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Sample records for acid hybridization probes

  1. 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

  2. Chemically modified nucleic acids as immunodetectable probes in hybridization experiments.

    PubMed Central

    Tchen, P; Fuchs, R P; Sage, E; Leng, M

    1984-01-01

    Guanine residues in nucleic acids can be modified by treatment with N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative in an in vitro nonenzymatic reaction. The modified nucleic acids (ribo or deoxyribo, single or double stranded) are recognized by specific antibodies. They can be immunoprecipitated or used as probes in hybridization experiments and detected by immunochemical techniques. Images PMID:6374657

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Studies towards the development of chemically synthesized non-radioactive biotinylated nucleic acid hybridization probes.

    PubMed Central

    Al-Hakim, A H; Hull, R

    1986-01-01

    Non-radioactive nucleic acid hybridization probes have been constructed in which the reporter group is long chain biotin chemically linked to a basic macromolecule (histone H1, cytochrome C or polyethyleneimine). The modified basic macromolecule which carries many biotin residues can, in turn, be covalently linked to nucleic acids (DNA) via the bifunctional cross-linking reagents, glutaraldehyde, 1,2,7,8-diepoxyoctane, bis (succinimidyl) suberate or bis (sulfonosuccinimidyl) suberate. This provides a very sensitive probe by which as little as between 10-50fg of target DNA can be visualized using dot-blot hybridization procedures in conjunction with avidin or streptavidin enzyme conjugates. PMID:3027670

  8. Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

    PubMed

    Fontenete, Sílvia; Carvalho, Daniel; Guimarães, Nuno; Madureira, Pedro; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-07-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms. PMID:26969040

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    PubMed

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH. PMID:23391931

  17. 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.

  18. Quantification of Syntrophic Fatty Acid-β-Oxidizing Bacteria in a Mesophilic Biogas Reactor by Oligonucleotide Probe Hybridization

    PubMed Central

    Hansen, Kaare H.; Ahring, Birgitte K.; Raskin, Lutgarde

    1999-01-01

    Small-subunit rRNA sequences were obtained for two saturated fatty acid-β-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. wolfei, 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-β-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-β-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, of which the majority was accounted for by the genus Syntrophomonas. PMID:10543784

  19. Molecular beacons: Probes that fluoresce upon hybridization

    SciTech Connect

    Tyagi, S.; Kramer, F.R.

    1996-03-01

    We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced. 23 refs., 6 figs.

  20. 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

  1. 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.

  2. Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

    PubMed Central

    Lehtola, Markku J.; Torvinen, Eila; Miettinen, Ilkka T.; Keevil, C. William

    2006-01-01

    Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply. PMID:16391126

  3. Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

    PubMed Central

    Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

    2000-01-01

    A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

  4. 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. PMID:23762388

  5. Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: The effect of pH, dextran sulfate and probe concentration.

    PubMed

    Rocha, Rui; Santos, Rita S; Madureira, Pedro; Almeida, Carina; Azevedo, Nuno F

    2016-05-20

    Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria. PMID:27021959

  6. 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.

  7. Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples ▿

    PubMed Central

    Almeida, C.; Azevedo, N. F.; Fernandes, R. M.; Keevil, C. W.; Vieira, M. J.

    2010-01-01

    A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 109 ± 5 × 108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 107 ± 5 × 106 CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity. PMID:20453122

  8. 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

  9. DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes.

    PubMed

    Hövelmann, Felix; Seitz, Oliver

    2016-04-19

    The increasing importance assigned to RNA dynamics in cells and tissues calls for probe molecules that enable fluorescence microscopy imaging in live cells. To achieve this goal, fluorescence dyes are conjugated with oligonucleotides so as to provide strong emission upon hybridization with the target molecule. The impressive 10(3)-fold fluorescence intensification observed when DNA stains such as thiazole orange (TO) interact with double-stranded DNA is intriguing and prompted the exploration of oligonucleotide conjugates. However, nonspecific interactions of DNA stains with polynucleotides tend to increase background, which would affect the contrast achievable in live-cell imaging. This Account describes the development of DNA-stain-labeled hybridization probes that provide high signal-to-background. We focus on our contributions in context with related advances from other laboratories. The emphasis will be on the requirements of RNA imaging in live cells. To reduce background, intercalator dyes such as TO were appended to peptide nucleic acid (PNA), which is less avidly recognized by DNA stains than DNA/RNA. Constraining the TO dye as a nucleobase surrogate in "forced intercalation (FIT) probes" improved the target specificity, presumably by helping to prevent unspecific interactions. The enforcement of TO intercalation between predetermined base pairs upon formation of the probe-target duplex provided for high brightness and enabled match/mismatch selectivity beyond stringency of hybridization. We show examples that highlight the use of PNA FIT probes in the imaging of mRNA, miRNA, and lncRNA in living cells. The "FIT approach" was recently extended to DNA probes. Signal brightness can become limiting when low-abundance targets ought to be visualized over cellular autofluorescence. We discuss strategies that further the brightness of signaling by FIT probes. Multilabeling with identical dyes does not solve the brightness issue. To avoid self-quenching, we

  10. Simulation-Guided DNA Probe Design for Consistently Ultraspecific Hybridization

    PubMed Central

    Wang, J. Sherry; Zhang, David Yu

    2015-01-01

    Hybridization of complementary sequences is one of the central tenets of nucleic acid chemistry; however, the unintended binding of closely related sequences limits the accuracy of hybridization-based approaches for analyzing nucleic acids. Thermodynamics-guided probe design and empirical optimization of reaction conditions have been used to enable discrimination of single nucleotide variants, but typically these approaches provide only an approximate 25-fold difference in binding affinity. Here we show that simulations of the binding kinetics are both necessary and sufficient to design nucleic acid probe systems with consistently high specificity as they enable the discovery of an optimal combination of thermodynamic parameters. Simulation-guided probe systems designed against 44 different target single nucleotide variants sequences showed between 200- and 3000-fold (median 890) higher binding affinity than their corresponding wildtype sequences. As a demonstration of the usefulness of this simulation-guided design approach we developed probes which, in combination with PCR amplification, we use to detect low concentrations of variant alleles (1%) in human genomic DNA. PMID:26100802

  11. 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.

  12. 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

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

    PubMed

    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

  14. Probing Compositional Variation within Hybrid Nanostructures

    SciTech Connect

    Yuhas, Benjamin D.; Habas, Susan E.; Fakra, Sirine C.; Mokari, Taleb

    2010-06-22

    We present a detailed analysis of the structural and magnetic properties of solution-grown PtCo-CdS hybrid structures in comparison to similar free-standing PtCo alloy nanoparticles. X-ray absorption spectroscopy is utilized as a sensitive probe for identifying subtle differences in the structure of the hybrid materials. We found that the growth of bimetallic tips on a CdS nanorod substrate leads to a more complex nanoparticle structure composed of a PtCo alloy core and thin CoO shell. The core-shell architecture is an unexpected consequence of the different nanoparticle growth mechanism on the nanorod tip, as compared to free growth in solution. Magnetic measurements indicate that the PtCo-CdS hybrid structures are superparamagnetic despite the presence of a CoO shell. The use of X-ray spectroscopic techniques to detect minute differences in atomic structure and bonding in complex nanosystems makes it possible to better understand and predict catalytic or magnetic properties for nanoscale bimetallic hybrid materials.

  15. Controlling microarray DNA hybridization efficiency by probe-surface distance and external surface electrostatics

    NASA Astrophysics Data System (ADS)

    Qamhieh, K.; Pettitt, B. Montgomery

    2015-03-01

    DNA microarrays are analytical devices designed to determine the composition of multicomponent solutions of nucleic acids, DNA or RNA. These devices are promising technology for diverse applications, including sensing, diagnostics, and drug/gene delivery. Here, we modify a hybridization adsorption isotherm to study the effects of probe-surface distance and the external electrostatic fields, on the oligonucleotide hybridization in microarray and how these effects are varies depending on surface probe density and target concentration. This study helps in our understanding on-surface hybridization mechanisms, and from it we can observe a significant effect of the probe-surface distance, and the external electrostatic fields, on the hybridization yield. In addition we present a simple new criteria to control the oligonucleotide hybridization efficiency by providing a chart illustrating the effects of all factors on the DNA-hybridization efficiency.

  16. 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

  17. 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.

  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. 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.

  20. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

    Fan, Wenhong (Inventor); Cassell, Alan M. (Inventor); Han, Jie (Inventor)

    2006-01-01

    Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

  1. Probing of Strain Mediated Hybrid Multiferroic Devices

    NASA Astrophysics Data System (ADS)

    Fohtung, Edwin; Kim, J.; Marsh, M.; Lei, Na; Chen, S.; Sinha, S.; Ravelosona, D.; Fullerton, Eric; Shpyrko, Oleg

    2012-02-01

    Smart materials for sensor technology, (non) volatile device memories for information technology, and ultrasound generators in medical imaging have one thing in common, their active elements consist of ferroelectrics (FE) driven by voltages or ferromagnetics (FM) driven by magnetization. In the quest to design high functionality devices to meet today's consumer technological demands, high focus has been given to multiferroic [1]. However, the coexistence of magnetic order and ferroelectric polarization combined in a single-phase material has proven to be rear as most of these materials tend to have low magnetic ordering temperatures and are often antiferromagnets, in which the magnetoelectric (ME) coupling effect is intrinsically small. We utilize an alternative approach to design multiferroic-hybrid devices based on FE-FM composites where the ME coupling emerges from strain-mediated interaction between individual phases [2]. We develop a nonlinear thermodynamic theory for strain-mediated direct ME effect and Bragg Ptychographic Coherent Diffraction Imaging (BCDI) serves as the unique tool of choice for sub-nanometer resolution nondestructive probing of the order parameters in the devices [1] N. Spaldin and M. Fiebig, Science 309, 391 (2005). [2] E. Fohtung et al., submitted (2012)

  2. Nucleic acid in-situ hybridization detection of infectious agents

    NASA Astrophysics Data System (ADS)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  3. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  4. RAPID IDENTIFICATION OF CANDIDA ALBICANS DIRECTLY FROM YEAST POSITIVE BLOOD CULTURE BOTTLES BY FLUORESCENCE IN SITU HYBRIDIZATION USING PNA PROBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for identification of Candida albicans directly from yeast-positive blood culture bottles is described. The test (C. albicans PNA FISH) is based on a fluorescein-labeled PNA probe targeting C. albicans 26...

  5. Selective Nucleic Acid Capture with Shielded Covalent Probes

    PubMed Central

    2013-01-01

    Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo). PMID:23745667

  6. Linear RNA amplification for the production of microarray hybridization probes.

    PubMed

    Klebes, Ansgar; Kornberg, Thomas B

    2008-01-01

    To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity. PMID:18641956

  7. Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.

    PubMed

    Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J; Parker, Heather; Winterbourn, Christine C; Salvesen, Guy S; Drag, Marcin

    2014-02-18

    The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1-S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

  8. Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

    PubMed Central

    Yilmaz, L. Safak; Loy, Alexander; Wright, Erik S.; Wagner, Michael; Noguera, Daniel R.

    2012-01-01

    Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu. PMID:22952791

  9. 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.

  10. Investigation of the hybrid molecular probe for intracellular studies

    PubMed Central

    Martinez, Karen; Medley, Colin D.; Yang, Chaoyong James; Tan, Weihong

    2009-01-01

    Monitoring gene expression in vivo is essential to the advancement of biological studies, medical diagnostics, and drug discovery. Adding to major efforts in developing molecular probes for mRNA monitoring, we have recently developed an alternative tool, the hybrid molecular probe (HMP). To optimize the probe, a series of experiments were performed to study the properties of HMP hybridization kinetics and stability. The results demonstrated the potential of the HMP as a prospective tool for use in both hybridization studies and in vitro and in vivo analyses. The HMP has shown no tendency to produce false positive signals, which is a major concern for living cell studies. Moreover, HMP has shown the ability to detect the mRNA expression of different genes inside single cells from both basal and stimulated genes. As an effective alternative to conventional molecular probes, the proven sensitivity, simplicity, and stability of HMPs show promise for their use in monitoring mRNA expression in living cells. PMID:18421445

  11. Base-acid hybrid water electrolysis.

    PubMed

    Chen, Long; Dong, Xiaoli; Wang, Fei; Wang, Yonggang; Xia, Yongyao

    2016-02-21

    A base-acid hybrid electrolytic system with a low onset voltage of 0.78 V for water electrolysis was developed by using a ceramic Li-ion exchange membrane to separate the oxygen-evolving reaction (OER) in a basic electrolyte solution containing the Li-ion and hydrogen-evolving reaction (HER) in an acidic electrolyte solution. PMID:26804323

  12. 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.

  13. 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.

  14. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

    SciTech Connect

    Jiang, X.; Estes, M.K.; Metcalf, T.G.; Melnick, J.L

    1986-10-01

    The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10/sup 4/ physical particlels of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hydbridization stringency, /sup 32/P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.

