second (15.3-41.6%) and LNA was third (18.1-28.9%). Mutant ROO-125/14 showed higher OL contents than parent (Rainbow). These results are expected to support the approval of ROO-125/14 in the National Uniform Varietal Yield Trials (NUVYT) as a new variety based on high oil quality.

  1. Galileo Probe Battery System

    NASA Technical Reports Server (NTRS)

    Dagarin, B. P.; Taenaka, R. K.; Stofel, E. J.

    1997-01-01

    The conclusions of the Galileo probe battery system are: the battery performance met mission requirements with margin; extensive ground-based and flight tests of batteries prior to probe separation from orbiter provided good prediction of actual entry performance at Jupiter; and the Li-SO2 battery was an important choice for the probe's main power.

  2. Use of a fiber optic probe for organic species determination

    DOEpatents

    Ekechukwu, Amy A.

    1996-01-01

    A fiber optic probe for remotely detecting the presence and concentration organic species in aqueous solutions. The probe includes a cylindrical housing with an organic species indicator, preferably diaminonaphthyl sulfonic acid adsorbed in a silica gel (DANS-modified gel), contained in the probe's distal end. The probe admits aqueous solutions to the probe interior for mixing within the DANS-modified gel. An optical fiber transmits light through the DANS-modified gel while the indicator reacts with organic species present in the solution, thereby shifting the location of the fluorescent peak. The altered light is reflected to a receiving fiber that carries the light to a spectrophotometer or other analysis device.

  3. Probing zeolites by vibrational spectroscopies.

    PubMed

    Bordiga, Silvia; Lamberti, Carlo; Bonino, Francesca; Travert, Arnaud; Thibault-Starzyk, Frédéric

    2015-10-21

    This review addresses the most relevant aspects of vibrational spectroscopies (IR, Raman and INS) applied to zeolites and zeotype materials. Surface Brønsted and Lewis acidity and surface basicity are treated in detail. The role of probe molecules and the relevance of tuning both the proton affinity and the steric hindrance of the probe to fully understand and map the complex site population present inside microporous materials are critically discussed. A detailed description of the methods needed to precisely determine the IR absorption coefficients is given, making IR a quantitative technique. The thermodynamic parameters of the adsorption process that can be extracted from a variable-temperature IR study are described. Finally, cutting-edge space- and time-resolved experiments are reviewed. All aspects are discussed by reporting relevant examples. When available, the theoretical literature related to the reviewed experimental results is reported to support the interpretation of the vibrational spectra on an atomic level.

  4. Ultrafast scanning probe microscopy

    DOEpatents

    Weiss, Shimon; Chemla, Daniel S.; Ogletree, D. Frank; Botkin, David

    1995-01-01

    An ultrafast scanning probe microscopy method for achieving subpicosecond-temporal resolution and submicron-spatial resolution of an observation sample. In one embodiment of the present claimed invention, a single short optical pulse is generated and is split into first and second pulses. One of the pulses is delayed using variable time delay means. The first pulse is then directed at an observation sample located proximate to the probe of a scanning probe microscope. The scanning probe microscope produces probe-sample signals indicative of the response of the probe to characteristics of the sample. The second pulse is used to modulate the probe of the scanning probe microscope. The time delay between the first and second pulses is then varied. The probe-sample response signal is recorded at each of the various time delays created between the first and second pulses. The probe-sample response signal is then plotted as a function of time delay to produce a cross-correlation of the probe sample response. In so doing, the present invention provides simultaneous subpicosecond-temporal resolution and submicron-spatial resolution of the sample.

  5. Ultrafast scanning probe microscopy

    DOEpatents

    Weiss, S.; Chemla, D.S.; Ogletree, D.F.; Botkin, D.

    1995-05-16

    An ultrafast scanning probe microscopy method is described for achieving subpicosecond-temporal resolution and submicron-spatial resolution of an observation sample. In one embodiment of the present claimed invention, a single short optical pulse is generated and is split into first and second pulses. One of the pulses is delayed using variable time delay means. The first pulse is then directed at an observation sample located proximate to the probe of a scanning probe microscope. The scanning probe microscope produces probe-sample signals indicative of the response of the probe to characteristics of the sample. The second pulse is used to modulate the probe of the scanning probe microscope. The time delay between the first and second pulses is then varied. The probe-sample response signal is recorded at each of the various time delays created between the first and second pulses. The probe-sample response signal is then plotted as a function of time delay to produce a cross-correlation of the probe sample response. In so doing, the present invention provides simultaneous subpicosecond-temporal resolution and submicron-spatial resolution of the sample. 6 Figs.

  6. Traversing probe system

    DOEpatents

    Mashburn, Douglas N.; Stevens, Richard H.; Woodall, Harold C.

    1977-01-01

    This invention comprises a rotatable annular probe-positioner which carries at least one radially disposed sensing probe, such as a Pitot tube having a right-angled tip. The positioner can be coaxially and rotatably mounted within a compressor casing or the like and then actuated to orient the sensing probe as required to make measurements at selected stations in the annulus between the positioner and compressor casing. The positioner can be actuated to (a) selectively move the probe along its own axis, (b) adjust the yaw angle of the right-angled probe tip, and (c) revolve the probe about the axis common to the positioner and casing. A cam plate engages a cam-follower portion of the probe and normally rotates with the positioner. The positioner includes a first-motor-driven ring gear which effects slidable movement of the probe by rotating the positioner at a time when an external pneumatic cylinder is actuated to engage the cam plate and hold it stationary. When the pneumatic cylinder is not actuated, this ring gear can be driven to revolve the positioner and thus the probe to a desired circumferential location about the above-mentioned common axis. A second motor-driven ring gear included in the positioner can be driven to rotate the probe about its axis, thus adjusting the yaw angle of the probe tip. The positioner can be used in highly corrosive atmosphere, such as gaseous uranium hexafluoride.

  7. Electrical resistivity probes

    DOEpatents

    Lee, Ki Ha; Becker, Alex; Faybishenko, Boris A.; Solbau, Ray D.

    2003-10-21

    A miniaturized electrical resistivity (ER) probe based on a known current-voltage (I-V) electrode structure, the Wenner array, is designed for local (point) measurement. A pair of voltage measuring electrodes are positioned between a pair of current carrying electrodes. The electrodes are typically about 1 cm long, separated by 1 cm, so the probe is only about 1 inch long. The electrodes are mounted to a rigid tube with electrical wires in the tube and a sand bag may be placed around the electrodes to protect the electrodes. The probes can be positioned in a borehole or on the surface. The electrodes make contact with the surrounding medium. In a dual mode system, individual probes of a plurality of spaced probes can be used to measure local resistance, i.e. point measurements, but the system can select different probes to make interval measurements between probes and between boreholes.

  8. Nucleic acid programmed polymeric nanomaterials for biological communication

    NASA Astrophysics Data System (ADS)

    Rush, Anthony Michael

    A number of nucleic acid-polymer conjugates were synthesized, resulting in amphiphilic polymer-nucleic acid conjugates with the capability to self-assemble into a range of discrete nanoscale architectures. These nanomaterials, termed DNA-polymer amphiphile nanoparticles (DPA NPs), were studied with respect to their enzymatic processing by both endo- and exonucleases and further deployed as antisense genetic regulatory elements in live cultured human cells. DPA NPs were designed to act as substrates for both non sequence-specific exonucleases and a sequence-specific endonuclease. In all cases, nucleic acids arranged in the corona of spherical nanoparticles exhibited increased resistance to nucleolytic cleavage as compared to native single- or double-stranded analogues. For the exonucleases studied (Exonuclease III from E. Coli and phosphodiesterase I from Crotalus adamanteus), nanoparticle display retarded enzymatic processing by roughly a factor of five. For the endonuclease studied (Nt.CviPII), nanoparticle display prohibited virtually all enzyme activity on oligonucleotides within the nanoparticle shell. To test the ability of these materials to regulate mRNA levels in live cultured human cells, LPA (LNA-polymer amphiphile) NPs were designed to be perfectly complementary to a 20-base region of mRNA encoding the anti-apoptosis protein survivin. In this study two key observations were made. The first observation is that packaging LNA into spherical micellar nanoparticles serves to dramatically enhance cellular uptake of LNA based on flow cytometry and fluorescence microscopy data. The second observation is that LPA NPs are capable of regulating mRNA levels by what is hypothesized to be activation of target mRNA for catalytic RNase H-mediated degradation. These materials represent a unique class of DNA delivery system capable of rendering nucleic acids with natural backbone chemistry resistant to nuclease degradation and further serving to deliver DNA into cells to

  9. Methods for making nucleotide probes for sequencing and synthesis

    SciTech Connect

    Church, George M; Zhang, Kun; Chou, Joseph

    2014-07-08

    Compositions and methods for making a plurality of probes for analyzing a plurality of nucleic acid samples are provided. Compositions and methods for analyzing a plurality of nucleic acid samples to obtain sequence information in each nucleic acid sample are also provided.

  10. High temperature probe

    DOEpatents

    Swan, Raymond A.

    1994-01-01

    A high temperature probe for sampling, for example, smokestack fumes, and is able to withstand temperatures of 3000.degree. F. The probe is constructed so as to prevent leakage via the seal by placing the seal inside the water jacket whereby the seal is not exposed to high temperature, which destroys the seal. The sample inlet of the probe is also provided with cooling fins about the area of the seal to provide additional cooling to prevent the seal from being destroyed. Also, a heated jacket is provided for maintaining the temperature of the gas being tested as it passes through the probe. The probe includes pressure sensing means for determining the flow velocity of an efficient being sampled. In addition, thermocouples are located in various places on the probe to monitor the temperature of the gas passing there through.

  11. An ultrafast reciprocating probe

    NASA Astrophysics Data System (ADS)

    Liu, Wenbin; Tan, Yi; Wang, Wenhao; Gao, Zhe

    2016-11-01

    For tokamak plasma diagnostics, an ultrafast reciprocating probe system driven by magnetic field coils, achieving a maximum velocity of 21 m/s, is introduced. The probes are attached with a driving hoop made of carbon steel and accelerated by three acceleration coils in series, then decelerated by two deceleration coils and buffer springs and return slowly. The coils with a current of about 1 kA generate a magnetic field of about 1 T. This probe system has been tested on the SUNIST (Sino-UNIted Spherical Tokamak) spherical tokamak. Radial profiles of the floating potential and other plasma parameters measured by this probe system are given.

  12. Atom probe tomography

    SciTech Connect

    Miller, M.K.; Forbes, R.G.

    2009-06-15

    This introductory tutorial describes the technique of atom probe tomography for materials characterization at the atomic level. The evolution of the technique from the initial atom probe field ion microscope to today's state-of-the-art three dimensional atom probe is outlined. An introduction is presented on the basic physics behind the technique, the operation of the instrument, and the reconstruction of the three-dimensional data. The common methods for analyzing the three-dimensional atom probe data, including atom maps, isoconcentration surfaces, proximity histograms, maximum separation methods, and concentration frequency distributions, are described.

  13. Single molecule probes of membrane structure: orientation of BODIPY probes in DPPC as a function of probe structure.

    PubMed

    Armendariz, Kevin P; Huckabay, Heath A; Livanec, Philip W; Dunn, Robert C

    2012-03-21

    Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir-Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe.

  14. Radio frequency-compensated Langmuir probe with auxiliary double probes

    SciTech Connect

    Oh, Se-Jin; Oh, Seung-Ju; Chung, Chin-Wook

    2010-09-15

    A radio frequency (rf) compensation design using auxiliary double probes connected in parallel with a main measurement probe was developed for Langmuir probe diagnostics. This probe structure can reduce the sheath impedance of the main probe. In our probe design, the sheath capacitance of the probe can be increased and its sheath resistance can be decreased with increasing dc bias differential voltage between the auxiliary double probes. The I-V characteristic curve and electron energy distribution functions measured by our probe system had sufficient rf compensation performance in inductively coupled plasmas.

  15. Radio frequency-compensated Langmuir probe with auxiliary double probes.

    PubMed

    Oh, Se-Jin; Oh, Seung-Ju; Chung, Chin-Wook

    2010-09-01

    A radio frequency (rf) compensation design using auxiliary double probes connected in parallel with a main measurement probe was developed for Langmuir probe diagnostics. This probe structure can reduce the sheath impedance of the main probe. In our probe design, the sheath capacitance of the probe can be increased and its sheath resistance can be decreased with increasing dc bias differential voltage between the auxiliary double probes. The I-V characteristic curve and electron energy distribution functions measured by our probe system had sufficient rf compensation performance in inductively coupled plasmas.

  16. Formative Assessment Probes

    ERIC Educational Resources Information Center

    Eberle, Francis; Keeley, Page

    2008-01-01

    Formative assessment probes can be effective tools to help teachers build a bridge between students' initial ideas and scientific ones. In this article, the authors describe how using two formative assessment probes can help teachers determine the extent to which students make similar connections between developing a concept of matter and a…

  17. PDV Probe Alignment Technique

    SciTech Connect

    Whitworth, T L; May, C M; Strand, O T

    2007-10-26

    This alignment technique was developed while performing heterodyne velocimetry measurements at LLNL. There are a few minor items needed, such as a white card with aperture in center, visible alignment laser, IR back reflection meter, and a microscope to view the bridge surface. The work was performed on KCP flyers that were 6 and 8 mils wide. The probes used were Oz Optics manufactured with focal distances of 42mm and 26mm. Both probes provide a spot size of approximately 80?m at 1550nm. The 42mm probes were specified to provide an internal back reflection of -35 to -40dB, and the probe back reflections were measured to be -37dB and -33dB. The 26mm probes were specified as -30dB and both measured -30.5dB. The probe is initially aligned normal to the flyer/bridge surface. This provides a very high return signal, up to -2dB, due to the bridge reflectivity. A white card with a hole in the center as an aperture can be used to check the reflected beam position relative to the probe and launch beam, and the alignment laser spot centered on the bridge, see Figure 1 and Figure 2. The IR back reflection meter is used to measure the dB return from the probe and surface, and a white card or similar object is inserted between the probe and surface to block surface reflection. It may take several iterations between the visible alignment laser and the IR back reflection meter to complete this alignment procedure. Once aligned normal to the surface, the probe should be tilted to position the visible alignment beam as shown in Figure 3, and the flyer should be translated in the X and Y axis to reposition the alignment beam onto the flyer as shown in Figure 4. This tilting of the probe minimizes the amount of light from the bridge reflection into the fiber within the probe while maintaining the alignment as near normal to the flyer surface as possible. When the back reflection is measured after the tilt adjustment, the level should be about -3dB to -6dB higher than the probes

  18. Inflatable traversing probe seal

    NASA Technical Reports Server (NTRS)

    Trimarchi, Paul A.

    1991-01-01

    An inflatable seal acts as a pressure-tight zipper to provide traversing capability for instrumentation rakes and probes. A specially designed probe segment with a teardrop cross-section in the vicinity of the inflatable seal minimizes leakage at the interface. The probe is able to travel through a lengthwise slot in a pressure vessel or wind tunnel section, while still maintaining pressure integrity. The design uses two commercially available inflatable seals, opposing each other, to cover the probe slot in a wind tunnel wall. Proof-of-concept tests were conducted at vessel pressures up to 30 psig, with seals inflated to 50 psig, showing no measurable leakage along the seal's length or around the probe teardrop cross-section. This seal concept can replace the existing technology of sliding face plate/O-ring systems in applications where lengthwise space is limited.

  19. Determination of the solution-bound conformation of an amino acid binding protein by NMR paramagnetic relaxation enhancement: use of a single flexible paramagnetic probe with improved estimation of its sampling space.

    PubMed

    Bermejo, Guillermo A; Strub, Marie-Paule; Ho, Chien; Tjandra, Nico

    2009-07-15

    We demonstrate the feasibility of elucidating the bound ("closed") conformation of a periplasmic binding protein, the glutamine-binding protein (GlnBP), in solution, using paramagnetic relaxation enhancements (PREs) arising from a single paramagnetic group. GlnBP consists of two globular domains connected by a hinge. Using the ligand-free ("open") conformation as a starting point, conjoined rigid-body/torsion-angle simulated annealing calculations were performed using backbone (1)H(N)-PREs as a major source of distance information. Paramagnetic probe flexibility was accounted for via a multiple-conformer representation. A conventional approach where the entire PRE data set is enforced at once during simulated annealing yielded poor results due to inappropriate conformational sampling of the probe. On the other hand, significant improvements in coordinate accuracy were obtained by estimating the probe sampling space prior to structure calculation. Such sampling is achieved by refining the ensemble of probe conformers with intradomain PREs only, keeping the protein backbone fixed in the open form. Subsequently, while constraining the probe to the previously found conformations, the domains are allowed to move relative to each other under the influence of the non-intradomain PREs, giving the hinge region torsional degrees of freedom. Thus, by partitioning the protocol into "probe sampling" and "backbone sampling" stages, structures significantly closer to the X-ray structure of ligand-bound GlnBP were obtained.

  20. alpha-Linolenic acid- and docosahexaenoic acid-enriched eggs from hens fed flaxseed: influence on blood lipids and platelet phospholipid fatty acids in humans.

    PubMed

    Ferrier, L K; Caston, L J; Leeson, S; Squires, J; Weaver, B J; Holub, B J

    1995-07-01

    This study was undertaken to examine the effects that consumption of eggs from hens fed diets containing flaxseed would have on plasma and platelet lipids of male volunteers. Feeding diets containing 0%, 10%, and 20% ground flaxseed to Leghorn pullets provided a marked progressive increase in n-3 fatty acid content as alpha-linolenic acid (alpha-LNA) (28, 261, and 527 mg/egg) and docosahexaenoic acid (DHA) (51, 81, and 87 mg/egg) but no alteration in the cholesterol concentration of the egg yolk. Twenty-eight male volunteers, divided into three groups, were fed four eggs per day for 2 wk according to a cyclic Latin-square design. No statistically significant changes were observed in total cholesterol, high-density-lipoprotein cholesterol, or plasma triglyceride concentrations. Significant increases in total n-3 fatty acids and in DHA content (which rose from 1.5 to 2.0% by wt or 33% overall), and a significant decrease in ratio of n-6 to n-3 fatty acids were found in platelet phospholipids of subjects consuming eggs from flaxseed-fed hens. Health and Welfare Canada in 1990 set recommended intakes for dietary n-3 fatty acids and for the ratio of n-6 to n-3 fatty acids, which are not being met currently by the overall population. Eggs modified by the inclusion of flaxseed in the laying hens' diet could provide an important nutritional source of n-3 fatty acid. PMID:7598070

  1. Design and investigation of a series of rhodamine-based fluorescent probes for optical measurements of pH.

    PubMed

    Best, Quinn A; Xu, Ruisong; McCarroll, Matthew E; Wang, Lichang; Dyer, Daniel J

    2010-07-16

    A series of structurally similar fluorescent probes (1-4), synthesized from rhodamine B, were designed to optically measure pH. Each probe had a unique "off-on" response as the solution went from basic to acidic. Probes 1-3 exhibited a spirocyclic quenching of the pyronin B fluorophore, whereas probe 4 was quenched by PET from the amine moiety.