  15. Selection of oligonucleotide probes and experimental conditions for multiplex hybridization experiments.

    PubMed

    Bains, W

    1994-01-01

    Different DNA probes hybridize under different conditions. I examine the constraints of the design of oligonucleotide probes that are meant to hybridize to different unique sites in human genomic DNA under a single set of hybridization conditions as a parallel array. In 522 kb of human genomic DNA, 75% of 12-base and 89% of 22-base are unique, as opposed to 90% and 100% as expected of unstructured DNA, and this is not due solely to repetitive elements in the DNA. Hybridization in TMAC to reduce A+T content effects on melting temperature allows only 90% of unique targets to be hybridized under one set of conditions if a 2 degrees C difference between matched and mismatched sequences is required. Standard hybridization conditions allow no more than 60% of unique probes to be used together. This suggests that probe, hybridization conditions, and instrument design for multiple-probe hybridization applications will be harder than previously suggested. PMID:7803130

  16. 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.

  17. 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.

  18. Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria

    SciTech Connect

    Devereux, R.; Kane, M.D.; Winfrey, J.; Stahl, D.A.

    1992-01-01

    A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. Four probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus multivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by hybridizations against closely related nontargeted species. In addition, each probe was screened by using a 'phylogrid' membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The value of these probes to studies in environmental microbiology was evaluated by hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.

  19. Comparative examination of probe labeling methods for microarray hybridization

    NASA Astrophysics Data System (ADS)

    Burke, David I.; Woodward, Karen; Setterquist, Robert A.; Kawasaki, Ernest S.

    2001-06-01

    For detection of differential gene expression, confocal laser based scanners are now capable of analyzing microarrays using one to five wavelengths. This allows investigators to choose among several labeling methods. Here we compare direct incorporation and indirect methods (amino-allyl and dendrimers) for labeling cDNA probes. We assessed reproducible sensitivity of each probe preparation method in two ways. First, by comparing hybridization intensities for limit of signal detection and second by measuring the lowest detectable concentration of a known ratio of mixed DNA (spikes). Limit of detection assay was done using arrays of mixed targets consisting of a serially diluted human specific gene fragment (HU1) and an undiluted DNA of chloramphenicol acetyl tranferase (CAT) gene. Then, individual single target arrays of CAT and HU1 DNA were used to determine the lowest detectable spike ratio of each labeling method. The results of this study will be presented and their significance for the analysis of microarrays will be discussed.

  20. An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

    PubMed

    Chen, Sherry Xi; Seelig, Georg

    2016-04-20

    Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology. PMID:27010123

  1. 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

  2. Fast ion measurement using a hybrid directional probe in the large helical device

    SciTech Connect

    Nagaoka, Kenichi; Watanabe, Kiyomasa Y.; Osakabe, Masaki; Takeiri, Yasuhiko; Minami, Takashi; Toi, Kazuo; Isobe, Mitsutaka; Nishiura, Masaki; Ito, Takafumi; Ogawa, Kunihiro

    2008-10-15

    A hybrid directional probe was newly installed in the large helical device for fast ion measurement. The collector of the probe mounts a thermocouple to estimate local power flux and can be also utilized as a collector of a conventional Langmuir probe; therefore, the hybrid directional probe can simultaneously measure both local power density flux and current flux at the same collector surface. The concept and design of the hybrid directional probe, the calibration of the power density measurement, and preliminary result of the fast ion measurement are presented.

  3. Probing DNA hybridization efficiency and single base mismatch by X-ray photoelectron spectroscopy.

    PubMed

    Liu, Zheng-Chun; Zhang, Xin; He, Nong-Yue; Lu, Zu-Hong; Chen, Zhen-Cheng

    2009-07-01

    We demonstrated the use of X-ray photoelectron spectroscopy (XPS) to study DNA hybridization. Target DNA labeled with hexachloro-fluorescein (HEX) was hybridized to DNA arrays with four different probes. Each probe dot of the hybridized arrays was detected with XPS. The XPS Cl2p peak areas were found to decrease with an increase in mismatched bases in DNA probes. The Cl2p core-level peak area ratio of a probe perfectly matched to one, two and three base-mismatched probes accorded well with the results of conventional fluorescent imaging, which shows that XPS is a potential tool for analyzing DNA arrays. The DNA arrays' hybridization efficiency was assessed by the molar ratio of chlorine to phosphorus in a DNA strand, which was determined from the relevant XPS Cl2p and P2p core-level peak areas after hybridization. This could provide a new method to detect DNA hybridization efficiency. PMID:19282155

  4. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    PubMed

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-01-01

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results. PMID:26345863

  5. Design and evaluation of peptide nucleic acid probes for specific identification of Candida albicans.

    PubMed

    Kim, Hyun-Joong; Brehm-Stecher, Byron F

    2015-02-01

    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

  6. 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

  7. Nucleic acid probes as a diagnostic method for tick-borne hemoparasites of veterinary importance.

    PubMed

    Figueroa, J V; Buening, G M

    1995-03-01

    An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures. PMID:7597795

  8. Quantification of Gordona amarae Strains in Foaming Activated Sludge and Anaerobic Digester Systems with Oligonucleotide Hybridization Probes

    PubMed Central

    de los Reyes, M. Fiorella; de los Reyes, Francis L.; Hernandez, Mark; Raskin, Lutgarde

    1998-01-01

    Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems. Gordona (formerly Nocardia) amarae often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four G. amarae strains were sequenced, and the resulting sequences were compared to the sequence of G. amarae type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for G. amarae, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional G. amarae isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, group 1, group 2, bacterial, mycolata, and Gordona rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain G. amarae strains may be regional or treatment plant specific and that previously uncharacterized G. amarae strains may be present in some systems. PMID:9647822

  9. Quantification of Gordona amarae strains in foaming activated sludge and anaerobic digester systems with oligonucleotide hybridization probes.

    PubMed

    de los Reyes, M F; de los Reyes, F L; Hernandez, M; Raskin, L

    1998-07-01

    Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems. Gordona (formerly Nocardia) amarae often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four G. amarae strains were sequenced, and the resulting sequences were compared to the sequence of G. amarae type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for G. amarae, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional G. amarae isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, group 1, group 2, bacterial, mycolata, and Gordona rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain G. amarae strains may be regional or treatment plant specific and that previously uncharacterized G. amarae strains may be present in some systems. PMID:9647822

  10. [Use of photo-anchoring of DNA probes for fluorescent in situ hybridization].

    PubMed

    Nasedkina, T V; Mal'kov, R B; Fedorova, L I; Godovikova, T S; Kolpashchikov, D M; Poletaev, A I

    1998-01-01

    A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization. PMID:9821246

  11. Hybridization and sequencing of nucleic acids using base pair mismatches

    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.

  12. The effects of multiple probes on the hybridization of target DNA on surfaces

    NASA Astrophysics Data System (ADS)

    Welling, Ryan C.; Knotts, Thomas A.

    2015-01-01

    DNA microarrays have disruptive potential in many fields including genetics and medicine, but the technology has yet to find widespread clinical use due to poor reliability. Microarrays work on the principle of hybridization and can only be as dependable as this process is reliable. As such, a significant amount of theoretical research has been done to understand hybridization on surfaces on the molecular level. Previous simulations of a target strand with a single, surface-tethered probe molecule have yielded valuable insights, but such is an ideal system and little is known about the effects of multiple probes—a situation that more closely approximates the real system. This work uses molecular simulation to determine the specific differences in duplex stability between one, three, six, and nine tethered probes on a surface. The results show that it is more difficult for a single target to hybridize to a probe as the number of probes on the surface increases due to crowding effects; however, once hybridized, the duplex is more stable than when fewer probes are present. The data also indicate that hybridization of a target to a probe on the face of a group of probes is more stable than hybridization to probes at the edge or center locations. Taken as a whole, the results offer new insights into the cause of the poor reproducibility exhibited by microarrays.

  13. Mismatch discrimination of lipidated DNA and LNA-probes (LiNAs) in hybridization-controlled liposome assembly.

    PubMed

    Jakobsen, Ulla; Vogel, Stefan

    2016-08-01

    Assays for mismatch discrimination and detection of single nucleotide variations by hybridization-controlled assembly of liposomes, which do not require tedious surface chemistry, are versatile for both DNA and RNA targets. We report herein a comprehensive study on different DNA and LNA (locked nucleic acids) probe designs, including membrane-anchoring requirements, studies on different probes and target lengths (including overhangs), DNA and RNA targets (including sequences associated with pathogens) for lipidated nucleic acids (LiNAs). Advantages and limitations of the liposome assembly based assay in the context of mismatch discrimination and SNP detection are presented. The advantages of membrane-anchored LiNA-probes compared to chemically attached probes on solid nanoparticles (e.g. gold nanoparticles) are described. Key functionalities such as non-covalent attachment of LiNA probes without the need for long spacers and the inherent mobility of membrane-anchored probes in lipid-bilayer membranes will be described for several different probe designs. PMID:27356098

  14. Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.

    PubMed

    Massey, Melissa; Ancona, Mario G; Medintz, Igor L; Algar, W Russ

    2015-12-01

    Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective

  15. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

    SciTech Connect

    Giraldez, R.A.; Harris, C.

    1994-09-01

    Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2 minutes, and detected with rhodamine/anti-digoxigenin-FITC/avidin for 15 minutes at 37{degrees}C. Slides were soaked 3X in 1XPBD and then counterstained with 15 {mu}L 0.05 {mu}g/mL DAP1/Antifade. 200 nuclei were scored for the presence of one green (X), two green (XX), one green and one red (XY), or a single red (Y) signal, using a fluorescent microscope equipped with a triple band pass filter. Greater than 90% of the hybridized nuclei from each of the 14 cases studied conformed to the sex chromosome pattern. The modal number in 9 cases showed two green signals (XX), and a green and a red signal (XY) in the other 5 cases; this was in complete agreement with the cytogenetic results.

  16. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    PubMed Central

    de los Reyes, F L; Ritter, W; Raskin, L

    1997-01-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems. PMID:9055425

  17. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    PubMed

    de los Reyes, F L; Ritter, W; Raskin, L

    1997-03-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems. PMID:9055425

  18. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    PubMed Central

    2011-01-01

    Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of

  19. HYBRIDIZATION OF DNA PROBES WITH WHOLE-COMMUNITY GENOMES FOR DETECTION OF GENES THAT ENCODE MICROBIAL RESPONSES TO POLLUTANTS: MER GENES AND HG2+ RESISTANCE

    EPA Science Inventory

    Nucleic acids extracted from microbial biomass were hybridized with probes representing four mer operons, to detect genes encoding adaptation to Hg2+. An enrichment in sequences similar to the mer genes of transposon 501 occurred during adaptation in a freshwater community. In an...

  20. Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.

    PubMed

    Orum, H; Nielsen, P E; Jørgensen, M; Larsson, C; Stanley, C; Koch, T

    1995-09-01

    Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of a target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His)6-PNA chimera exhibits strong binding to chelated Ni2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His)6-PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni(2+)-NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touchstones of any nucleic acid purification scheme. We show that the specificity of the (His)6-PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His)6-PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure. PMID:7495562

  1. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  2. DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe.

    PubMed

    Maruyama, Tatsuo; Park, Lian-Chun; Shinohara, Toshimitsu; Goto, Masahiro

    2004-01-01

    We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm. PMID:14715007

  3. Nucleic acid hybridization for detection of cell culture-amplified adenovirus.

    PubMed Central

    Huang, C; Deibel, R

    1988-01-01

    A number of recombinant plasmids containing genomic segments of adenovirus were constructed. Seven cloned probes, as well as total adenovirus type 2 (Ad2) and Ad16 genomic DNA, were tested by a nucleic acid hybridization technique for sensitivity and specificity in detecting adenoviruses in infected cells. Adenovirus DNA was spotted onto a nitrocellulose filter and hybridized with 32P-labeled DNA probes. The probes, total Ad2 genomic DNA, and plasmid pAd2-H (containing the hexon gene from Ad2 DNA) all detected 10 reference serotypes of five genomic subgroups (A through E) with similar sensitivities. However, plasmid pAd2-H required less preparation time than did total Ad2 DNA. Probes pAd2-F (containing the fiber gene from Ad2) and pAd16-BD (containing the BamHI D fragment from Ad16) hybridized only with reference serotypes from the homologous subgroups (C and B, respectively). Of 101 patient isolates amplified in cells, pAd2-H detected 100% of all isolates from both the homologous and the heterologous subgroups. The detection rates for pAd2-F were 100% (subgroup C) and 3.6% (subgroups A, B, and D), and those for pAd16-BD were 100% (subgroup B) and 9.4% (subgroups A, C, and D). A commercial biotinylated product (Pathogene II) was also included in this study for comparison. Images PMID:3230138

  4. Probing hybrid modified gravity by stellar motion around Galactic Center

    NASA Astrophysics Data System (ADS)

    Borka, D.; Capozziello, S.; Jovanović, P.; Borka Jovanović, V.