  2. Preparation and Characterization of Fluorescence Probe from Assembly Hydroxyapatite Nanocomposite

    PubMed Central

    2010-01-01

    A new nanocomposite fluorescence probe with thioglycolic acid (TA) functional layers embedded inside the hydroxyapatite nanoribbon spherulites has been synthesized. The fluorescence intensity of the novel probe is about 1.5–3.3-fold increase compared with the probe containing no TA. When used to detect cadmium ion, the most of original assembly nanoribbon spherulites structure in the novel probe is found to have been damaged to new flake structures. The mechanism of determining cadmium ion in alcohol solution has been studied. The present systematic study provides significant information on the effect of assembly nanostructure on the metal-enhanced fluorescence phenomenon. PMID:20672031

  3. Directly labeled fluorescent DNA probes for chromosome mapping

    SciTech Connect

    Marrone, B.L.; Deaven, L.L.; Chen, D.J.; Park, Min S.; MacInnes, M.A.; Salzman, G.C.; Yoshida, T.M.

    1995-12-31

    A new strategy is briefly described for employing nucleic acid probes that are directly labeled with fluorochromes in fluorescence in situ hybridization techniques. These probes will permit the detection, quantitation, and high-precision spatial analysis of multiple DNA sequences along a single chromosome using video-enhanced fluorescence microscopy and digital image processing and analysis. Potential advantages of direct labeled DNA probes for fluorescence in situ hybridization far surpass currently available, indirect DNA probe labeling techniques in ease of use, versatility, and increased signal- to-noise ratio.

  4. Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics.

    PubMed

    Kor, Kamalodin; Turner, Anthony P F; Zarei, Kobra; Atabati, Morteza; Beni, Valerio; Mak, Wing Cheung

    2016-02-01

    A single-probe strip test for the rapid and sensitive detection of miRNA-21 mimics is reported herein. Highly specific structurally responsive bi-functional, thiol and biotin, DNA/LNA oligonucleotide probes (molecular beacons-MB) were designed and conjugated with gold nanoparticles (AuNPs) (i.e. biotin-MB-AuNPs). The proposed design had the ability to modulate the accessibility of the biotin group as a function of the presence of a miRNA target allowing the interaction of the boilable with the streptavidin test zone only in the presence of the miRNA-21 mimics. For quantitative evaluation, images of the strip tests were recorded using a flatbed scanner (Epson Perfection V370 Photo). The colour intensities of the test zones of the strip tests were analysed with the ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity. The response of the strip test was linear over the range 0.5 to 20 nM miRNA-21 (limit of detection of 115 pM) and showed good reproducibility (intra and inter CVs below 8%); furthermore, the assay was shown to be highly selective, discriminating other interference miRNAs mimics (e.g. miRNA-221 and miRNA-205). Finally, the proposed strip test was used for detection of miRNA-21 mimics in spiked serum samples, demonstrating its potential for point-of-care clinical applications. Main advantages of the single-probe strip test design are its versatility, simplicity and robustness, which can be easily extended to other miRNA targets by tuning the sequence of the single probe. Furthermore, the use of the structurally responsive single probe is particularly relevant in the case of short-length targets, such as miRNA, whereas a conventional sandwich approach might require a careful control of assay conditions such as hybridization temperature and salt concentration.

  5. Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus).

    PubMed

    Xu, Houguo; Dong, Xiaojing; Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

  6. Regulation of Tissue LC-PUFA Contents, Δ6 Fatty Acyl Desaturase (FADS2) Gene Expression and the Methylation of the Putative FADS2 Gene Promoter by Different Dietary Fatty Acid Profiles in Japanese Seabass (Lateolabrax japonicus)

    PubMed Central

    Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53±0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter. PMID:24498178

  7. Arrays of probes for positional sequencing by hybridization

    DOEpatents

    Cantor, Charles R.; Prezetakiewiczr, Marek; Smith, Cassandra L.; Sano, Takeshi

    2008-01-15

    This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

  8. ALEX neutral beam probe

    SciTech Connect

    Pourrezaei, K.

    1982-01-01

    A neutral beam probe capable of measuring plasma space potential in a fully 3-dimensional magnetic field geometry has been developed. This neutral beam was successfully used to measure an arc target plasma contained within the ALEX baseball magnetic coil. A computer simulation of the experiment was performed to refine the experimental design and to develop a numerical model for scaling the ALEX neutral beam probe to other cases of fully 3-dimensional magnetic field. Based on this scaling a 30 to 50 keV neutral cesium beam probe capable of measuring space potential in the thermal barrier region of TMX Upgrade was designed.

  9. BEAM CONTROL PROBE

    DOEpatents

    Chesterman, A.W.

    1959-03-17

    A probe is described for intercepting a desired portion of a beam of charged particles and for indicating the spatial disposition of the beam. The disclosed probe assembly includes a pair of pivotally mounted vanes moveable into a single plane with adjacent edges joining and a calibrated mechanical arrangement for pivoting the vancs apart. When the probe is disposed in the path of a charged particle beam, the vanes may be adjusted according to the beam current received in each vane to ascertain the dimension of the beam.

  10. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  11. Foldable polymers as probes

    DOEpatents

    Li, Alexander D. Q.; Wang, Wei

    2007-07-03

    Disclosed herein are novel probes, which can be used to detect and identify target molecules of interest in a sample. The disclosed probes can be used to monitor conformational changes induced by molecular recognition events in addition to providing signaling the presence and/or identity of a target molecule. Methods, including solid phase synthesis techniques, for making probe molecules that exhibit changes in their optical properties upon target molecule binding are described in the disclosure. Also disclosed herein are novel chromophore moieties, which have tailored fluorescent emission spectra.

  12. Foldable polymers as probes

    DOEpatents

    Li, Alexander D. Q.; Wang, Wei

    2009-07-07

    Disclosed herein are novel probes, which can be used to detect and identify target molecules of interest in a sample. The disclosed probes can be used to monitor conformational changes induced by molecular recognition events in addition to providing signaling the presence and/or identity of a target molecule. Methods, including solid phase synthesis techniques, for making probe molecules that exhibit changes in their optical properties upon target molecule binding are described in the disclosure. Also disclosed herein are novel chromophore moieties, which have tailored fluorescent emission spectra.

  13. Transient enthalpy probe development

    NASA Astrophysics Data System (ADS)

    Bennett, Brian K.

    A reliable diagnostic probe has been developed to measure the local enthalpy in high-pressure, arc heated test streams that simulate atmospheric reentry conditions. The probe employs the double sonic-throat technique and is designed for the sweep (transient) mode to survive the severe heating environment. Tests in the high-pressure arc heater facilities show that, under certain conditions, the enthalpy probe measurements are in good agreement with enthalpy profiles inferred from heat flux measurements using the theory of Fay and Riddell (1958).

  14. Chemical sensing flow probe

    DOEpatents

    Laguna, George R.; Peter, Frank J.; Butler, Michael A.

    1999-01-01

    A new chemical probe determines the properties of an analyte using the light absorption of the products of a reagent/analyte reaction. The probe places a small reaction volume in contact with a large analyte volume. Analyte diffuses into the reaction volume. Reagent is selectively supplied to the reaction volume. The light absorption of the reaction in the reaction volume indicates properties of the original analyte. The probe is suitable for repeated use in remote or hostile environments. It does not require physical sampling of the analyte or result in significant regent contamination of the analyte reservoir.

  15. Chemical sensing flow probe

    DOEpatents

    Laguna, G.R.; Peter, F.J.; Butler, M.A.

    1999-02-16

    A new chemical probe determines the properties of an analyte using the light absorption of the products of a reagent/analyte reaction. The probe places a small reaction volume in contact with a large analyte volume. Analyte diffuses into the reaction volume. Reagent is selectively supplied to the reaction volume. The light absorption of the reaction in the reaction volume indicates properties of the original analyte. The probe is suitable for repeated use in remote or hostile environments. It does not require physical sampling of the analyte or result in significant regent contamination of the analyte reservoir. 7 figs.

  16. Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen.

    PubMed

    Maia, Margarida R G; Chaudhary, Lal C; Figueres, Lauren; Wallace, R John

    2007-05-01

    Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12-18:2) substantially. The most common product was vaccenic acid (trans-11-18:1), produced by species related to Butyrivibrio fibrisolvens. alpha-Linolenic acid (LNA; cis-9,cis-12,cis-15-18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n - 3)) and docosahexaenoic acid (DHA; 22:6(n - 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 microg ml(-1), nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11-18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.

  17. Use of a fiber optic probe for organic species determination

    DOEpatents

    Ekechukwu, A.A.

    1996-12-10

    A fiber optic probe is described for remotely detecting the presence and concentration organic species in aqueous solutions. The probe includes a cylindrical housing with an organic species indicator, preferably diaminonaphthyl sulfonic acid adsorbed in a silica gel (DANS-modified gel), contained in the probe`s distal end. The probe admits aqueous solutions to the probe interior for mixing within the DANS-modified gel. An optical fiber transmits light through the DANS-modified gel while the indicator reacts with organic species present in the solution, thereby shifting the location of the fluorescent peak. The altered light is reflected to a receiving fiber that carries the light to a spectrophotometer or other analysis device. 5 figs.

  18. Technology for Entry Probes

    NASA Technical Reports Server (NTRS)

    Cutts, James A.; Arnold, James; Venkatapathy, Ethiraj; Kolawa, Elizabeth; Munk, Michelle; Wercinski, Paul; Laub, Bernard

    2005-01-01

    A viewgraph describing technologies for entry probes is presented. The topics include: 1) Entry Phase; 2) Descent Phase; 3) Long duration atmospheric observations; 4) Survivability at high temperatures; and 5) Summary.

  19. An Ultrasonographic Periodontal Probe

    NASA Astrophysics Data System (ADS)

    Bertoncini, C. A.; Hinders, M. K.