    2016-06-01

    We consider possible signatures for the so called hybrid gravity within the Galactic Central Parsec. This modified theory of gravity consists of a superposition of the metric Einstein-Hilbert Lagrangian with an f(R) term constructed à la Palatiniand can be easily reduced to an equivalent scalar-tensor theory. Such an approach is introduced in order to cure the shortcomings related to f(R) gravity, in general formulated either in metric or in metric-affine frameworks. Hybrid gravity allows to disentangle the further gravitational degrees of freedom with respect to those of standard General Relativity. The present analysis is based on the S2 star orbital precession around the massive compact dark object at the Galactic Center where the simulated orbits in hybrid modified gravity are compared with astronomical observations. These simulations result with constraints on the range of hybrid gravity interaction parameter ϕ0, showing that in the case of S2 star it is between -0.0009 and -0.0002. At the same time, we are also able to obtain the constraints on the effective mass parameter mϕ, and found that it is between -0.0034 and -0.0025 AU-1 for S2 star. Furthermore, the hybrid gravity potential induces precession of S2 star orbit in the same direction as General Relativity. In previous papers, we considered other types of extended gravities, like metric power law f(R)∝Rn gravity, inducing Yukawa and Sanders-like gravitational potentials, but it seems that hybrid gravity is the best among these models to explain different gravitational phenomena at different astronomical scales.

  5. Fiber-based hybrid probe for non-invasive cerebral monitoring in neonatology

    NASA Astrophysics Data System (ADS)

    Rehberger, Matthias; Giovannella, Martina; Pagliazzi, Marco; Weigel, Udo; Durduran, Turgut; Contini, Davide; Spinelli, Lorenzo; Pifferi, Antonio; Torricelli, Alessandro; Schmitt, Robert

    2015-07-01

    Improved cerebral monitoring systems are needed to prevent preterm infants from long-term cognitive and motor restrictions. Combining advanced near-infrared diffuse spectroscopy measurement technologies, time-resolved spectroscopy (TRS) and diffuse correlation spectroscopy (DCS) will introduce novel indicators of cerebral oxygen metabolism and blood flow for neonatology. For non-invasive sensing a fiber-optical probe is used to send and receive light from the infant head. In this study we introduce a new fiber-based hybrid probe that is designed for volume production. The probe supports TRS and DCS measurements in a cross geometry, thus both technologies gain information on the same region inside the tissue. The probe is highly miniaturized to perform cerebral measurements on heads of extreme preterm infants down to head diameters of 6cm. Considerations concerning probe production focus on a reproducible accuracy in shape and precise optical alignment. In this way deviations in measurement data within a series of probes should be minimized. In addition to that, requirements for clinical use like robustness and hygiene are considered. An additional soft-touching sleeve made of FDA compatible silicone allows for a flexible attachment with respect to the individual anatomy of each patient. We present the technical concept of the hybrid probe and corresponding manufacturing methods. A prototype of the probe is shown and tested on tissue phantoms as well as in vivo to verify its operational reliability.

  6. Hybride magnetic nanostructure based on amino acids functionalized polypyrrole

    SciTech Connect

    Nan, Alexandrina Bunge, Alexander; Turcu, Rodica

    2015-12-23

    Conducting polypyrrole is especially promising for many commercial applications because of its unique optical, electric, thermal and mechanical properties. We report the synthesis and characterization of novel pyrrole functionalized monomers and core-shell hybrid nanostructures, consisting of a conjugated polymer layer (amino acids functionalized pyrrole copolymers) and a magnetic nanoparticle core. For functionalization of the pyrrole monomer we used several amino acids: tryptophan, leucine, phenylalanine, serine and tyrosine. These amino acids were linked via different types of hydrophobic linkers to the nitrogen atom of the pyrrole monomer. The magnetic core-shell hybrid nanostructures are characterized by various methods such as FTIR spectroscopy, transmission electron microscopy (TEM) and magnetic measurements.

  7. Hybride magnetic nanostructure based on amino acids functionalized polypyrrole

    NASA Astrophysics Data System (ADS)

    Nan, Alexandrina; Bunge, Alexander; Turcu, Rodica

    2015-12-01

    Conducting polypyrrole is especially promising for many commercial applications because of its unique optical, electric, thermal and mechanical properties. We report the synthesis and characterization of novel pyrrole functionalized monomers and core-shell hybrid nanostructures, consisting of a conjugated polymer layer (amino acids functionalized pyrrole copolymers) and a magnetic nanoparticle core. For functionalization of the pyrrole monomer we used several amino acids: tryptophan, leucine, phenylalanine, serine and tyrosine. These amino acids were linked via different types of hydrophobic linkers to the nitrogen atom of the pyrrole monomer. The magnetic core-shell hybrid nanostructures are characterized by various methods such as FTIR spectroscopy, transmission electron microscopy (TEM) and magnetic measurements.

  8. Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via Replica Molding for Facile Nucleic Acid Hybridization Assays

    PubMed Central

    Lewis, Christina L.; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-01-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of probe and target DNA, femtomole sensitivity, and sequence specificity. Combined these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  9. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    PubMed

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  10. Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

    PubMed Central

    Poulsen, Lena; Søe, Martin Jensen; Snakenborg, Detlef; Møller, Lisbeth Birk; Dufva, Martin

    2008-01-01

    Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 × 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 × SSC, while probes placed away from the surface required 0.35 × SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis. PMID:18805905

  11. Field-hardened optical waveguide hybrid integrated-circuit multisensor chemical probe and its chemistry

    NASA Astrophysics Data System (ADS)

    Pollina, Richard J.; Himka, Roger L.; Saini, Devinder P.; McGibbon, Alan; Klainer, Stanley M.

    1997-05-01

    A single probe containing three hybrid integrated-circuit, optical waveguide, chemical-biochemical sensors (chip sensors) has been developed. Each chip sensor contains two hybrid waveguides -- one for sensing and one for reference. The sense waveguide is coated with a species-specific or group-specific chemistry or biochemistry. The reference waveguide is coated with a version of the sense chemistry or biochemistry, which is not sensitive to the analyte. The integrated structure is encapsulated and contains a single fixed light source, two detectors (reference and sense), and an optical train. The design is amenable to fluorescence, absorption, and refraction measurements. The three chip sensors are individually mounted in a probe that contains all of the electronics and computing capability necessary to collect and process the output information from each chip sensor. Only the surface of the individual chips are exposed to the target analytes. The probe is rugged, intrinsically safe, and can operate under 75 m (250 ft) of water.

  12. The theory of the deoxyribonucleic acid - ribonucleic acid hybridization reaction.

    PubMed Central

    Thomou, H; Katsanos, N A

    1976-01-01

    A general equation is derived describing data of DNA-RNA hybridization in the presence of a competing self-annealing reaction of RNA. The well known double-reciprocal relation and the Scatchard equation are shown to be limiting cases of this general equation. Some new hybridization data at various temperatures are presented and analysed by using the new equation. The results can only be explained if we assume that the behavior of DNA towards single RNA molecules is the same as that towards the annealed form, (RNA12. The variation of the equilibrium constant of the hybridization reaction with temperature is small, indicating a small heat of reaction. The maximum amount of hybridized RNA at equilibrium appears to be independent of temperature. PMID:1275887

  13. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.

    PubMed

    Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Ökten, Hatice E; Noguera, Daniel R

    2014-08-01

    Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu). PMID:24928876

  14. Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense

    PubMed Central

    Chen, Guofu; Zhang, Chunyu; Zhang, Baoyu; Wang, Guangce; Lu, Douding; Xu, Zhong; Yan, Peishen

    2011-01-01

    Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1–D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future. PMID:22022408

  15. Hybrid semiconducting polymer nanoparticles as polarization-sensitive fluorescent probes

    PubMed Central

    Zeigler, Maxwell B.; Sun, Wei; Rong, Yu; Chiu, Daniel T.

    2013-01-01

    Much work has been done on collapsed chains of conjugated semiconducting polymers and their applications as fluorescent probes or sensors. On surfaces spin-coated with semiconducting polymers, excitation energy transfer along the polymer backbone can be used to quickly and efficiently funnel energy to chromophores with localized energy minima. If each chromophore is immobilized within its matrix, this can result in large fluorescence anisotropy. Through nanoprecipitation of a matrix polymer blended at low mass ratios with short-chain, hydrophobic, fluorescent semiconducting polymers, we take advantage of this large fluorescence anisotropy to make polarization-sensitive nanoparticles. These nanoparticles are small at approximately 7 nm in diameter; exhibit a high quantum yield of 0.75; and are easily functionalized to bind to protein targets. By exciting the nanoparticles with polarized light on a wide-field fluorescence microscope, we are able to monitor not only protein location, but also changes in their orientation. PMID:23895535

  16. Detection and classification of Trypanosoma cruzi by DNA hybridization with nonradioactive probes.

    PubMed

    Solari, A; Venegas, J; Gonzalez, E; Vasquez, C

    1991-01-01

    Total or kinetoplast DNA (kDNA) from 72 isolates and clones of Trypanosoma cruzi as well as from nine related trypanosomatids were analyzed by dot hybridization using nonradioactive kDNA or cloned minicircle fragments as probes. Biotinylated-kDNA probes generated by nick-translation proved reliable for distinguishing Zymodeme 1 and Zymodeme 2bol of T. cruzi parasites. In contrast, digoxigenin-labeled kDNA obtained by random-priming did not distinguish among T. cruzi isolates but did distinguish among New World leishmanias. Cloned minicircle fragments labeled with digoxigenin gave the same results as digoxigenin-labeled kDNA, except for a 10-fold decrease in sensitivity. Digoxigenin-labeled DNA probes proved useful in unambiguously detecting T. cruzi from different geographic regions of America. However, T. rangeli and T. cruzi marinkellei were not distinguished by these probes. PMID:1667933

  17. D-region blunt probe data analysis using hybrid computer techniques

    NASA Technical Reports Server (NTRS)

    Burkhard, W. J.

    1973-01-01

    The feasibility of performing data reduction techniques with a hybrid computer was studied. The data was obtained from the flight of a parachute born probe through the D-region of the ionosphere. A presentation of the theory of blunt probe operation is included with emphasis on the equations necessary to perform the analysis. This is followed by a discussion of computer program development. Included in this discussion is a comparison of computer and hand reduction results for the blunt probe launched on 31 January 1972. The comparison showed that it was both feasible and desirable to use the computer for data reduction. The results of computer data reduction performed on flight data acquired from five blunt probes are also presented.

  18. Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

    PubMed Central

    Wang, Jingjing; Morabito, Kenneth; Tang, Jay X.; Tripathi, Anubhav

    2013-01-01

    The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein. PMID:24404041

  19. Photoinitiator Nucleotide for Quantifying Nucleic Acid Hybridization

    PubMed Central

    Johnson, Leah M.; Hansen, Ryan R.; Urban, Milan; Kuchta, Robert D.; Bowman, Christopher N.

    2010-01-01

    This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′-deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm2. Film heights of 10–20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm2 while below the detection limit of ~10 EITC-nucleotides/μm2, no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges. PMID:20337438

  20. 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...

  1. A highly sensitive aptasensor for OTA detection based on hybridization chain reaction and fluorescent perylene probe.

    PubMed

    Wang, Bin; Wu, Yuanya; Chen, Yanfen; Weng, Bo; Xu, Liqun; Li, Changming

    2016-07-15

    An optical aptasensor was developed for ultrasensitive detection of ochratoxin A (OTA) based on hybridization chain reaction (HCR) amplification strategy and fluorescent perylene probe (PAPDI)/DNA composites. Dendritic DNA concatamers were synthesized by HCR strategy and modified on magnetic nanoparticles through aptamer as medium. A large amount of PAPDI probe aggregated under the induction of DNA concatamers and caused fluorescence quenching. In the presence of OTA, the PAPDI/DNA composites were released from magnetic nanoparticles due to the strong affinity between aptamer and OTA. In ethanol, PAPDI monomers disaggregated and produced strong fluorescence. The present method displays excellent sensitivity and selectivity towards OTA. PMID:26938491

  2. Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridization.

    PubMed Central

    Raskin, L; Poulsen, L K; Noguera, D R; Rittmann, B E; Stahl, D A

    1994-01-01

    The microbial community structure of anaerobic biological reactors was evaluated by using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens. Phylogenetically defined groups of methanogens were quantified and visualized, respectively, by hybridization of 32P- and fluorescent-dye-labeled probes to the 16S rRNAs from samples taken from laboratory acetate-fed chemostats, laboratory municipal solid waste digestors, and full-scale sewage sludge digestors. Methanosarcina species, members of the order Methanobacteriales, and Methanosaeta species were the most abundant methanogens present in the chemostats, the solid-waste digestors, and the sewage sludge digestors, respectively. PMID:7517129

  3. Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting.

    PubMed

    Westerlund, Kristina; Honarvar, Hadis; Tolmachev, Vladimir; Eriksson Karlström, Amelie

    2015-08-19

    In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63

  4. Detection of viral genomes in the liver by in situ hybridization using 35S-, bromodeoxyuridine-, and biotin-labeled probes

    SciTech Connect

    Niedobitek, G.; Finn, T.; Herbst, H.; Stein, H.

    1989-03-01

    Methods employing /sup 35/S-, biotin-, and bromodeoxyuridine (BrdUrd)-labeled DNA probes were compared for the detection of hepatitis B virus (HBV) and cytomegalovirus (CMV) in the liver. The results demonstrate that: 1) HBV can be detected reliably only by the use of radiolabeled probes, whereas methods employing nonradioactive probes obviously are not sensitive enough for this virus. The use of /sup 35/S-labeled probes shortens the exposure times considerably in comparison to tritiated probes. 2) Biotin-labeled probes are of limited value for in situ hybridization on liver tissues because the presence of endogenous avidin-binding activity often leads to false positive results. 3) Brd-Urd-labeled probes are a useful alternative to biotinylated probes for the detection of CMV. In comparison with biotinylated probes, BrdUrd-labeled probes produce a specific signal of similar staining intensity in the absence of background staining in the liver.

  5. Probing interactions between plant virus movement proteins and nucleic acids.

    PubMed

    Tzfira, Tzvi; Citovsky, Vitaly

    2008-01-01

    Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP-nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for detection and characterization of MP binding to DNA and RNA molecules. We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays. PMID:18370264

  6. Identification of cDNAs by direct hybridization using cosmid probes

    SciTech Connect

    Lennon, G.G.; Lieuallen, K.

    1993-12-01

    The goal of this effort is to quickly obtain as many chromosome-specific cDNAs with as much map and sequence detail as possible. Many techniques have been proposed to isolate and identify genes within defined genomic regions; the technique discussed here is direct hybridization of a relatively complex genomic probe, an entire cosmid clone, to cDNA libraries. This method continues to be a straightforward technique with a fair number of successes.

  7. Electric field directed nucleic acid hybridization on microchips.

    PubMed Central

    Edman, C F; Raymond, D E; Wu, D J; Tu, E; Sosnowski, R G; Butler, W F; Nerenberg, M; Heller, M J

    1997-01-01

    Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, beta-alanine and gamma-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization. PMID:9396795

  8. Synthesis and Characterization of Hybrid Hyaluronic Acid-Gelatin Hydrogels

    PubMed Central

    Camci-Unal, Gulden; Cuttica, Davide; Annabi, Nasim; Demarchi, Danilo; Khademhosseini, Ali

    2013-01-01

    Biomimetic hybrid hydrogels have generated broad interest in tissue engineering and regenerative medicine. Hyaluronic acid (HA) and gelatin (hydrolyzed collagen) are naturally derived polymers and biodegradable under physiological conditions. Moreover, collagen and HA are major components of the extracellular matrix (ECM) in most of the tissues (e.g. cardiovascular, cartilage, neural). When used as a hybrid material, HA-gelatin hydrogels may enable mimicking the ECM of native tissues. Although HA-gelatin hybrid hydrogels are promising biomimetic substrates, their material properties have not been thoroughly characterized in the literature. Herein, we generated hybrid hydrogels with tunable physical and biological properties by using different concentrations of HA and gelatin. The physical properties of the fabricated hydrogels including swelling ratio, degradation, and mechanical properties were investigated. In addition, in vitro cellular responses in both two and three dimensional (2D and 3D) culture conditions were assessed. It was found that the addition of gelatin methacrylate (GelMA) into HA methacrylate (HAMA) promoted cell spreading in the hybrid hydogels. Moreover, the hybrid hydrogels showed significantly improved mechanical properties compared to their single component analogs. The HAMA-GelMA hydrogels exhibited remarkable tunability behavior and may be useful for cardiovascular tissue engineering applications. PMID:23419055

  9. Synthesis and characterization of hybrid hyaluronic acid-gelatin hydrogels.

    PubMed

    Camci-Unal, Gulden; Cuttica, Davide; Annabi, Nasim; Demarchi, Danilo; Khademhosseini, Ali

    2013-04-01

    Biomimetic hybrid hydrogels have generated broad interest in tissue engineering and regenerative medicine. Hyaluronic acid (HA) and gelatin (hydrolyzed collagen) are naturally derived polymers and biodegradable under physiological conditions. Moreover, collagen and HA are major components of the extracellular matrix (ECM) in most of the tissues (e.g., cardiovascular, cartilage, neural). When used as a hybrid material, HA-gelatin hydrogels may enable mimicking the ECM of native tissues. Although HA-gelatin hybrid hydrogels are promising biomimetic substrates, their material properties have not been thoroughly characterized in the literature. Herein, we generated hybrid hydrogels with tunable physical and biological properties by using different concentrations of HA and gelatin. The physical properties of the fabricated hydrogels including swelling ratio, degradation, and mechanical properties were investigated. In addition, in vitro cellular responses in both two and three-dimensional culture conditions were assessed. It was found that the addition of gelatin methacrylate (GelMA) into HA methacrylate (HAMA) promoted cell spreading in the hybrid hydogels. Moreover, the hybrid hydrogels showed significantly improved mechanical properties compared to their single component analogs. The HAMA-GelMA hydrogels exhibited remarkable tunability behavior and may be useful for cardiovascular tissue engineering applications. PMID:23419055

  10. Platinum porous nanoparticles hybrid with metal ions as probes for simultaneous detection of multiplex cancer biomarkers.

    PubMed

    Wang, Zifeng; Liu, Na; Ma, Zhanfang

    2014-03-15

    In this work, platinum porous nanoparticles (PtPNPs) absorbed metal ions as electrochemical signals were fabricated. Clean-surface PtPNPs were prepared by a surfactant-free method and decorated with amino groups via 2-aminoethanethiol. Amino capped PtPNPs complexation with Cd(2+) and Cu(2+) to form PtPNPs-Cd(2+) and PtPNPs-Cu(2+) hybrids, respectively. Anti-CEA and Anti-AFP separately labeled with PtPNPs-Cd(2+) and PtPNPs-Cu(2+) were used as distinguishable signal tags for capturing antigens. The metal ions were detected in a single run through differential pulse voltammetry (DPV) without acid dissolution, electric potentials and peak heights of which reflected the identity and concentrations of the corresponding antigen. Ionic liquid reduced graphene oxide (IL-rGO) modified glassy carbon electrode (GCE) was used as a substrate, which was rich in amino groups to immobilize antibodies by glutaraldehyde through cross-link between aldehyde groups and amino groups. Using the proposed probes and platform, a novel sandwich-type electrochemical immunosensor for simultaneous detecting carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was successfully developed. This immunoassay possessed good linearity from 0.05 ng mL(-1) to 200 ng mL(-1) for both CEA and AFP. The detection limit of CEA was 0.002 ng mL(-1) and that of AFP was 0.05 ng mL(-1) (S/N=3). Furthermore, analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). It suggested that the proposed electrochemical immunoassay provided a potential application of clinical screening for early-stage cancers. PMID:24176967

  11. Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes

    SciTech Connect

    McDonnell, P.J.; McDonnell, J.M.; Kessis, T.; Green, W.R.; Shah, K.V.

    1987-11-01

    Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated /sup 35/S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.

  12. Design and Application of Hybrid Magnetic Field-Eddy Current Probe

    NASA Technical Reports Server (NTRS)

    Wincheski, Buzz; Wallace, Terryl; Newman, Andy; Leser, Paul; Simpson, John

    2013-01-01

    The incorporation of magnetic field sensors into eddy current probes can result in novel probe designs with unique performance characteristics. One such example is a recently developed electromagnetic probe consisting of a two-channel magnetoresistive sensor with an embedded single-strand eddy current inducer. Magnetic flux leakage maps of ferrous materials are generated from the DC sensor response while high-resolution eddy current imaging is simultaneously performed at frequencies up to 5 megahertz. In this work the design and optimization of this probe will be presented, along with an application toward analysis of sensory materials with embedded ferromagnetic shape-memory alloy (FSMA) particles. The sensory material is designed to produce a paramagnetic to ferromagnetic transition in the FSMA particles under strain. Mapping of the stray magnetic field and eddy current response of the sample with the hybrid probe can thereby image locations in the structure which have experienced an overstrain condition. Numerical modeling of the probe response is performed with good agreement with experimental results.

  13. Microwave probe diagnostic for the lower hybrid multijunction antenna on TdeV

    SciTech Connect

    Jacquet, P.; Demers, Y.; Chaudron, G.A.; Glaude, V.; Cote, A.; Dube, A.; Mireault, R.; Robert, A.; Vachon, L.

    1997-02-01

    On the TdeV tokamak a microwave probe diagnostic enables direct measurement of the electromagnetic fields in ten reduced waveguides of the lower hybrid current drive multijunction antenna. In each instrumented reduced waveguide, the local field is measured at two different locations by probes through coupling holes located in the narrow wall of the waveguides. The amplitude and phase of the signals are measured with a multichannel heterodyne circuit and are used to calculate the incident and reflected fields at the antenna mouth. The probes are under vacuum and they are bakeable up to the maximum operating temperature of the antenna ({ital T}=350{degree}C). They are calibrated at room temperature but the evolution of their characteristic with temperature is taken into account in the data analysis. Typical accuracies of the field measurements at the grill mouth are: {plus_minus}9{percent} for the amplitude and {plus_minus}6{degree} for the phase. The probe diagnostic has been operating reliably for the last two years and the probes do not appear to perturb the operation of the antenna nor to reduce its power handling capability. Comparisons of the probe measurements with calculations from the multijunction antenna modeling code SWAN show that the code is accurate for low rf power densities at the antenna mouth. {copyright} {ital 1997 American Institute of Physics.}

  14. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

    PubMed Central

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-01-01

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection. PMID:23211753

  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. Natural cinnamic acids, synthetic derivatives and hybrids with antimicrobial activity.

    PubMed

    Guzman, Juan David

    2014-01-01

    Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC) of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships. PMID:25429559

  17. Activity-Based Probe for N-Acylethanolamine Acid Amidase.

    PubMed

    Romeo, Elisa; Ponzano, Stefano; Armirotti, Andrea; Summa, Maria; Bertozzi, Fabio; Garau, Gianpiero; Bandiera, Tiziano; Piomelli, Daniele

    2015-09-18

    N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme. PMID:26102511

  18. 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.

  19. 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.

  20. 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.

  1. GENUS- AND GROUP-SPECIFIC HYBRIDIZATION PROBES FOR DETERMINATIVE AND ENVIRONMENTAL STUDIES OF SULFATE-REDUCING BACTERIA

    EPA Science Inventory

    A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. our probes were genus specific and id...

  2. High accuracy plasma density measurement using hybrid Langmuir probe and microwave interferometer method

    SciTech Connect

    Deline, C.; Gilchrist, B. E.; Dobson, C.; Jones, J. E.; Chavers, D. G.

    2007-11-15

    High spatial resolution plasma density measurements have been taken as part of an investigation into magnetic nozzle physics at the NASA/MSFC Propulsion Research Center. These measurements utilized a Langmuir triple probe scanned across the measurement chord of either of two stationary rf interferometers. By normalizing the scanned profile to the microwave interferometer line-integrated density measurement for each electrostatic probe measurement, the effect of shot-to-shot variation of the line-integrated density can be removed. In addition, by summing the voltage readings at each radial position in a transverse scan, the line density can be reconstituted, allowing the absolute density to be determined, assuming that the shape of the profile is constant from shot to shot. The spatial and temporal resolutions of this measurement technique depend on the resolutions of the scanned electrostatic probe and the interferometer. The measurement accuracy is 9%-15%, which is on the order of the accuracy of the rf interferometer. The measurement technique was compared directly with both scanning rf interferometer and standard Langmuir probe theory. The hybrid technique compares favorably with the scanning rf interferometer, and appears more accurate than probe theory alone. Additionally, our measurement technique is generally applicable even for nonaxisymmetric plasmas.

  3. High accuracy plasma density measurement using hybrid Langmuir probe and microwave interferometer method.

    PubMed

    Deline, C; Gilchrist, B E; Dobson, C; Jones, J E; Chavers, D G

    2007-11-01

    High spatial resolution plasma density measurements have been taken as part of an investigation into magnetic nozzle physics at the NASA/MSFC Propulsion Research Center. These measurements utilized a Langmuir triple probe scanned across the measurement chord of either of two stationary rf interferometers. By normalizing the scanned profile to the microwave interferometer line-integrated density measurement for each electrostatic probe measurement, the effect of shot-to-shot variation of the line-integrated density can be removed. In addition, by summing the voltage readings at each radial position in a transverse scan, the line density can be reconstituted, allowing the absolute density to be determined, assuming that the shape of the profile is constant from shot to shot. The spatial and temporal resolutions of this measurement technique depend on the resolutions of the scanned electrostatic probe and the interferometer. The measurement accuracy is 9%-15%, which is on the order of the accuracy of the rf interferometer. The measurement technique was compared directly with both scanning rf interferometer and standard Langmuir probe theory. The hybrid technique compares favorably with the scanning rf interferometer, and appears more accurate than probe theory alone. Additionally, our measurement technique is generally applicable even for nonaxisymmetric plasmas. PMID:18052471

  4. Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization.

    PubMed

    Kavanagh, Paul; Leech, Dónal

    2006-04-15

    The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach. PMID:16615783

  5. Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization

    SciTech Connect

    Morotomi, M.; Ohno, T.; Mutai, M.