    2010-02-01

    Periodontal disease, commonly known as gum disease, affects millions of people. The current method of detecting periodontal pocket depth is painful, invasive, and inaccurate. As an alternative to manual probing, an ultrasonographic periodontal probe is being developed to use ultrasound echo waveforms to measure periodontal pocket depth, which is the main measure of periodontal disease. Wavelet transforms and pattern classification techniques are implemented in artificial intelligence routines that can automatically detect pocket depth. The main pattern classification technique used here, called a binary classification algorithm, compares test objects with only two possible pocket depth measurements at a time and relies on dimensionality reduction for the final determination. This method correctly identifies up to 90% of the ultrasonographic probe measurements within the manual probe's tolerance.

  20. Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

    PubMed Central

    Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

    2014-01-01

    Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p < 0.05) the α-linolenic acid (C18:3n-3) concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05) decreased the arachidonic acid (C20:4n-6) and conjugated linolenic acid (CLA) c-9 t-11 content in the ST muscle. There was a significant (p < 0.05) upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD) gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression. PMID:25255382

  1. Production of Conjugated Linoleic and Conjugated α-Linolenic Acid in a Reconstituted Skim Milk-Based Medium by Bifidobacterial Strains Isolated from Human Breast Milk

    PubMed Central

    Villar-Tajadura, María Antonia; Rodríguez-Alcalá, Luis Miguel; Martín, Virginia; Gómez de Segura, Aránzazu; Rodríguez, Juan Miguel; Fontecha, Javier

    2014-01-01

    Eight bifidobacterial strains isolated from human breast milk have been tested for their abilities to convert linoleic acid (LA) and α-linolenic acid (LNA) to conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), respectively. These bioactive lipids display important properties that may contribute to the maintenance and improvement human health. Three selected Bifidobacterium breve strains produced CLA from LA and CLNA from LNA in MRS (160–170 and 210–230 μg mL−1, resp.) and, also, in reconstituted skim milk (75–95 and 210–244 μg mL−1, resp.). These bifidobacterial strains were also able to simultaneously produce both CLA (90–105 μg mL−1) and CLNA (290–320 μg mL−1) in reconstituted skim milk. Globally, our findings suggest that these bifidobacterial strains are potential candidates for the design of new fermented dairy products naturally containing very high concentrations of these bioactive lipids. To our knowledge, this is the first study describing CLNA production and coproduction of CLA and CLNA by Bifidobacterium breve strains isolated from human milk in reconstituted skim milk. PMID:25110689

  2. Production of conjugated linoleic and conjugated α-linolenic acid in a reconstituted skim milk-based medium by bifidobacterial strains isolated from human breast milk.

    PubMed

    Villar-Tajadura, María Antonia; Rodríguez-Alcalá, Luis Miguel; Martín, Virginia; Gómez de Segura, Aránzazu; Rodríguez, Juan Miguel; Requena, Teresa; Fontecha, Javier

    2014-01-01

    Eight bifidobacterial strains isolated from human breast milk have been tested for their abilities to convert linoleic acid (LA) and α-linolenic acid (LNA) to conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), respectively. These bioactive lipids display important properties that may contribute to the maintenance and improvement human health. Three selected Bifidobacterium breve strains produced CLA from LA and CLNA from LNA in MRS (160-170 and 210-230 μg mL(-1), resp.) and, also, in reconstituted skim milk (75-95 and 210-244 μg mL(-1), resp.). These bifidobacterial strains were also able to simultaneously produce both CLA (90-105 μg mL(-1)) and CLNA (290-320 μg mL(-1)) in reconstituted skim milk. Globally, our findings suggest that these bifidobacterial strains are potential candidates for the design of new fermented dairy products naturally containing very high concentrations of these bioactive lipids. To our knowledge, this is the first study describing CLNA production and coproduction of CLA and CLNA by Bifidobacterium breve strains isolated from human milk in reconstituted skim milk.

  3. Oligodeoxynucleotide Probes for Detecting Intact Cells

    NASA Technical Reports Server (NTRS)

    Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

    2004-01-01

    A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

  4. Molecular beacon-metal nanowire interface: effect of probe sequence and surface coverage on sensor performance.

    PubMed

    Cederquist, Kristin B; Stoermer Golightly, Rebecca; Keating, Christine D

    2008-08-19

    We report the effect of surface coverage and sequence on the performance of 5' thiolated, 3' fluorophore-labeled DNA hairpin probes bound to Au/Ag striped ("barcoded") metal nanowires. Coverage was controlled by varying probe concentration, buffer ionic strength, and by addition of short hydroxy-terminated alkanethiol diluent molecules during probe assembly onto the nanowire surface. Surface dilution of the surface-bound probes with a omega-hydroxyl alkanethiol, a commonly accepted practice in the surface-bound DNA literature, did not appreciably improve sensor performance as compared to similar probe coverages without hydroxyalkanethiol diluents; this finding underscores the differences between the molecular beacon probes used here and more traditional nonfluorescent, random coil probes. We found that intermediate probe coverage of approximately 10 (12) molecules/cm (2) gave the best discrimination between presence and absence of a target sequence. Because we are interested in multiplexed assays, we also compared several beacon probe sequences having different stabilities for secondary structure formation in solution; we found that both probe surface coverage and sensor performance varied for different probe sequences. When five different molecular beacon probes, each bound to barcoded nanowires, were used in a multiplexed, wash-free assay for target oligonucleotides corresponding to viral nucleic acid sequences, these differences in probe performance did not prevent accurate target identification. We anticipate that the findings described here will also be relevant to other applications involving molecular beacons or other structured nucleic acid probes immobilized on metal surfaces.

  5. Model for resonant plasma probe.

    SciTech Connect

    Warne, Larry Kevin; Johnson, William Arthur; Hebner, Gregory Albert; Jorgenson, Roy E.; Coats, Rebecca Sue

    2007-04-01

    This report constructs simple circuit models for a hairpin shaped resonant plasma probe. Effects of the plasma sheath region surrounding the wires making up the probe are determined. Electromagnetic simulations of the probe are compared to the circuit model results. The perturbing effects of the disc cavity in which the probe operates are also found.

  6. Chromosome-specific DNA Repeat Probes

    SciTech Connect

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  7. Surgical force detection probe

    NASA Technical Reports Server (NTRS)

    Tcheng, Ping; Roberts, Paul; Scott, Charles; Prass, Richard

    1991-01-01

    The development progress of a precision electro-mechanical instrument which allows the detection and documentation of the forces and moment applied to human tissue during surgery (under actual operation room conditions), is reported. The pen-shaped prototype probe which measures 1/2 inch in diameter and 7 inches in length was fabricated using an aerodynamic balance. The aerodynamic balance, a standard wind tunnel force and moment sensing transducer, measures the forces and the moments transmitted through the surgeon's hand to the human tissue during surgery. The prototype probe which was fabricated as a development tool was tested successfully. The final version of the surgical force detection probe will be designed based on additional laboratory tests in order to establish the full scale loads. It is expected that the final product will require a simplified aerodynamic balance with two or three force components and one moment component with lighter full scale loads. A signal conditioner was fabricated to process and display the outputs from the prototype probe. This unit will be interfaced with a PC-based data system to provide automatic data acquisition, data processing, and graphics display. The expected overall accuracy of the probe is better than one percent full scale.

  8. Convective heat flow probe

    DOEpatents

    Dunn, James C.; Hardee, Harry C.; Striker, Richard P.

    1985-01-01

    A convective heat flow probe device is provided which measures heat flow and fluid flow magnitude in the formation surrounding a borehole. The probe comprises an elongate housing adapted to be lowered down into the borehole; a plurality of heaters extending along the probe for heating the formation surrounding the borehole; a plurality of temperature sensors arranged around the periphery of the probe for measuring the temperature of the surrounding formation after heating thereof by the heater elements. The temperature sensors and heater elements are mounted in a plurality of separate heater pads which are supported by the housing and which are adapted to be radially expanded into firm engagement with the walls of the borehole. The heat supplied by the heater elements and the temperatures measured by the temperature sensors are monitored and used in providing the desired measurements. The outer peripheral surfaces of the heater pads are configured as segments of a cylinder and form a full cylinder when taken together. A plurality of temperature sensors are located on each pad so as to extend along the length and across the width thereof, with a heating element being located in each pad beneath the temperature sensors. An expansion mechanism driven by a clamping motor provides expansion and retraction of the heater pads and expandable packer-type seals are provided along the probe above and below the heater pads.

  9. Convective heat flow probe

    DOEpatents

    Dunn, J.C.; Hardee, H.C.; Striker, R.P.

    1984-01-09

    A convective heat flow probe device is provided which measures heat flow and fluid flow magnitude in the formation surrounding a borehole. The probe comprises an elongate housing adapted to be lowered down into the borehole; a plurality of heaters extending along the probe for heating the formation surrounding the borehole; a plurality of temperature sensors arranged around the periphery of the probe for measuring the temperature of the surrounding formation after heating thereof by the heater elements. The temperature sensors and heater elements are mounted in a plurality of separate heater pads which are supported by the housing and which are adapted to be radially expanded into firm engagement with the walls of the borehole. The heat supplied by the heater elements and the temperatures measured by the temperature sensors are monitored and used in providing the desired measurements. The outer peripheral surfaces of the heater pads are configured as segments of a cylinder and form a full cylinder when taken together. A plurality of temperature sensors are located on each pad so as to extend along the length and across the width thereof, with a heating element being located in each pad beneath the temperature sensors. An expansion mechanism driven by a clamping motor provides expansion and retraction of the heater pads and expandable packet-type seals are provided along the probe above and below the heater pads.

  10. Pioneer Venus large probe neutral mass spectrometer

    NASA Technical Reports Server (NTRS)

    Hoffman, J.

    1982-01-01

    The deuterium hydrogen abundance ratio in the Venus atmosphere was measured while the inlets to the Pioneer Venus large probe mass spectrometer were coated with sulfuric acid from Venus' clouds. The ratio is (1.6 + or - 0.2) x 10 to the minus two power. It was found that the 100 fold enrichment of deuterium means that Venus outgassed at least 0.3% of a terrestrial ocean and possibly more.