    1988-05-01

    A dot blot hybridization procedure with /sup 32/P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.

  6. Quantum Dot-Bead-DNA Probe-Based Hybridization Fluorescence Assays on Microfluidic Chips.

    PubMed

    Ankireddy, Seshadri Reddy; Kim, Jongsung

    2015-10-01

    The development of chip-based, quantum dot (QD)-bead-DNA conjugate probes for hybridization detection is a prime research focus in the field of microfluidics. QD-Bead-DNA probe-based hybridization detection methods are often called "bead-based assays," and their success is substantially influenced by the dispensing and manipulation capabilities of microfluidic technology. Met was identified as a prognostic marker in different cancers including lung, renal, liver, head and neck, stomach, and breast. In this report, the cancer causing Met gene was detected with QDs attached to polystyrene microbeads. We constructed a microfluidic platform using a flexible PDMS polymer. The chip consists of two channels, with two inlets and two outlets. The two channels were integrated with QD-bead-DNA probes for simultaneous detection of wild type target DNA and mutant DNA, containing three nucleotide changes compared to the wild type sequence. The fluorescence quenching ability of QDs within the channels of microfluidic chips were compared for both DNAs. PMID:26726440

  7. Visualization of NRAS RNA G-Quadruplex Structures in Cells with an Engineered Fluorogenic Hybridization Probe.

    PubMed

    Chen, Shuo-Bin; Hu, Ming-Hao; Liu, Guo-Cai; Wang, Jin; Ou, Tian-Miao; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2016-08-24

    The RNA G-quadruplex is an important secondary structure formed by guanine-rich RNA sequences. However, its folding studies have mainly been studied in vitro. Accurate identification of RNA G-quadruplex formation within a sequence of interest remains difficult in cells. Herein, and based on the guanine-rich sequence in the 5'-UTR of NRAS mRNA, we designed and synthesized the first G-quadruplex-triggered fluorogenic hybridization (GTFH) probe, ISCH-nras1, for the unique visualization of the G-quadruplexes that form in this region. ISCH-nras1 is made up of two parts: The first is a fluorescent light-up moiety specific to G-quadruplex structures, and the second is a DNA molecule that can hybridize with a sequence that is adjacent to the guanine-rich sequence in the NRAS mRNA 5'-UTR. Further evaluation studies indicated that ISCH-nras1 could directly and precisely detect the targeted NRAS RNA G-quadruplex structures, both in vitro and in cells. Thus, this GTFH probe was a useful tool for directly investigating the folding of G-quadruplex structures within an RNA of interest and represents a new direction for the design of smart RNA G-quadruplex probes. PMID:27508892

  8. 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.

  9. 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.

  10. Monitoring of microbial communities by flow cytometry and rRNA-targeted hybridization probes

    NASA Astrophysics Data System (ADS)

    Wallner, Guenter; Amann, Rudolf

    1995-10-01

    Flow cytometry in combination with ribosomal RNA (rRNA) based fluorescence in situ hybridization is a new technique for the analysis of microbial communities. Oligonucleotide probes directed against ribosomal RNA allow the identification of species or groups of micro- organisms. Combined with flow cytometry, up to several thousand cells per second can be classified. In addition to the identification and specific enumeration of micro-organisms, further information on the distribution of cell size, DNA and ribosome content -- and therefore an assessment of activity -- within the entire community or subpopulations can be obtained. This technique is much more accurate, informative, and rapid than classical culture-dependent methods. Data of activated sludge samples hybridized with fluorescein labeled oligonucleotides and counterstained with the DNA-specific dye Hoechst 33342 are presented as examples for its applicability to complex microbial communities.

  11. Guided assembly of metal and hybrid conductive probes using floating potential dielectrophoresis.

    PubMed

    Puigmartí-Luis, Josep; Stadler, Johannes; Schaffhauser, Daniel; del Pino, Angel Pérez; Burg, Brian R; Dittrich, Petra S

    2011-03-01

    We present the site-selective, parallel and reproducible formation of conductive gold and tetrathiafulvalene-gold (TTF-Au) hybrid micro- and nanowires from their respective ion salt and cation-radical solutions. While the formation of micro- and nanowires by means of dielectrophoresis with directly coupled electrodes has been thoroughly investigated in recent studies, we present here the first relevant example of metal and hybrid wire assembly obtained by floating potential dielectrophoresis. In this configuration, the assembly of micro- and nanowires is achieved by capacitively coupling a large electrode (bias electrode) to a conductive substrate (p-doped Si) separated by an insulating oxide layer. In contrast to former studies, this allows parallel production of micro- and nanowires with only one pair of electrodes connected to a sine wave generator. We further demonstrate that these structures are suitable probes for localized surface enhanced Raman spectroscopy (SERS). PMID:21225055

  12. Probing hybridization of a single energy level coupled to superconducting leads

    NASA Astrophysics Data System (ADS)

    van Zanten, D. M. T.; Balestro, F.; Courtois, H.; Winkelmann, C. B.

    2015-11-01

    Electron transport through a quantum dot coupled to superconducting leads shows a sharp conductance onset when a quantum dot orbital level crosses the superconducting coherence peak of one lead. We study superconducting single electron transistors in the weak coupling limit by connecting individual gold nanoparticles with aluminum leads formed by electromigration. We show that the transport features close to the conductance onset threshold can be accurately described by the quantum dot levels' hybridization with the leads, which is strongly enhanced by the divergent density of states at the superconducting gap edge. This highlights the importance of electron cotunneling effects in spectroscopies with superconducting probes.

  13. Guided assembly of metal and hybrid conductive probes using floating potential dielectrophoresis

    NASA Astrophysics Data System (ADS)

    Puigmartí-Luis, Josep; Stadler, Johannes; Schaffhauser, Daniel; Del Pino, Ángel Pérez; Burg, Brian R.; Dittrich, Petra S.

    2011-03-01

    We present the site-selective, parallel and reproducible formation of conductive gold and tetrathiafulvalene-gold (TTF-Au) hybrid micro- and nanowires from their respective ion salt and cation-radical solutions. While the formation of micro- and nanowires by means of dielectrophoresis with directly coupled electrodes has been thoroughly investigated in recent studies, we present here the first relevant example of metal and hybrid wire assembly obtained by floating potential dielectrophoresis. In this configuration, the assembly of micro- and nanowires is achieved by capacitively coupling a large electrode (bias electrode) to a conductive substrate (p-doped Si) separated by an insulating oxide layer. In contrast to former studies, this allows parallel production of micro- and nanowires with only one pair of electrodes connected to a sine wave generator. We further demonstrate that these structures are suitable probes for localized surface enhanced Raman spectroscopy (SERS).We present the site-selective, parallel and reproducible formation of conductive gold and tetrathiafulvalene-gold (TTF-Au) hybrid micro- and nanowires from their respective ion salt and cation-radical solutions. While the formation of micro- and nanowires by means of dielectrophoresis with directly coupled electrodes has been thoroughly investigated in recent studies, we present here the first relevant example of metal and hybrid wire assembly obtained by floating potential dielectrophoresis. In this configuration, the assembly of micro- and nanowires is achieved by capacitively coupling a large electrode (bias electrode) to a conductive substrate (p-doped Si) separated by an insulating oxide layer. In contrast to former studies, this allows parallel production of micro- and nanowires with only one pair of electrodes connected to a sine wave generator. We further demonstrate that these structures are suitable probes for localized surface enhanced Raman spectroscopy (SERS). Electronic supplementary

  14. Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans.

    PubMed

    Seo, Beomseok; Lee, Junho

    2016-01-01

    Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)(1). Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT(2). Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads. PMID:27583462

  15. Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

    PubMed Central

    Glazer, Marc I.; Fidanza, Jacqueline A.; McGall, Glenn H.; Trulson, Mark O.; Forman, Jonathan E.; Frank, Curtis W.

    2007-01-01

    We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (ka, kd, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for

  16. Bipolar lead-acid battery for hybrid vehicles

    NASA Astrophysics Data System (ADS)

    Saakes, M.; Woortmeijer, R.; Schmal, D.

    Within the framework of the European project bipolar lead-acid power source (BILAPS), a new production route is being developed for the bipolar lead-acid battery. The performance targets are 500 W kg -1, 30 Wh kg -1 and 100 000 power-assist life cycles (PALCs). The operation voltage of the battery can be, according to the requirements, 12, 36 V or any other voltage. Tests with recently developed 4 and 12 V prototypes, each of 30 Ah capacity have demonstrated that the PALC can be operated using 10 C discharge and 9 C charge peaks. The tests show no overvoltage or undervoltage problems during three successive test periods of 16 h with 8 h rest in between. The temperature stabilizes during these tests at 40-45 °C using a thermal-management system. The bipolar lead acid battery is operated at an initial 50% state-of-charge. During the tests, the individual cell voltages display only very small differences. Tests are now in progress to improve further the battery-management system, which has been developed at the cell level, during the period no PALCs are run in order to improve the hybrid behaviour of the battery. The successful tests show the feasibility of operating the bipolar lead-acid battery in a hybrid mode. The costs of the system are estimated to be much lower than those for nickel-metal-hydride or Li-ion based high-power systems. An additional advantage of the lead-acid system is that recycling of lead-acid batteries is well established.

  17. Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy

    PubMed Central

    Dutta, Samrat; Armitage, Bruce A.; Lyubchenko, Yuri L.

    2016-01-01

    Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplex. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. (2011) Journal of Organic Chemistry 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γMPPNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that γMPPNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 0.030 ± 0.01 sec-1 for γMPPNA-DNA hybrid duplex vs. 0.375 ± 0.18 sec-1 for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γMPPNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γMPPNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes. PMID:26898903

  18. Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

    PubMed

    Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

    2016-03-15

    Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes. PMID:26898903

  19. Use of peptide nucleic acid probes for rapid detection and enumeration of viable bacteria in recreational waters and beach sand.

    PubMed

    Esiobu, Nwadiuto

    2006-01-01

    Environmental monitoring and public health risk assessments require methods that are rapid and quantitative with defined sensitivity and specificity thresholds. Although several molecular techniques have been developed to rapidly detect bacteria in complex matrices, the challenge to simultaneously detect and enumerate only viable cells remains a limiting factor to their routine application. This chapter describes the use of peroxidase-labeled peptide nucleic acid (PNA) probes to simultaneously detect and count live Staphylococcus aureus, a human pathogen in sea water and beach sand. Mixed bacteria from the environmental sample were immobilized on polyvinylidene difluoride membrane filters and allowed to form microcolonies during a 5-h incubation on Tryptic soy agar plates. PNA probes targeting species-specific regions of the 16S rRNA sequences of S. aureus were then used to hybridize the target bacteria in situ. Probes were detected by capturing chemiluminiscence on instant (e.g., Polaroid) films. Each viable cell (i.e., rRNA producing) is detected as a light spot from its microcolony on the film after scanning the image into a computer. This rapid in situ hybridization technique is simple and highly sensitive and could be developed into portable kits for monitoring pathogens and indicators in the environment. PMID:16957353

  20. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes.

    PubMed

    Hwang, Gyoyeon; Lee, Hansol; Lee, Jiyeon

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. PMID:26449454

  1. Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification

    SciTech Connect

    Wimpee, C.F.; Nadeau, T.L.; Nealson, K.H. )

    1991-05-01

    By using two highly conserved regions of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, a cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.

  2. Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

    PubMed Central

    Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2012-01-01

    A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. Addition of thermostable RNase H resulted in the cleavage of the RNA loop which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9x above background), resulting in a ~2–2.8 fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time PCR reactions by measuring enhancement of donor fluorescence upon R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  3. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

    Jacroux, Thomas; Rieck, Daniel C; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  4. Reactive Microcontact Printing of DNA Probes on (DMA-NAS-MAPS) Copolymer-Coated Substrates for Efficient Hybridization Platforms.

    PubMed

    Castagna, Rossella; Bertucci, Alessandro; Prasetyanto, Eko Adi; Monticelli, Marco; Conca, Dario Valter; Massetti, Matteo; Sharma, Parikshit Pratim; Damin, Francesco; Chiari, Marcella; De Cola, Luisa; Bertacco, Riccardo

    2016-04-01

    High-performing hybridization platforms fabricated by reactive microcontact printing of DNA probes are presented. Multishaped PDMS molds are used to covalently bind oligonucleotides over a functional copolymer (DMA-NAS-MAPS) surface. Printed structures with minimum width of about 1.5 μm, spaced by 10 μm, are demonstrated, with edge corrugation lower than 300 nm. The quantification of the immobilized surface probes via fluorescence imaging gives a remarkable concentration of 3.3 × 10(3) oligonucleotides/μm(2), almost totally active when used as probes in DNA-DNA hybridization assays. Indeed, fluorescence and atomic force microscopy show a 95% efficiency in target binding and uniform DNA hybridization over printed areas. PMID:26972953

  5. 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.