  11. Ice-Borehole Probe

    NASA Technical Reports Server (NTRS)

    Behar, Alberto; Carsey, Frank; Lane, Arthur; Engelhardt, Herman

    2006-01-01

    An instrumentation system has been developed for studying interactions between a glacier or ice sheet and the underlying rock and/or soil. Prior borehole imaging systems have been used in well-drilling and mineral-exploration applications and for studying relatively thin valley glaciers, but have not been used for studying thick ice sheets like those of Antarctica. The system includes a cylindrical imaging probe that is lowered into a hole that has been bored through the ice to the ice/bedrock interface by use of an established hot-water-jet technique. The images acquired by the cameras yield information on the movement of the ice relative to the bedrock and on visible features of the lower structure of the ice sheet, including ice layers formed at different times, bubbles, and mineralogical inclusions. At the time of reporting the information for this article, the system was just deployed in two boreholes on the Amery ice shelf in East Antarctica and after successful 2000 2001 deployments in 4 boreholes at Ice Stream C, West Antarctica, and in 2002 at Black Rapids Glacier, Alaska. The probe is designed to operate at temperatures from 40 to +40 C and to withstand the cold, wet, high-pressure [130-atm (13.20-MPa)] environment at the bottom of a water-filled borehole in ice as deep as 1.6 km. A current version is being outfitted to service 2.4-km-deep boreholes at the Rutford Ice Stream in West Antarctica. The probe (see figure) contains a sidelooking charge-coupled-device (CCD) camera that generates both a real-time analog video signal and a sequence of still-image data, and contains a digital videotape recorder. The probe also contains a downward-looking CCD analog video camera, plus halogen lamps to illuminate the fields of view of both cameras. The analog video outputs of the cameras are converted to optical signals that are transmitted to a surface station via optical fibers in a cable. Electric power is supplied to the probe through wires in the cable at a

  12. Multispectral imaging probe

    DOEpatents

    Sandison, David R.; Platzbecker, Mark R.; Descour, Michael R.; Armour, David L.; Craig, Marcus J.; Richards-Kortum, Rebecca

    1999-01-01

    A multispectral imaging probe delivers a range of wavelengths of excitation light to a target and collects a range of expressed light wavelengths. The multispectral imaging probe is adapted for mobile use and use in confined spaces, and is sealed against the effects of hostile environments. The multispectral imaging probe comprises a housing that defines a sealed volume that is substantially sealed from the surrounding environment. A beam splitting device mounts within the sealed volume. Excitation light is directed to the beam splitting device, which directs the excitation light to a target. Expressed light from the target reaches the beam splitting device along a path coaxial with the path traveled by the excitation light from the beam splitting device to the target. The beam splitting device directs expressed light to a collection subsystem for delivery to a detector.

  13. Multispectral imaging probe

    DOEpatents

    Sandison, D.R.; Platzbecker, M.R.; Descour, M.R.; Armour, D.L.; Craig, M.J.; Richards-Kortum, R.

    1999-07-27

    A multispectral imaging probe delivers a range of wavelengths of excitation light to a target and collects a range of expressed light wavelengths. The multispectral imaging probe is adapted for mobile use and use in confined spaces, and is sealed against the effects of hostile environments. The multispectral imaging probe comprises a housing that defines a sealed volume that is substantially sealed from the surrounding environment. A beam splitting device mounts within the sealed volume. Excitation light is directed to the beam splitting device, which directs the excitation light to a target. Expressed light from the target reaches the beam splitting device along a path coaxial with the path traveled by the excitation light from the beam splitting device to the target. The beam splitting device directs expressed light to a collection subsystem for delivery to a detector. 8 figs.

  14. A curved vitrectomy probe.

    PubMed

    Chalam, K V; Shah, Vinay A; Tripathi, Ramesh C

    2004-01-01

    A curved vitrectomy probe for better accessibility of the peripheral retina in phakic eyes is described. The specially designed curved vitrectomy probe has a 20-gauge pneumatic cutter. The radius of curvature at the shaft is 19.4 mm and it is 25 mm long. The ora serrata is accessed through a 3.0- or 4.0-mm sclerotomy in phakic eyes without touching the crystalline lens. Use of this instrument avoids inadvertent trauma to the clear lens in phakic eyes requiring vitreous base excision. This curved vitrectomy instrument complements wide-angle viewing systems and endoscopes for safe surgical treatment of peripheral retinal pathology in phakic eyes. PMID:15185799

  15. Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

    PubMed

    Kiewnick, Sebastian; Frey, Jürg E; Braun-Kiewnick, Andrea

    2015-09-01

    Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

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

    PubMed

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

    2009-12-01

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

  17. Lactam nonanic acid, a new substance from Cleome viscosa with allelopathic and antimicrobial properties.

    PubMed

    Jana, Anirban; Biswas, Suparna Mandal

    2011-03-01

    Cleome viscosa L. (Capparidaceae) is well known for its medicinal properties. Lactam nonanoic acid (LNA) [2-amino-9-(4-oxoazetidin-2-yl)-nonanoic acid; C12H22N2O3, mol. wt. 242] has been isolated and purified from the root exudates of Cleome viscosa. The aqueous solution of this pure compound has been tested on bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and fungi (Aspergillus fumigatus, A. niger and A. tamarii). At a dosage of 500 ppm and above, P. aeruginosa and S. aureus were totally inhibited while E. coli remained unaffected. On the other hand, growth of A. niger and A. tamarii was stimulated while there was no effect on A. fumigatus. This pure compound showed concentration-dependent inhibitory activity on rice, gram and mustard seeds. PMID:21451245

  18. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    PubMed Central

    Samuelsen, Simone V.; Solov’yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

    2016-01-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies. PMID:27775006

  19. Neurochemistry and electroanalytical probes.

    PubMed

    Troyer, Kevin P; Heien, Michael L A V; Venton, B Jill; Wightman, R Mark

    2002-10-01

    Electroanalytical techniques have been applied to monitoring chemical events including neurotransmitter release during rodent behaviour and the release of zeptomoles of molecules from single cells. Transgenic mice models have been developed and studied to identify specific cell types in vitro. Characterization and surface modification of electroanalytical probes has enhanced the selectivity and sensitivity of measurements.

  20. Cervical Neoplasia Probe Control

    1997-01-24

    This software, which consists of a main executive and several subroutines, performs control of the optics, image acquisition, and Digital Signal Processing (DSP) of this image, of an optical based medical instrument that performs fluoresence detection of precancerous lesions (neoplasia) of the human cervix. The hardware portion of this medical instrument is known by the same name Cervical Neoplasia Probe (CNP)

  1. The Phoenix Pluto Probe

    NASA Technical Reports Server (NTRS)

    Gunning, George R.; Spapperi, Jeff; Wilkinson, Jeffrey P.; Eldred, Jim; Labij, Dennis; Strinni, Meredith

    1990-01-01

    A design proposal for an unmanned probe to Pluto is presented. The topics covered include: (1) scientific instrumentation; (2) mission management, planning, and costing; (3) power and propulsion system; (4) structural subsystem; (5) command, control, and communication; and (6) attitude and articulation control.

  2. Ultrasonic search wheel probe

    DOEpatents

    Mikesell, Charles R.

    1978-01-01

    A device is provided for reducing internal reflections from the tire of an ultrasonic search wheel probe or from within the material being examined. The device includes a liner with an anechoic chamber within which is an ultrasonic transducer. The liner is positioned within the wheel and includes an aperture through which the ultrasonic sound from the transducer is directed.

  3. Endocavity Ultrasound Probe Manipulators

    PubMed Central

    Stoianovici, Dan; Kim, Chunwoo; Schäfer, Felix; Huang, Chien-Ming; Zuo, Yihe; Petrisor, Doru; Han, Misop

    2014-01-01

    We developed two similar structure manipulators for medical endocavity ultrasound probes with 3 and 4 degrees of freedom (DoF). These robots allow scanning with ultrasound for 3-D imaging and enable robot-assisted image-guided procedures. Both robots use remote center of motion kinematics, characteristic of medical robots. The 4-DoF robot provides unrestricted manipulation of the endocavity probe. With the 3-DoF robot the insertion motion of the probe must be adjusted manually, but the device is simpler and may also be used to manipulate external-body probes. The robots enabled a novel surgical approach of using intraoperative image-based navigation during robot-assisted laparoscopic prostatectomy (RALP), performed with concurrent use of two robotic systems (Tandem, T-RALP). Thus far, a clinical trial for evaluation of safety and feasibility has been performed successfully on 46 patients. This paper describes the architecture and design of the robots, the two prototypes, control features related to safety, preclinical experiments, and the T-RALP procedure. PMID:24795525

  4. Experimenting with Temperature Probes.

    ERIC Educational Resources Information Center

    Roth, Wolff-Michael

    1989-01-01

    Presented are four activities which are designed to familiarize children with the multiple uses of computers and help them learn about heat and temperature using temperature probes. Included are the tempering effect of water, heat capacity, caloric content of foods, and weather. Hardware and software are discussed. (CW)

  5. Probing the Solar System

    ERIC Educational Resources Information Center

    Wilkinson, John

    2013-01-01

    Humans have always had the vision to one day live on other planets. This vision existed even before the first person was put into orbit. Since the early space missions of putting humans into orbit around Earth, many advances have been made in space technology. We have now sent many space probes deep into the Solar system to explore the planets and…

  6. Laboratory plasma probe studies

    NASA Technical Reports Server (NTRS)

    Heikkila, W. J.

    1975-01-01

    Diagnostic experiments performed in a collisionless plasma using CO2 as the working gas are described. In particular, simultaneous measurements that have been performed by means of Langmuir- and RF-probes are presented. A resonance occurring above the parallel resonance in the frequency characteristic of a two electrode system is interpreted as being due to the resonant excitation of electroacoustic waves.

  7. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities

    PubMed Central

    Huete-Stauffer, Tamara M.; Arandia-Gorostidi, Nestor; Alonso-Sáez, Laura; Morán, Xosé Anxelu G.

    2016-01-01

    Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures. PMID:27242747

  8. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities.

    PubMed

    Huete-Stauffer, Tamara M; Arandia-Gorostidi, Nestor; Alonso-Sáez, Laura; Morán, Xosé Anxelu G

    2016-01-01

    Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures. PMID:27242747

  9. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities.

    PubMed

    Huete-Stauffer, Tamara M; Arandia-Gorostidi, Nestor; Alonso-Sáez, Laura; Morán, Xosé Anxelu G

    2016-01-01

    Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.

  10. Calibration Fixture For Anemometer Probes

    NASA Technical Reports Server (NTRS)

    Lewis, Charles R.; Nagel, Robert T.

    1993-01-01

    Fixture facilitates calibration of three-dimensional sideflow thermal anemometer probes. With fixture, probe oriented at number of angles throughout its design range. Readings calibrated as function of orientation in airflow. Calibration repeatable and verifiable.

  11. Modular Rake of Pitot Probes

    NASA Technical Reports Server (NTRS)

    Dunlap, Timothy A.; Henry, Michael W.; Homyk, Raymond P.

    2004-01-01

    The figure presents selected views of a modular rake of 17 pitot probes for measuring both transient and steady-state pressures in a supersonic wind tunnel. In addition to pitot tubes visible in the figure, the probe modules contain (1) high-frequency dynamic-pressure transducers connected through wires to remote monitoring circuitry and (2) flow passages that lead to tubes that, in turn, lead to remote steady-state pressure transducers. Prior pitot-probe rakes were fabricated as unitary structures, into which the individual pitot probes were brazed. Repair or replacement of individual probes was difficult, costly, and time-consuming because (1) it was necessary to remove entire rakes in order to unbraze individual malfunctioning probes and (2) the heat of unbrazing a failed probe and of brazing a new probe in place could damage adjacent probes. In contrast, the modules in the present probe are designed to be relatively quickly and easily replaceable with no heating and, in many cases, without need for removal of the entire rake from the wind tunnel. To remove a malfunctioning probe, one first removes a screw-mounted V-cross-section cover that holds the probe and adjacent probes in place. Then one removes a screw-mounted cover plate to gain access to the steady-state pressure tubes and dynamicpressure wires. Next, one disconnects the tube and wires of the affected probe. Finally, one installs a new probe in the reverse of the aforementioned sequence. The wire connections can be made by soldering, but to facilitate removal and installation, they can be made via miniature plugs and sockets. The connections between the probe flow passages and the tubes leading to the remote pressure sensors can be made by use of any of a variety of readily available flexible tubes that can be easily pulled off and slid back on for removal and installation, respectively.

  12. Hybridization-based biosensor containing hairpin probes and use thereof

    DOEpatents

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2010-10-12

    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  13. Heavy ion beam probing

    SciTech Connect

    Hickok, R L

    1980-07-01

    This report consists of the notes distributed to the participants at the IEEE Mini-Course on Modern Plasma Diagnostics that was held in Madison, Wisconsin in May 1980. It presents an overview of Heavy Ion Beam Probing that briefly describes the principles and discuss the types of measurements that can be made. The problems associated with implementing beam probes are noted, possible variations are described, estimated costs of present day systems, and the scaling requirements for large plasma devices are presented. The final chapter illustrates typical results that have been obtained on a variety of plasma devices. No detailed calculations are included in the report, but a list of references that will provide more detailed information is included.

  14. Probing properties of cold radiofrequency plasma with polymer probe

    NASA Astrophysics Data System (ADS)

    Bormashenko, E.; Chaniel, G.; Multanen, V.

    2015-01-01

    The probe intended for the characterization of cold plasma is introduced. The probe allows the estimation of Debye length of cold plasma. The probe is based on the pronounced modification of surface properties (wettability) of polymer films by cold plasmas. The probe was tested with the cold radiofrequency inductive air plasma discharge. The Debye length and the concentration of charge carriers were estimated for various gas pressures. The reported results coincide reasonably with the corresponding values established by other methods. The probe makes possible measurement of characteristics of cold plasmas in closed chambers.

  15. Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids.

    PubMed

    Asari, Masaru; Okuda, Katsuhiro; Yajima, Daisuke; Maseda, Chikatoshi; Hoshina, Chisato; Omura, Tomohiro; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

    2015-04-01

    Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics.

  16. Gravity Probe B Inspection

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The space vehicle Gravity Probe B (GP-B) is the relativity experiment developed at Stanford University to test two extraordinary predictions of Albert Einstein's general theory of relativity. The experiment will measure, very precisely, the expected tiny changes in the direction of the spin axes of four gyroscopes contained in an Earth-orbiting satellite at a 400-mile altitude. So free are the gyroscopes from disturbance that they will provide an almost perfect space-time reference system. They will measure how space and time are very slightly warped by the presence of the Earth, and, more profoundly, how the Earth's rotation very slightly drags space-time around with it. These effects, though small for the Earth, have far-reaching implications for the nature of matter and the structure of the Universe. GP-B is among the most thoroughly researched programs ever undertaken by NASA. This is the story of a scientific quest in which physicists and engineers have collaborated closely over many years. Inspired by their quest, they have invented a whole range of technologies that are already enlivening other branches of science and engineering. In this photograph, engineer Gary Reynolds is inspecting the inside of the probe neck during probe thermal repairs. GP-B is scheduled for launch in April 2004 and managed for NASA by the Marshall Space Flight Center. Development of the GP-B is the responsibility of Stanford University along with major subcontractor Lockheed Martin Corporation. (Image credit to Russ Leese, Gravity Probe B, Stanford University)

  17. Space Probe Launch

    NASA Technical Reports Server (NTRS)

    1970-01-01

    Managed by Marshall Space Flight Center, the Space Tug was a reusable multipurpose space vehicle designed to transport payloads to different orbital inclinations. Utilizing mission-specific combinations of its three primary modules (crew, propulsion, and cargo) and a variety of supplementary kits, the Space Tug was capable of numerous space applications. This 1970 artist's concept depicts the Tug's propulsion module launching a space probe into lunar orbit.

  18. Icing Sensor Probe

    NASA Technical Reports Server (NTRS)

    Emery, Edward; Kok, Gregory L.

    2002-01-01

    Aircraft icing is a serious safety problem for the general aviation and some commuter transport airplanes. There has been tremendous growth in the commuter aviation industry in the last few years, Since these type of aircraft generally operate at lower altitudes they consequently spend a far greater proportion of their time operating in icing conditions. For the past thirty years airborne and ground based facilities have relied primarily on two types of cloud physics instrumentation to measure the characteristics of icing clouds: hot wire liquid water content probes and laser based particle sizing probes for the measurement of water droplet size. The instrumentation is severely limited by the technology that was developed during the 1970's and is quite large in size. The goal of this research is to develop one instrument with a wide bandwidth, better response time, higher resolution, user selectability, and small and lightweight. NASA Glenn Research Center, Droplet Measurement Technology, and Meteorology Society of Canada have developed a collaborative effort to develop such an instrument. This paper describes the development and test results of the prototype Icing Sensor Probe.

  19. Einstein Inflationary Probe (EIP)

    NASA Technical Reports Server (NTRS)

    Hinshaw, Gary

    2004-01-01

    I will discuss plans to develop a concept for the Einstein Inflation Probe: a mission to detect gravity waves from inflation via the unique signature they impart to the cosmic microwave background (CMB) polarization. A sensitive CMB polarization satellite may be the only way to probe physics at the grand-unified theory (GUT) scale, exceeding by 12 orders of magnitude the energies studied at the Large Hadron Collider. A detection of gravity waves would represent a remarkable confirmation of the inflationary paradigm and set the energy scale at which inflation occurred when the universe was a fraction of a second old. Even a strong upper limit to the gravity wave amplitude would be significant, ruling out many common models of inflation, and pointing to inflation occurring at much lower energy, if at all. Measuring gravity waves via the CMB polarization will be challenging. We will undertake a comprehensive study to identify the critical scientific requirements for the mission and their derived instrumental performance requirements. At the core of the study will be an assessment of what is scientifically and experimentally optimal within the scope and purpose of the Einstein Inflation Probe.