    PubMed

    Esiobu, Nwadiuto; Mohammed, Renuka; Echeverry, Andrea; Green, Melissa; Bonilla, Tonya; Hartz, Aaron; McCorquodale, Don; Rogerson, Andrew

    2004-05-01

    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, Staphylococcus aureus and Pseudomonas aeruginosa, was achieved within 6-8 h of processing. Following 5 h of incubation on TSA, soybean peroxidase-labeled peptide nucleic acid probes (Boston Probes, Boston, MA) targeting species-specific 16S rRNA sequences of P. aeruginosa and S. aureus were used to hybridize microcolonies of the target species in-situ. In addition, a universal probe for 16S rRNA sequences was used to target the eubacteria. Probes were detected after a light generating reaction with a chemiluminescent substrate and their presence recorded on Polaroid film. The probes showed limited cross-reactivity with mixed indigenous bacteria extracted from seawater and sand by shaking with phosphate-buffered saline (PBS). Specificity and cross-reactivity was tested on the reference bacterial genera Pseudomonas, Staphylococcus, Vibrio, Shigella, Salmonella, Acinetobacter, Enterobacter, Escherichia and Citrobacter. These tests confirmed that the probes were specific for the microorganisms of interest and were unaffected by high salt levels. The results of the PNA chemiluminescent in situ hybridization were compared with traditional plate count methods (PCM) for total 'freshwater' eubacteria, S. aureus and P. aeruginosa. Counts of eubacteria and S. aureus were comparable with numbers obtained from traditional plate counts but levels of P. aeruginosa were higher with PNA than with PCM. It is possible that PNA is more sensitive than PCM because it can detect microcolonies on the agar surface that never fully develop with the plate count method. We conclude that the in situ hybridization technique used here represents an important potential tool for the rapid monitoring of novel indicator organisms in

  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. PMID:27472344

  7. In situ hybridization of phytoplankton using fluorescently labeled rRNA probes.

    PubMed

    Groben, René; Medlin, Linda

    2005-01-01

    Phytoplankton are one of the major components of ecosystem processes and play an important role in many biogeochemical cycles in the marine and freshwater environment. Despite their importance, many microalgae are poorly described and little is known of broad spatial and temporal scale trends in their abundance and distribution. Reasons for this are that microalgae are often small, lack distinct morphological features, and are unculturable, which make analyses difficult. It is now possible by using molecular biological techniques to advance our knowledge of aquatic biodiversity and to understand how biodiversity supports ecosystem structure, dynamics, and resilience. We present in this chapter a brief review of the progress that has been made in analyzing microalgae from populations to the species level. The described methods range from DNA fingerprinting techniques, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs), to microsatellites, which are used in population studies, to sequence analysis, which help to reconstruct the evolutionary history of organisms and to examine relationships at various taxonomic levels. Special emphasis is given to the application of molecular probes for the identification and characterization of microalgal taxa. The fast and secure identification of phytoplankton, especially of toxic species, is important from an ecological and economical point of view and whole-cell hybridization with specific fluorochrome-labeled probes followed by fluorescence microscopy or flow cytometry offers a fast method for this purpose. In this context, we present a detailed protocol for fluorescence in situ hybridization (FISH) of ribosomal RNA (rRNA) probes that can be applied to many algal cell types and discuss practical considerations of its use. PMID:15865974

  8. 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

  9. Artificial mismatch hybridization

    DOEpatents

    Guo, Zhen; Smith, Lloyd M.

    1998-01-01

    An improved nucleic acid hybridization process is provided which employs a modified oligonucleotide and improves the ability to discriminate a control nucleic acid target from a variant nucleic acid target containing a sequence variation. The modified probe contains at least one artificial mismatch relative to the control nucleic acid target in addition to any mismatch(es) arising from the sequence variation. The invention has direct and advantageous application to numerous existing hybridization methods, including, applications that employ, for example, the Polymerase Chain Reaction, allele-specific nucleic acid sequencing methods, and diagnostic hybridization methods.

  10. An unnatural amino acid based fluorescent probe for phenylalanine ammonia lyase.

    PubMed

    Tian, Zhenlin; Zhu, Weiping; Xu, Yufang; Qian, Xuhong

    2014-08-21

    A fluorescent probe (2a-LP) based on an unnatural amino acid (UAA) is developed for the detection of phenylalanine ammonia lyase (PAL). In the presence of PAL, 2a-LP is catalytically deaminated to ortho-amino-transcinnamic acid (o-a-CA), which shows a remarkable “off–on” fluorescence signal. Thus, the probe 2a-LP enables direct visualization of the PAL activity in tomato under UV illumination and has potential in vitro assays. PMID:24971756

  11. Synthesis of Ag(2) S-Ag nanoprisms and their use as DNA hybridization probes.

    PubMed

    Liu, Bing; Ma, Zhanfang

    2011-06-01

    A simple synthetic route to prepare Ag(2) S-Ag nanoprisms consists of the facile addition of Na(2) S to a solution of triangular Ag nanoprisms. The resulting Ag(2) S-Ag nanoparticles are more stable in solution than the original Ag nanoprisms, and two surface plasmon resonance (SPR) bands of the original Ag nanoprisms still remain. In addition, the SPR bands of the Ag(2) S-Ag nanoprisms are tunable over a wide range. The Ag(2) S-Ag nanoprisms can be directly bioconjugated via well-established stable Ag(2) S surface chemistry with readily available sulfur coupling agents. The nanoprisms are used in the hybridization of functionalized oligonucleotides, and show promise as probes for future biosensing applications. PMID:21538868

  12. "Inosaminoacids": novel inositol-amino acid hybrid structures accessed by microbial arene oxidation.

    PubMed

    Pilgrim, Sarah; Kociok-Köhn, Gabriele; Lloyd, Matthew D; Lewis, Simon E

    2011-04-28

    Microbial 1,2-dihydroxylation of sodium benzoate permits the rapid construction of novel inositol-amino acid hybrid structures. Both β- and γ-amino acids are accessible by means of an acylnitroso Diels-Alder cycloaddition. PMID:21409268

  13. In situ detection of freshwater fungi in an alpine stream by new taxon-specific fluorescence in situ hybridization probes.

    PubMed

    Baschien, Christiane; Manz, Werner; Neu, Thomas R; Marvanová, Ludmila; Szewzyk, Ulrich

    2008-10-01

    New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes. PMID:18776035

  14. Creatinyl amino acids: new hybrid compounds with neuroprotective activity.

    PubMed

    Burov, Sergey; Leko, Maria; Dorosh, Marina; Dobrodumov, Anatoliy; Veselkina, Olga

    2011-09-01

    Prolonged oral creatine administration resulted in remarkable neuroprotection in experimental models of brain stroke. However, because of its polar nature creatine has poor ability to penetrate the blood-brain barrier (BBB) without specific creatine transporter (CRT). Thus, synthesis of hydrophobic derivatives capable of crossing the BBB by alternative pathway is of great importance for the treatment of acute and chronic neurological diseases including stroke, traumatic brain injury and hereditary CRT deficiency. Here we describe synthesis of new hybrid compounds-creatinyl amino acids, their neuroprotective activity in vivo and stability to degradation in different media. The title compounds were synthesized by guanidinylation of corresponding sarcosyl peptides or direct creatine attachment using isobutyl chloroformate method. Addition of lipophilic counterion (p-toluenesulfonate) ensures efficient creatine dissolution in DMF with simultaneous protection of guanidino group towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents, permits to simplify synthetic procedure and adapt it to large-scale production. The biological activity of creatinyl amino acids was tested in vivo on ischemic stroke and NaNO(2) -induced hypoxia models. One of the most effective compounds-creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. PMID:21644247

  15. Detection of Cryptosporidium parvum in raw milk by PCR and oligonucleotide probe hybridization.

    PubMed Central

    Laberge, I; Ibrahim, A; Barta, J R; Griffiths, M W

    1996-01-01

    Cryptosporidium spp. are potential contaminants of food. Suspected cases of food-borne cryptosporidiosis are rarely confirmed because of the limited numbers of oocysts in the samples and the lack of sensitive detection methods adaptable to food. PCR was investigated as a means of overcoming this problem. A PCR assay was designed for the specific amplification of a previously sequenced portion of an oocyst protein gene fragment of Cryptosporidium parvum (N. C. Lally, G. D. Baird, S. J. McQuay, F. Wright, and J. J. Oliver, Mol. Biochem. Parasitol. 56:69-78, 1992) and compared with the primer set of Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991). The PCR products were hybridized with digoxigenin-labeled internal probes and detected by chemiluminescence to enhance sensitivity. The two sets of primers were compared with regard to their sensitivity and specificity by using a variety of human and animal isolates of C. parvum and related parasites. Both assays enabled the detection of 1 to 10 oocysts in 20 ml of artificially contaminated raw milk. The assay based on the PCR set and probe of Laxer et al. detected DNAs from Eimeria acervulina and Giardia intestinalis. The new assay has good specificity for C. parvum bovine isolates and hence has a better potential for monitoring the prevalence of C. parvum in raw milk and other environmental samples. PMID:8795214

  16. Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes

    NASA Technical Reports Server (NTRS)

    Wu, H.; George, K.; Yang, T. C.

    1998-01-01

    PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

  17. Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

    PubMed

    Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

    2013-01-01

    Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. PMID:23797655

  18. Molecular epidemiology of Vibrio cholerae O1 isolated in Nepal by southern hybridization with a cholera toxin gene probe.

    PubMed

    Yamamoto, K; Shrestha, J; Iida, T; Yoh, M; Honda, T

    1995-06-01

    A cholera epidemic broke out in 1992 due to Vibrio cholerae O1 biotype El Tor in the eastern and southern belt of Nepal mainly among the Bhutanese refugees. Restriction fragment profiles (RFP) of DNA fragments of V. cholerae O1 isolates hybridized with an enzyme-labelled oligonucleotide probe for cholera toxin gene (ctx) by Southern Hybridization were compared. The probe hybridized with the 13- and 8-kb fragments of PstI-digested total DNA in all isolates observed in the epidemic. This RFP in the Nepalese strain was not observed in the strains isolated during other epidemics but was observed in the strains isolated from the exported marine products from Taiwan and Thailand. PMID:7594311

  19. Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

    SciTech Connect

    Park, J.S.; Kurman, R.J.; Kessis, T.D.; Shah, K.V. )

    1991-01-01

    A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

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

    SciTech Connect

    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.

  1. 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

  2. A comparison of two methods for colorimetric in situ hybridization using paraffin-embedded tissue sections and digoxigenin-labeled hybridization probes.

    PubMed

    Marcino, Joe

    2013-06-01

    Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90-100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals. PMID:23697605

  3. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  4. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method.

    PubMed

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6-99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  5. A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

    PubMed

    Behr, T; Koob, C; Schedl, M; Mehlen, A; Meier, H; Knopp, D; Frahm, E; Obst, U; Schleifer, K; Niessner, R; Ludwig, W

    2000-12-01

    Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants. PMID:11249027

  6. Identification of species in tribe Brassiceae by dot-blot hybridization using species-specific ITS1 probes.

    PubMed

    Tonosaki, K; Nishio, Takeshi

    2010-10-01

    Simple, reliable methods for identification of species are required for management of many species and lines in a plant gene bank. Species-specific probes were designed from published sequences of the ITS1 region in rDNA of 16 species in Brassica and its related genera, and used as probes for dot-blot hybridization with plant genomic DNA. All the probes detected species-specific signals at dot-blots of genomic DNAs of the 16 species in Brassica, Diplotaxis, Eruca, and Raphanus. Signals of the Brassica digenomic species in the U's triangle, i.e., B. napus, B. juncea, and B. carinata, were detected by the probes of their parental monogenomic species, i.e., B. rapa, B. nigra, and B. oleracea. The probe for B. oleracea showed signals of B. balearica, B. cretica, B. incana, B. insularis, and B. macrocarpa, which have the C genome as B. oleracea. Eruca vesicaria DNA was detected by the probe for E. sativa, which has been classified as a subspecies of E. vescaria. DNA of leaf tissue extracted by an alkaline solution and seed DNA prepared by the NaI method can be used directly for dot-blotting. Misidentification of species was revealed in 20 accessions in the Tohoku University Brassica Seed Bank. These results indicate dot-blot hybridization to be a simple and efficient technique for identification of plant species in a gene bank. PMID:20683723

  7. Detecting Microbial Nucleic Acids within Nematode Bodies: A Photo Essay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a taxa-specific, fluorescence in situ hybridization (FISH) technique to localize microbial nucleic acids within nematode bodies. This technique involves hybridization of a nucleic acid probe to target microbial sequences. Hybridization is detected microscopically, as the probes have f...

  8. Adenovirus Type 2-Simian Virus 40 Hybrid Population: Evidence for a Hybrid Deoxyribonucleic Acid Molecule and the Absence of Adenovirus-Encapsidated Circular Simian Virus 40 Deoxyribonucleic Acid

    PubMed Central

    Crumpacker, Clyde S.; Levin, Myron J.; Wiese, William H.; Lewis, Andrew M.; Rowe, Wallace P.