  20. Nanoscale thermal probing

    PubMed Central

    Yue, Yanan; Wang, Xinwei

    2012-01-01

    Nanoscale novel devices have raised the demand for nanoscale thermal characterization that is critical for evaluating the device performance and durability. Achieving nanoscale spatial resolution and high accuracy in temperature measurement is very challenging due to the limitation of measurement pathways. In this review, we discuss four methodologies currently developed in nanoscale surface imaging and temperature measurement. To overcome the restriction of the conventional methods, the scanning thermal microscopy technique is widely used. From the perspective of measuring target, the optical feature size method can be applied by using either Raman or fluorescence thermometry. The near-field optical method that measures nanoscale temperature by focusing the optical field to a nano-sized region provides a non-contact and non-destructive way for nanoscale thermal probing. Although the resistance thermometry based on nano-sized thermal sensors is possible for nanoscale thermal probing, significant effort is still needed to reduce the size of the current sensors by using advanced fabrication techniques. At the same time, the development of nanoscale imaging techniques, such as fluorescence imaging, provides a great potential solution to resolve the nanoscale thermal probing problem. PMID:22419968

  1. Comparative evaluation of probing depth and clinical attachment level using a manual probe and Florida probe

    PubMed Central

    Kour, Amandeep; Kumar, Ashish; Puri, Komal; Khatri, Manish; Bansal, Mansi; Gupta, Geeti

    2016-01-01

    Background: To compare and evaluate the intra- and inter-examiner efficacy and reproducibility of the first-generation manual (Williams) probe and the third-generation Florida probe in terms of measuring pocket probing depth (PD) and clinical attachment level (CAL). Materials and Methods: Forty subjects/4000 sites were included in this comparative, cross-sectional study. Group- and site-wise categorizations were done. Based on gingival index, PD, and CAL, patients were divided into four groups, i.e., periodontally healthy, gingivitis, mild to moderate periodontitis, and severe periodontitis. Further, based on these parameters, a total of 4000 sites, with 1000 sites in each category randomly selected from these 40 patients, were taken. Full mouth PD and CAL measurements were recorded with two probes, by Examiner 1 and on Ramfjord teeth by Examiner 2. Results: Full mouth and Ramfjord teeth group- and site-wise PD obtained with the manual probe by both the examiners were statistically significantly deeper than that obtained with the Florida probe. The full mouth and Ramfjord teeth mean CAL measurement by Florida probe was higher as compared to manual probe in mild to moderate periodontitis group and sites, whereas in severe periodontitis group and sites, manual probe recorded higher CAL as compared to Florida probe. Conclusion: Mean PD and CAL measurements were deeper with the manual probe as compared to the Florida probe in all the groups and sites, except for the mild-moderate periodontitis group and sites where the CAL measurements with the manual probe were less than the Florida probe. Manual probe was more reproducible and showed less interexaminer variability as compared to the Florida probe. PMID:27563204

  2. Nanostar probes for tip-enhanced spectroscopy

    NASA Astrophysics Data System (ADS)

    Kim, Woong; Kim, Nara; Park, Joon Won; Kim, Zee Hwan

    2015-12-01

    To overcome the current limit of tip-enhanced spectroscopy that is based on metallic nano-probes, we developed a new scanning probe with a metallic nanostar, a nanoparticle with sharp spikes. A Au nanoparticle of 5 nm was first attached to the end of a tip through DNA-DNA hybridization and mechanical pick-up. The nanoparticle was converted to a nanostar with a core diameter of ~70 nm and spike lengths between 50 nm and 80 nm through the reduction of Au3+ with ascorbic acid in the presence of Ag+. Fabrication yields of such tips exceeded 60%, and more than 80% of such tips showed a mechanical durability sufficient for use in scanning microscopy. Effectiveness of the new probes for tip-enhanced Raman scattering (TERS) and tip-enhanced fluorescence (TEF) was confirmed. The probes exhibited the necessary enhancement for TEF, and the tip-on and tip-off ratios varied between 5 and 100. This large tip-to-tip variability may arise from the uncontrolled orientation of the apexes of the spike with respect to the sample surface, which calls for further fabrication improvement. The result overall supports a new fabrication approach for the probe that is effective for tip-enhanced spectroscopy.To overcome the current limit of tip-enhanced spectroscopy that is based on metallic nano-probes, we developed a new scanning probe with a metallic nanostar, a nanoparticle with sharp spikes. A Au nanoparticle of 5 nm was first attached to the end of a tip through DNA-DNA hybridization and mechanical pick-up. The nanoparticle was converted to a nanostar with a core diameter of ~70 nm and spike lengths between 50 nm and 80 nm through the reduction of Au3+ with ascorbic acid in the presence of Ag+. Fabrication yields of such tips exceeded 60%, and more than 80% of such tips showed a mechanical durability sufficient for use in scanning microscopy. Effectiveness of the new probes for tip-enhanced Raman scattering (TERS) and tip-enhanced fluorescence (TEF) was confirmed. The probes exhibited

  3. The Effectiveness of Predict-Observe-Explain Technique in Probing Students' Understanding about Acid-Base Chemistry: A Case for the Concepts of pH, pOH, and Strength

    ERIC Educational Resources Information Center

    Kala, Nesli; Yaman, Fatma; Ayas, Alipasa

    2013-01-01

    The present study describes high school students' conceptions about acids and bases in terms of pH, pOH, microscopic level, strength, and concentration. A total of 27 high school students participated in the study. The data was collected using 3 POE tasks and a semi-structured interview. The data analysis demonstrated that most of the students had…

  4. Insect E-probe Diagnostic Nucleic acid Analysis (EDNA): the application of a novel bioinformatic tool to detection of vectors and pathogens in individual insect and simulated insect trap metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant pathogen detection takes many forms. In simple cases, researchers are attempting to detect a known pathogen from a known host utilizing targeted nucleic acid or antigenic assays. However, in more complex scenarios researchers may not know the identity of a pathogen, or they may need to screen ...

  5. Metrological scanning probe microscope

    NASA Astrophysics Data System (ADS)

    Dorozhovets, N.; Hausotte, T.; Manske, E.; Jäger, G.; Hofmann, N.

    2006-04-01

    Today's technological progress calls for metrologically accurate object measurement, positioning and scanning with nanometre precision and over large measuring ranges. In order to meet that requirement a nanopositioning and nanomeasuring machine (NPM machine) was developed at the Institute of Process Measurement and Sensor Technology of the Technische Universitaet Ilmenau. This device is capable of highly exact long-range positioning and measurement of objects with a resolution of less than 0.1 nm. Due to the structure of the machine many different probe systems can be installed, including scanning probe microscopes (SPMs). A few SPMs have outstanding metrological characteristics and many commercial microscopes only perform as image acquisition tools. Commercial SPMs use piezoelectric actuators in order to move either the sample or the probe. The position measurement sometimes results from the applied voltage to the piezoelectric actuators or from the strain gauge or capacitive displacement sensor data. This means that they suffer from hysteresis, creep, nonlinear characteristics and Abbe offsets. For an accurate measurement the position of the cantilever must be measured in addition to the torsion and bending. The best solution is a combined detection system with a single laser beam. This system has been realized with a special interferometer system, in which the measuring beam is focused on the cantilever backside using a lens. The reflected beam is split with a part being detected by a quadrant photo-diode and the other part being fed back into the interferometer for position measurement. The quadrant photo-diode is used to detect the cantilever torsion and bending.

  6. Crystallization and X-ray diffraction analysis of an ‘all-locked’ nucleic acid duplex derived from a tRNASer microhelix

    PubMed Central

    Behling, Katja; Eichert, André; Fürste, Jens P.; Betzel, Christian; Erdmann, Volker A.; Förster, Charlotte

    2009-01-01

    Modified nucleic acids are of great interest with respect to their nuclease resistance and enhanced thermostability. In therapeutical and diagnostic applications, such molecules can substitute for labile natural nucleic acids that are targeted against particular diseases or applied in gene therapy. The so-called ‘locked nucleic acids’ contain modified sugar moieties such as 2′-­O,4′-­C-methylene-bridged β-d-ribofuranose and are known to be very stable nucleic acid derivatives. The structure of locked nucleic acids in single or multiple LNA-substituted natural nucleic acids and in LNA–DNA or LNA–RNA heteroduplexes has been well investigated, but the X-ray structure of an ‘all-locked’ nucleic acid double helix has not been described to date. Here, the crystallization and X-ray diffraction data analysis of an ‘all-locked’ nucleic acid helix, which was designed as an LNA originating from a tRNASer microhelix RNA structure, is presented. The crystals belonged to space group C2, with unit-cell parameters a = 77.91, b = 40.74, c = 30.06 Å, β = 91.02°. A high-resolution and a low-resolution data set were recorded, with the high-resolution data showing diffraction to 1.9 Å resolution. The crystals contained two double helices per asymmetric unit, with a Matthews coefficient of 2.48 Å3 Da−1 and a solvent content of 66.49% for the merged data. PMID:19652346

  7. Vacuum probe surface sampler

    NASA Technical Reports Server (NTRS)

    Zahlava, B. A. (Inventor)

    1973-01-01

    A vacuum probe surface sampler is described for rapidly sampling relatively large surface areas which possess relatively light loading densities of micro-organism, drug particles or the like. A vacuum head with a hollow handle connected to a suitable vacuum source is frictionally attached to a cone assembly terminating in a flared tip adapted to be passed over the surface to be sampled. A fine mesh screen carried by the vacuum head provides support for a membrane filter which collects the microorganisms or other particles. The head assembly is easily removed from the cone assembly without contacting the cone assembly with human hands.

  8. Experimental probes of axions

    SciTech Connect

    Chou, Aaron S.; /Fermilab

    2009-10-01

    Experimental searches for axions or axion-like particles rely on semiclassical phenomena resulting from the postulated coupling of the axion to two photons. Sensitive probes of the extremely small coupling constant can be made by exploiting familiar, coherent electromagnetic laboratory techniques, including resonant enhancement of transitions using microwave and optical cavities, Bragg scattering, and coherent photon-axion oscillations. The axion beam may either be astrophysical in origin as in the case of dark matter axion searches and solar axion searches, or created in the laboratory from laser interactions with magnetic fields. This note is meant to be a sampling of recent experimental results.

  9. Atom Probe Tomography 2012

    NASA Astrophysics Data System (ADS)

    Kelly, Thomas F.; Larson, David J.

    2012-08-01

    In the world of tomographic imaging, atom probe tomography (APT) occupies the high-spatial-resolution end of the spectrum. It is highly complementary to electron tomography and is applicable to a wide range of materials. The current state of APT is reviewed. Emphasis is placed on applications and data analysis as they apply to many fields of research and development including metals, semiconductors, ceramics, and organic materials. We also provide a brief review of the history and the instrumentation associated with APT and an assessment of the existing challenges in the field.

  10. Sequence-specific, self-reporting hairpin inversion probes.

    PubMed

    Browne, Kenneth A

    2005-02-16

    Sequence-specific probes for detecting target nucleic acids are the cornerstone of the genomics revolution (e.g., microarrays) and of molecular diagnostics. Molecular beacons are self-reporting, nucleic acid probes whose structure includes complementary terminal arm sequences and a loop that is complementary to a target sequence; fluorescence detection is by changes in proximity of fluorophore and quencher pairs attached on opposite arms. However, molecular beacon design is not as simple as attaching arbitrary arm sequences onto previously designed linear probes. The stem arms can also interact with flanking target sequences, changing the hybridization specificity; constantly adapting the arms to avoid such interactions, if not desired, increases design complexity. Herein, I report the use of inversion linkages in probe backbones leading to stem arms of sequence polarity opposite to that of the target-binding region, thereby eliminating potential hybridization of the arms with the target. Using two microbial sequence categories, thermal denaturation and target titration analyses demonstrate that these new hairpin inversion probes retain closed-state stability comparable to that of molecular beacons, contain easily designed arm sequences that do not interact with targets, and, therefore, can be used universally with optimized linear probe sequences.

  11. Egg yolk as a source of long-chain polyunsaturated fatty acids in infant feeding.

    PubMed

    Simopoulos, A P; Salem, N

    1992-02-01

    In this paper we compare the fatty acid content of egg yolks from hens fed four different feeds as a source of docosahexaenoic acid to supplement infant formula. Greek eggs contain more docosahexaenoic acid (DHA, 22:6 omega 3) and less linoleic acid (LA, 18:2 omega 6) and alpha-linolenic acid (LNA, 18:3 omega 3) than do fish-meal or flax eggs. Two to three grams of Greek egg yolk may provide an adequate amount of DHA and arachidonic acid for a preterm neonate. Mean intake of breast milk at age 1 mo provides 250 mg long-chain omega 3 fatty acids. This amount can be obtained from less than 1 yolk of a Greek egg (0.94), greater than 1 yolk of flax eggs (1.6) and fish-meal eggs (1.4), or 8.3 yolks of supermarket eggs. With proper manipulation of the hens' diets, eggs could be produced with fatty acid composition similar to that of Greek eggs.

  12. Fiber optic current probe

    NASA Astrophysics Data System (ADS)

    Wyntjes, G.; Fox, R.

    1984-02-01

    This report documents the results of Phase 1 research into a new type of Fiber Optic Current probe, suitable for high voltage, high current applications. The probe uses a stabilized two frequency HeNe laser to read the magnitude and sign of magnetic field induced circular birefringence in an optical fiber wound around a conductor. Measurements of both alternating and direct currents were demonstrated with a breadboard system. The system was tested at low voltages with currents of up to 4500 amperes peak and with up to 28 turns of optical fiber around the conductor. The response was found to increase linearly with the number of fiber turns. Experimental determinations of the system's frequency response and dynamic range were not possible due to our inability to generate large, fast current transients. The predicted frequency response is 100 kHz with an ability to read transient amplitudes of 300 times the nominal line current. Several single-mode fibers were used to form transducers, and the optimum fiber for further development was identified. The 2-frequency interrogation technique described worked entirely as predicted, and should be applicable to magnetic field measurements in general (i.e., charged particle beams, Tokamaks, antenna patterns, EMP testing, etc.).

  13. Novel live imaging techniques of cellular functions and in vivo tumors based on precise design of small molecule-based 'activatable' fluorescence probes.

    PubMed

    Urano, Yasuteru

    2012-12-01

    Recently established rational design strategies for novel fluorescence probes, especially those based on photoinduced electron transfer and spirocyclization were reviewed. Based on these design strategies, various novel fluorescence probes were successfully developed including those for reactive oxygen species, reporter enzymes. Furthermore, in vivo cancer imaging techniques based on rationally designed activatable probes such as cancer-specific antibodies tagged with acidic-pH activatable fluorescence probes and peptidase activatable fluorescence probes were also discussed.

  14. Molecular beacons: an optimal multifunctional biological probe.

    PubMed

    Li, Yongsheng; Zhou, Xiaoyan; Ye, Duyun

    2008-09-01

    Molecular beacon technology is set up based on fluorescence resonance energy transfer (FRET) and the complementary pairing principles. These fluorescent molecular probes, which are very highly specific and sensitive, have now become one important tool in medical and biological researches. This review introduces the molecular beacons structure, principle, the main impact factors, the labeling of the molecular beacons, and research progress on molecular beacons fluorescent-label in the polymerase chain reaction (PCR), DNA sequence analysis, gene dynamic detection in living cells, protein (enzyme)-nucleic acid interactions and applications in clinical medicine.

  15. Rapid antigen testing for group A Streptococcus by DNA probe.

    PubMed

    Heelan, J S; Wilbur, S; Depetris, G; Letourneau, C

    1996-02-01

    The Gen-probe group A Streptococcus direct test (GASD), a nucleic acid probe assay for detecting GAS from throat swabs, has recently been developed. The test uses an acridium ester-labeled DNA probe which is complementary to the rRNA of Streptococcus pyogenes. In this study, 318 single culturette throat swabs were tested by this method using culture as a "gold standard." After plating onto trypticase soy agar plates with 5% sheep blood, swabs were stored at 4 degrees C for no more than 72 h before the probe assay was performed. Our patient population consisted of symptomatic outpatients seen in the Memorial Hospital Emergency Department and in the Family Care Center. After discrepancy testing, sensitivity, specificity, and positive and negative predictive values were 91.4%, 97%, 91.4%, and 97%. The GASD is a rapid, easy-to-perform method for batch screening for streptococcal pharyngitis.

  16. DNA/DNA in situ hybridization with enzyme linked probes

    SciTech Connect

    Grillo, S.; Mosher, M.; Charles, P.; Henry, S.; Taub, F.

    1987-05-01

    A non-radioactive in situ nucleic acid hybridization method which requires no antibodies, haptens, avidin or biotin intermediateries is presented. Horseradish peroxidase (HRP) labeled nucleic acid probes are hybridized in situ for 2 hours or less, followed by brief washing of hybridized cells and the direct detection of in situ hybrids with diaminobenzidine (DAB). Application of this method to the detection of Human Papilloma Virus (HPV) in human cells is shown.

  17. Lipid metabolic dose response to dietary alpha-linolenic acid in monk parrot (Myiopsitta monachus).

    PubMed

    Petzinger, Christina; Heatley, J J; Bailey, Christopher A; Bauer, John E

    2014-03-01

    Monk parrots (Myiopsitta monachus) are susceptible to atherosclerosis, a progressive disease characterized by the formation of plaques in the arteries accompanied by underlying chronic inflammation. The family of n-3 fatty acids, especially eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), have consistently been shown to reduce atherosclerotic risk factors in humans and other mammals. Some avian species have been observed to convert α-linolenic acid (18:3n-3, ALA) to EPA and DHA (Htin et al. in Arch Geflugelk 71:258-266, 2007; Petzinger et al. in J Anim Physiol Anim Nutr, 2013). Therefore, the metabolic effects of including flaxseed oil, as a source of ALA, in the diet at three different levels (low, medium, and high) on the lipid metabolism of Monk parrots was evaluated through measuring plasma total cholesterol (TC), free cholesterol (FC), triacylglycerols (TAG), and phospholipid fatty acids. Feed intake, body weight, and body condition score were also assessed. Thus the dose and possible saturation response of increasing dietary ALA at constant linoleic acid (18:2n-6, LNA) concentration on lipid metabolism in Monk parrots (M. monachus) was evaluated. Calculated esterified cholesterol in addition to plasma TC, FC, and TAG were unaltered by increasing dietary ALA. The high ALA group had elevated levels of plasma phospholipid ALA, EPA, and docosapentaenoic acid (DPAn-3, 22:5n-3). The medium and high ALA groups had suppressed plasma phospholipid 20:2n-6 and adrenic acid (22:4n-6, ADA) compared to the low ALA group. When the present data were combined with data from a previous study (Petzinger et al. in J Anim Physiol Anim Nutr, 2013) a dose response to dietary ALA was observed when LNA was constant. Plasma phospholipid ALA, EPA, DPAn-3, DHA, and total n-3 were positively correlated while 20:2n-6, di-homo-gamma-linoleic acid (20:3n-6Δ7), arachidonic acid (20:4n-6), ADA, and total n-6 were inversely correlated with dietary en% ALA.

  18. Fixture For Calibrating Pressure Probe

    NASA Technical Reports Server (NTRS)

    Ashby, George C., Jr.; Vasquez, Peter; Horsley, Lewis A.; Bowman, John T.; Zumbrun, Henry N.; Eves, John W.

    1994-01-01

    Fixture in form of specially designed clamshell housing enables in situ calibration of pressure transducer mounted in body of pressure probe in wind tunnel. Includes two metal half shells machined with necks and matching cavities, when put together, define larger neck and cavity accommodating probe. Probe secured to bottom half shell by use of clamp before installing top half shell: necessary to follow sequence to protect probe during assembly. Clamshell calibration fixture attached to pressure probe in few minutes, making it possible to calibrate pressure transducer at convenient times. Calibrations performed before and after wind-tunnel runs each day, between runs in event of delays or suspected malfunctions, and essentially any other time, without having to remove probe from wind tunnel.

  19. Development and application of multiple-probe scanning probe microscopes.

    PubMed

    Nakayama, Tomonobu; Kubo, Osamu; Shingaya, Yoshitaka; Higuchi, Seiji; Hasegawa, Tsuyoshi; Jiang, Chun-Sheng; Okuda, Taichi; Kuwahara, Yuji; Takami, Kazuhiro; Aono, Masakazu

    2012-04-01

    In the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequently modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.

  20. Development and Application of Multiple-Probe Scanning Probe Microscopes

    SciTech Connect

    Nakayama, T.; Kubo, O.; Shingaya, Y.; Higuchi, S.; Hasegawa, T.; Jiang, C. S.; Okuda, T.; Kuwahara, Y.; Takami, K.; Aono, M.

    2012-04-03

    the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequently modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.