    1970-01-01

    The deoxyribonucleic acid (DNA) from the adenovirus-encapsidated particles of the adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid population plaque variant (Ad2++ HEY), known to yield SV40 virus with high efficiency, was studied by equilibrium density centrifugation followed by ribonucleic acid-DNA hybridization employing virus-specific complementary ribonucleic acids synthesized in vitro. These techniques establish linkage between the Ad2 and SV40 components in the adenovirus-encapsidated particles of this population. The linkage is alkali-resistant and presumably covalent; thus, the Ad2 DNA and SV40 DNA are present in a hybrid molecule. Velocity centrifugation studies in alkaline sucrose gradients eliminated the possibility that supercoiled circular SV40 DNA is present in the adenovirus capsids. The DNA obtained from the adenovirus-encapsidated particles of the Ad2++ HEY population appears to consist of nonhybrid Ad2 DNA and Ad2-SV40 hybrid DNA molecules. PMID:4322081

  9. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  10. High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.

    PubMed

    Roy, Subhadeep; Tanious, Farial A; Wilson, W David; Ly, Danith H; Armitage, Bruce A

    2007-09-18

    Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed. PMID:17718513

  11. Facile Probe Design: Fluorescent Amphiphilic Nucleic Acid Probes without Quencher Providing Telomerase Activity Imaging Inside Living Cells.

    PubMed

    Jia, Yongmei; Gao, Pengcheng; Zhuang, Yuan; Miao, Mao; Lou, Xiaoding; Xia, Fan

    2016-06-21

    Nowadays, the probe with fluorophore but no quencher is promising for its simple preparation, environmental friendliness, and wide application scope. This study designs a new amphiphilic nucleic acid probe (ANAP) based on aggregation-caused quenching (ACQ) effect without any quencher. Upon binding with targets, the dispersion of hydrophobic part (conjugated fluorene, CF) in ANAP is enhanced as a signal-on model for proteins, nucleic acids, and small molecules detection or the aggregation of CF is enhanced as a signal-off model for ion detection. Meanwhile, because of the high specificity of ANAP, a one-step method is developed powerfully for monitoring the telomerase activity not only from the cell extracts but also from 50 clinic urine samples (positive results from 45 patients with bladder cancer and negative results from 5 healthy people). ANAPs can also readily enter into cells and exhibit a good performance for distinguishing natural tumor cells from the tumor cells pretreated by telomerase-related drugs or normal cells. In contrast to our previous results ( Anal. Chem. 2015 , 87 , 3890 - 3894 ), the present CF is a monomer which is just the structure unit of the previous fluorescent polymer. Since the accurate molecular structure and high DNA/CF ratio of the present CF, these advanced experiments obtain an easier preparation of probes, an improved sensitivity and specificity, and broader detectable targets. PMID:27223599

  12. Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization.

    PubMed

    Singh, M; Singh, R P

    1996-06-01

    A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY(O), but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY(O)-specific probe. As little as 1 pg of purified PVY(O)-RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY(O) amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY(O) concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and did not result in reduced detection of PVY(O) by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America. PMID:8795005

  13. Identification of bovine Neospora parasites by PCR amplification and specific small-subunit rRNA sequence probe hybridization.

    PubMed

    Ho, M S; Barr, B C; Marsh, A E; Anderson, M L; Rowe, J D; Tarantal, A F; Hendrickx, A G; Sverlow, K; Dubey, J P; Conrad, P A

    1996-05-01

    Neospora is a newly recognized genus of pathogenic coccidia, closely related to Toxoplasma gondii, that can cause abortion or congenital disease in a variety of domestic animal hosts. On the basis of the small-subunit rRNA gene sequences of Neospora spp. and other apicomplexa coccidia, oligonucleotide primers COC-1 and COC-2 were used for PCR amplification of conserved sequences of approximately 300 bp in size. A Neospora-specific chemiluminescent probe hybridized to Southern blots of amplification products from Neospora DNA but not to Southern blots with amplified DNA from the other coccidian parasites tested. A Toxoplasma-specific probe whose sequence differed from that of the probe for Neospora spp. by a single base pair was used to distinguish these parasites by specific Southern blot hybridization. The PCR system detected as few as one Neospora tachyzoite in the culture medium or five tachyzoites in samples of whole blood or amniotic fluid spiked with Neospora parasites. In addition, Neospora PCR products were successfully amplified from whole blood and amniotic fluid samples of experimentally infected bovine and rhesus macaque fetuses. These results indicate that this PCR and probe hybridization system could be a valuable adjunct to serology and immunohistochemistry for the diagnosis of Neospora infections in bovine or primate fetuses. PMID:8727903

  14. Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids.

    PubMed

    Yang, Li; Li, Bosheng; Zheng, Xiao-yu; Li, Jigang; Yang, Mei; Dong, Xinnian; He, Guangming; An, Chengcai; Deng, Xing Wang

    2015-01-01

    Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen. PMID:26065719

  15. Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids

    PubMed Central

    Yang, Li; Li, Bosheng; Zheng, Xiao-yu; Li, Jigang; Yang, Mei; Dong, Xinnian; He, Guangming; An, Chengcai; Deng, Xing Wang

    2015-01-01

    Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen. PMID:26065719

  16. Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization.

    PubMed Central

    Forghani, B; Yu, G J; Hurst, J W

    1991-01-01

    We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector insert procedure. We cloned over 85% of the VZV genome and obtained 18 recombinants. Plasmids containing the B, F, G, H, and J fragments of VZV DNA were labeled by the nick translation method with biotin-11-dUTP as the dTTP analog. Additionally, the B fragment was cleaved with RE AvaI, subcloned into the plasmid pGEM-4 transcription vector, and subsequently linearized with REs PstI and EcoRI. RNA was transcribed with T7 or SP6 polymerase, with a substitution of allylamine-UTP as the UTP analog, and labeled with epsilon-caproylamidobiotin-N-hydroxysuccinimide ester. The DNA and RNA probes were used under full-stringency conditions for in situ hybridization with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate-Nitro Blue Tetrazolium as the substrate. When tested under comparable conditions, the RNA probe was slightly more sensitive than was the DNA probe: both probes showed homology only with VZV-infected cells and clinical tissues and not with the other herpesviruses. Probes prepared from variable regions of the genome (fragments F and J) performed as well as did those from conserved regions (fragments B. G. and H). Biotinylated probes have distinct advantages over isotopic probes and retain their full potency for more than 2 years when stored properly. Images PMID:1645371

  17. Structure and DNA Hybridization Properties of Mixed Nucleic Acid/Maleimide-Ethylene Glycol Monolayers

    SciTech Connect

    Lee,C.; Nguyen, P.; Grainger, D.; Gamble, L.; Castner, D.

    2007-01-01

    The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s {yields} {pi}* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the 'high-density' probe surface than on the 'high-efficiency' probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.

  18. Identification of triclosan-degrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography.

    PubMed

    Lolas, Ihab Bishara; Chen, Xijuan; Bester, Kai; Nielsen, Jeppe Lund

    2012-11-01

    Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions of enrichment culture incubated with (13)C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting the Methylobacillus group was designed and applied to the enrichment culture incubated with (14)C-labelled triclosan for MAR-FISH. The MAR-FISH results confirmed a positive uptake of carbon from (14)C-labelled triclosan by the Methylobacillus. The high representation of Methylobacillus in the (13)C-labelled DNA clone library and its observed utilization of (14)C-labelled triclosan by MAR-FISH reveal that these micro-organisms are the primary consumers of triclosan in the enrichment culture. The results from this study show that the combination of SIP and MAR-FISH can shed light on the networks of uncultured micro-organisms involved in degradation of organic micro-pollutants. PMID:22956759

  19. 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. PMID:26753813

  20. Poly(o-phenylenediamine) colloid-quenched fluorescent oligonucleotide as a probe for fluorescence-enhanced nucleic acid detection.

    PubMed

    Tian, Jingqi; Li, Hailong; Luo, Yonglan; Wang, Lei; Zhang, Yingwei; Sun, Xuping

    2011-02-01

    In this Letter, we demonstrate that chemical oxidation polymerization of o-phenylenediamine (OPD) by potassium bichromate at room temperature results in the formation of submicrometer-scale poly(o-phenylenediamine) (POPD) colloids. Such colloids can absorb and quench dye-labeled single-stranded DNA (ssDNA) very effectively. In the presence of a target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the POPD surface, leading to recovery of dye fluorescence. With the use of an oligonucleotide (OND) sequence associated with human immunodeficiency virus (HIV) as a model system, we demonstrate the proof of concept that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch. PMID:21186809

  1. 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

    2016-04-22

    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

  2. Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

    PubMed

    Smyth, J A; Weston, J; Moffett, D A; Todd, D

    2001-11-01

    Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent. PMID:11724137

  3. Evaluating a hybrid three-dimensional metrology system: merging data from optical and touch probe devices

    NASA Astrophysics Data System (ADS)

    Gerde, Janice R.; Christens-Barry, William A.

    2011-08-01

    In a project to meet requirements for CBP Laboratory analysis of footwear under the Harmonized Tariff Schedule of the United States (HTSUS), a hybrid metrology system comprising both optical and touch probe devices has been assembled. A unique requirement must be met: To identify the interface-typically obscured in samples of concern-of the "external surface area upper" (ESAU) and the sole without physically destroying the sample. The sample outer surface is determined by discrete point cloud coordinates obtained using laser scanner optical measurements. Measurements from the optically inaccessible insole region are obtained using a coordinate measuring machine (CMM). That surface similarly is defined by point cloud data. Mathematically, the individual CMM and scanner data sets are transformed into a single, common reference frame. Custom software then fits a polynomial surface to the insole data and extends it to intersect the mesh fitted to the outer surface point cloud. This line of intersection defines the required ESAU boundary, thus permitting further fractional area calculations to determine the percentage of materials present. With a draft method in place, and first-level method validation underway, we examine the transformation of the two dissimilar data sets into the single, common reference frame. We also will consider the six previously-identified potential error factors versus the method process. This paper reports our on-going work and discusses our findings to date.

  4. Gibberellic Acid-induced Phase Change in Hedera helix as Studied by Deoxyribonucleic Acid-Ribonucleic Acid Hybridization 1

    PubMed Central

    Rogler, Charles E.; Dahmus, Michael E.

    1974-01-01

    Applications of gibberellic acid to the mature form of Hedera helix induce morphological reversions to the juvenile form of growth. The juvenile forms produced are stable with time and differ dramatically from the mature in phenotype. DNA-RNA hybridization techniques have been used to study the RNA populations of juvenile, mature and gibberellic acid-treated mature apices. Hybridization competition experiments using RNA extracted by a hot phenol technique and uniformly labeled in vitro with 3H dimethylsulfate show no qualitative differences between the species of RNA present in juvenile and mature apices. However, differences are observed in the frequency distribution of RNA species using both uniformly labeled or pulse-labeled RNA as a reference. RNA extracted from gibberellic acid-treated mature buds was a less effective competitor than control mature RNA and the difference observed was comparable to that observed between mature and juvenile RNA. These results indicate that at least part of the molecular basis of phase change and gibberellic acid action may involve an alteration in the rate of transcription of certain genes in the apices of the mature form. RNA extracted using the hot phenol procedure contained a fraction of rapidly labeled RNA which was not extractable with cold phenol. When RNA extracted only with cold phenol was used in competition experiments sequences unique to the juvenile were detected and sequences unique to the mature were not detected. Implications of these results in relation to possible post-transcriptional control mechanisms are discussed. PMID:16658844

  5. Babesia gibsoni: detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis.

    PubMed

    Yamasaki, Masahiro; Kobayashi, Yusuke; Nakamura, Kensuke; Sasaki, Noboru; Murakami, Masahiro; Rajapakshage, Bandula Kumara Wickramasekara; Ohta, Hiroshi; Yamato, Osamu; Maede, Yoshimitsu; Takiguchi, Mitsuyoshi

    2011-01-01

    In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis. PMID:20637756

  6. Mfold web server for nucleic acid folding and hybridization prediction

    PubMed Central

    Zuker, Michael

    2003-01-01

    The abbreviated name, ‘mfold web server’, describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as ‘MFOLDROOT’. PMID:12824337

  7. Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.

    PubMed Central

    Woo, T H; Patel, B K; Smythe, L D; Symonds, M L; Norris, M A; Dohnt, M F

    1997-01-01

    Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected. PMID:9399509

  8. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

  9. Synthesis and Monolayer Behaviors of Novel Hybrid Corynomycolic Acids Containing Semifluoroalkyl Groups.

    PubMed

    Kawase, Tokuzo; Tamaki, Kazuki; Oida, Tatsuo

    2016-01-01

    In this work, novel hybrid-type corynomycolic acids [hybrid-OH and hybrid-COOH, with semifluoroalkyl groups (Rf-(CH2)n-: Rf = C4F9, n = 6 and Rf = C6F13, n = 3) located on the carbon atoms attached to the hydroxyl and carboxylic acid groups (C-OH and C-COOH), respectively] were successfully synthesized. The behaviors and formation of hybrid corynomycolic acid monolayers at the air-water interface were investigated by surface tension and surface pressure-area (π-A) measurements to clarify the effects of the Rf chain length, position of the semifluoroalkyl group, and surfactant molecule stereochemistry. Compared to dialkyl corynomycolic acid, both the critical micelle concentration (CMC) and the surface tension at the CMC (γCMC) of hybrid corynomycolic acids were reduced by the presence of the Rf group. With respect to the surface tension versus log concentration (γ vs. log C) isotherms, all syn-isomers of the hybrid-OH and hybrid-COOH acids showed two break points, while the anti-isomers showed only one break point. These different isotherms can be explained in terms of the steric repulsion between the two hydrophilic groups (OH and COO(-)), which depend on the stereochemistry of the surfactant. No effect of the location of the semifluoroalkyl group was observed. With respect to the formation of a monolayer film, four parameters-the lift-off area (AL), zero-pressure molecular area (A0), maximum of the Gibbs elastic modulus [EG (max)], and monolayer collapse pressure (πc)-were measured. Both AL and A0 of all hybrid corynomycolic acids were larger than the corresponding dialkyl acids due to the bulky and rigid Rf groups. Interestingly, syn- and anti-hybrids had almost identical isotherms on compression, although the values of πc of anti-hybrids were higher than those of syn-isomers. In addition, the values of EG (max) of hybrid-COOHs were slightly larger than those of the corresponding hybrid-OHs. Using the nascent soap method (agent-in-oil method), we found that

  10. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.

    PubMed

    Shokry, Ibrahim M; Callanan, John J; Sousa, John; Tao, Rui

    2015-01-01

    3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete. PMID:26437182

  11. Photodegradation and inhibition of drug-resistant influenza virus neuraminidase using anthraquinone-sialic acid hybrids.

    PubMed

    Aoki, Yusuke; Tanimoto, Shuho; Takahashi, Daisuke; Toshima, Kazunobu

    2013-02-11

    The anthraquinone-sialic acid hybrids designed effectively degraded not only non-drug-resistant neuraminidase but also drug-resistant neuraminidase, which is an important target of anti-influenza therapy. Degradation was achieved using long-wavelength UV radiation in the absence of any additives and under neutral conditions. Moreover, the hybrids efficiently inhibited neuraminidase activities upon photo-irradiation. PMID:23282898

  12. Catalytic performance of hybrid nanocatalyst for levulinic acid production from glucose

    NASA Astrophysics Data System (ADS)

    Ya'aini, Nazlina; Amin, Nor Aishah Saidina

    2012-11-01

    Levulinic acid is one of the potential and versatile biomass-derived chemicals. Product analysis via HPLC revealed that the heterogeneous dehydration of glucose over hybrid nanocatalyst exhibited better performance compared to single catalyst. Hybrid nanocatalyst containing H-Y zeolite and CrCl3 could substitute homogenous acid catalyst for attaining high levulinic acid yield. Different CrC3 and H-Y zeolite weight ratios of 1:1, 1:2 and 2:1 were prepared according to the wetness impregnation method. The hybrid catalyst with a 1:1 weight ratio performed better compared to others with the highest levulinic acid yield reported (93.5%) at 140 °C, 180 min reaction time, 0.1 g catalyst loading and 0.1 g glucose feed. Characterization results revealed that properties such as surface area, mesoporosity and acidic strength of the catalyst have significant effects on glucose dehydration for levulinic acid production.

  13. Chromosome painting in plants: in situ hybridization with a DNA probe from a specific microdissected chromosome arm of common wheat.

    PubMed Central

    Vega, M; Abbo, S; Feldman, M; Levy, A A

    1994-01-01

    We report here on the successful painting of a specific plant chromosome within its own genome. Isochromosomes for the long arm of chromosome 5 of the wheat B genome (5BL) were microdissected from first meiotic metaphase spreads of a monoisosomic 5BL line of the common wheat Triticum aestivum cv. Chinese Spring. The dissected isochromosomes were amplified by degenerate oligonucleotide-primed PCR in a single tube reaction. The amplified DNA was used as a complex probe mixture for fluorescent in situ hybridization on first meiotic metaphase spreads of lines carrying 5BL as a distinctive marker. Hybridization signals were observed, specifically, along the entire 5BL. In some of the cells, labeling was also detected in two bivalents, presumably those of the 5B "homoeologues" (partial homologues) found in common wheat (5A and 5D). The probe also revealed discrete domains in tapetal nuclei at interphase, further supporting the probe's high specificity. These data suggest that chromosome and homoeologous group-specific sequences are more abundant in 5BL than genome-specific sequences. Chromosome-painting probes, such as the one described here for 5BL, can facilitate the study of chromosome evolution in polyploid wheat. Images PMID:7991581

  14. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    PubMed

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. PMID:26879193

  15. Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH).

    PubMed

    Urzì, Clara; La Cono, Violetta; Stackebrandt, Erko

    2004-07-01

    Bacteria of the family of Geodermatophilaceae are actively involved in the decay processes [Urzì, C. and Realini, M. (1998) Int Biodeterior Biodegrad 42: 45-54; Urzì, C., Salamone, P., Schumann, P., and Stackebrandt, E. (2000) Int J Syst Evol Microbiol 50: 529-536] of stone monuments. Characterization of isolates includes phenotypic, chemotaxonomic and genetic analysis often requiring long-term procedures. The use of specific probes for members of Geodermatophilaceae family could be useful for the easy detection of those strains colonizing rock surfaces and involved in the biodeterioration. Two 16S rRNA-targeted oligonucleotide probes were designed for the specific detection of members of the family Geodermatophilaceae using fluorescence in situ hybridization (FISH); one probe specific for members of the two genera Geodermatophilus/Blastococcus and the second for members of the genus Modestobacter. PMID:15186346

  16. 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

  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. 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 ...

  19. Phagocytosis of hybrid molecular nanosomal compositions containing oxidized dextrans conjugated with isonicotinic acid hydrazide by macrophages.

    PubMed

    Shkurupy, V A; Arkhipov, S A; Troitsky, A V; Luzgina, N G; Zaikovskaja, M V; Ufimceva, E G; Iljine, D A; Akhramenko, E S; Gulyaeva, E P; Bistrova, T N

    2009-12-01

    We studied phagocytic activity of macrophages towards hybrid molecular nanosomal compositions consisting of 150-800-nm nanoliposomes containing oxidized dextrans with a molecular weight of 35 and 60 kDa obtained by chemical ("permanganate") and radiochemical oxidation of dextran conjugated with isonicotinic acid hydrazide (dextrazides, intracellular prolonged antituberculous drugs). Phagocytic activity of macrophages towards hybrid molecular nanosomal compositions containing dextrazides obtained by chemical oxidation of dextrans is higher than activity towards hybrid molecular nanosomal compositions containing dextrazides prepared by radiochemical oxidation and depends on the size of hybrid molecular nanosomal compositions and molecular weight of oxidized dextrans. PMID:21116494

  20. Label-free nucleic acids detection based on DNA templated silver nanoclusters fluorescent probe.

    PubMed

    Zhao, Haiyan; Wang, Lei; Zhu, Jing; Wei, Haiping; Jiang, Wei

    2015-06-01

    Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3'-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3'-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10(-10) mol L(-1) HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples. PMID:25863386

  1. Rapid detection of sequence variation in Clostridium difficile genes using LATE-PCR with multiple mismatch-tolerant hybridization probes.

    PubMed

    Pierce, Kenneth E; Khan, Huma; Mistry, Rohit; Goldenberg, Simon D; French, Gary L; Wangh, Lawrence J

    2012-11-01

    A novel molecular assay for Clostridium difficile was developed using Linear-After-The-Exponential polymerase chain reaction (LATE-PCR). Single-stranded DNA products generated by LATE-PCR were detected and distinguished by hybridization to fluorescent mismatch-tolerant probes, as the temperature was lowered after amplification in 5(°)C intervals between 65°C and 25°C. Single-tube multiplex reactions for tcdA, tcdB, tcdC, and cdtB (binary toxin) sequences were initially optimized using synthetic targets and were subsequently done using genomic DNA; each target was detected and characterized by hybridization to one or more probes of a different fluorescent color. In the case of tcdC, three probes, each labeled with a Quasar fluorophore, hybridize to different locations with known mutations, including the deletion at nucleotide 117 in ribotype 027 strains and the premature stop codon mutation at nucleotide 184 in ribotype 078 strains, each of which is associated with hypervirulent infections. These and other tcdC mutations were distinguished from the reference sequence, as well as from each other by changes in the fluorescent contour generated from the combined Quasar-labeled probes. Specific variations in tcdA and tcdB were also identified in the multiplex assay, including those that identified strains lacking toxin A production. This single closed-tube assay generates substantially more information about virulent C. difficile than currently available commercial assays and could be further expanded to provide strain typing. PMID:22982259

  2. Lead-acid batteries in micro-hybrid vehicles

    NASA Astrophysics Data System (ADS)

    Albers, Joern; Meissner, Eberhard; Shirazi, Sepehr

    More and more vehicles hit the European automotive market, which comprise some type of micro-hybrid functionality to improve fuel efficiency and reduce emissions. Most carmakers already offer at least one of their vehicles with an optional engine start/stop system, while some other models are sold with micro-hybrid functions implemented by default. But these car concepts show a wide variety in detail-the term "micro-hybrid" may mean a completely different functionality in one vehicle model compared to another. Accordingly, also the battery technologies are not the same. There is a wide variety of batteries from standard flooded and enhanced flooded to AGM which all are claimed to be "best choice" for micro-hybrid applications. A technical comparison of micro-hybrid cars available on the European market has been performed. Different classes of cars with different characteristics have been identified. Depending on the scope and characteristics of micro-hybrid functions, as well as on operational strategies implemented by the vehicle makers, the battery operating duties differ significantly between these classes of vehicles. Additional laboratory investigations have been carried out to develop an understanding of effects observed in batteries operated in micro-hybrid vehicles pursuing different strategies, to identify limitations for applications of different battery technologies.

  3. Optimization of levulinic acid from lignocellulosic biomass using a new hybrid catalyst.

    PubMed

    Ya'aini, Nazlina; Amin, Nor Aishah Saidina; Asmadi, Mohd

    2012-07-01

    Conversion of glucose, empty fruit bunch (efb) and kenaf to levulinic acid over a new hybrid catalyst has been investigated in this study. The characterization and catalytic performance results revealed that the physico-chemical properties of the new hybrid catalyst comprised of chromium chloride and HY zeolite increased the levulinic acid production from glucose compared to the parent catalysts. Optimization of the glucose conversion process using two level full factorial designs (2(3)) with two center points reported 55.2% of levulinic acid yield at 145.2 °C, 146.7 min and 12.0% of reaction temperature, reaction time and catalyst loading, respectively. Subsequently, the potential of efb and kenaf for producing levulinic acid at the optimum conditions was established after 53.2% and 66.1% of efficiencies were reported. The observation suggests that the hybrid catalyst has a potential to be used in biomass conversion to levulinic acid. PMID:22609656

  4. Modeling aqueous ozone/UV process using oxalic acid as probe chemical.

    PubMed

    Garoma, Temesgen; Gurol, Mirat D

    2005-10-15

    A kinetic model that describes the removal of organic pollutants by an ozone/UV process is described. Oxalic acid, which reacts with a very low rate constant with ozone and relatively high rate constant with hydroxyl radical (OH*), was used as the probe chemical to model the process. The model was verified by experimental data on concentrations of oxalic acid and hydrogen peroxide (H202) under various experimental conditions, i.e., ozone gas dosage, UV light intensity, and varying oxalic acid concentrations. PMID:16295862

  5. Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

    PubMed Central

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

    2010-01-01

    Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging. PMID:20885944

  6. Visualization and enumeration of marine planktonic archaea and bacteria by using polyribonucleotide probes and fluorescent in situ hybridization.

    PubMed

    DeLong, E F; Taylor, L T; Marsh, T L; Preston, C M

    1999-12-01

    Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4',6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50-56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10(5)/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or "ghosts," as was suggested in a previous report. PMID:10584017

  7. Emission wavelength-dependent decay of the 9-anthroyloxy-fatty acid membrane probes.

    PubMed Central

    Matayoshi, E D; Kleinfeld, A M

    1981-01-01

    Using the phase-modulation technique, we have measured the fluorescence decay of 2- and 12-(9-anthroyloxy)-stearic acid (2- and 12-AS) and 16-(9-anthroyloxy)-palmitic acid (16-AP) bound to egg phosphatidylcholine vesicles or dissolved in nonpolar solvents. Heterogeneity analysis demonstrates that the decay is generally not monoexponential and exhibits large component variations across it emission spectrum. The mean decay time increases (and in parallel, the steady-state polarization decreases) monotonically with increasing wavelength from values at the blue end. The decay at the red side of the emission spectrum contains an exponential term with a negative amplitude, indicating that emission occurs from intermediates created in the excited-state. This behavior is interpreted as arising from intramolecular fluorophore relaxation occurring on the time scale of the fluorescence lifetime. We believe this to be the first study of wavelength-dependent fluorescent emission which is dominated by an intramolecular relaxation process. Although the three probes exhibit qualitatively similar effects, the emission band variations are greatest for 2-AS and smallest for 16-AP. The differences among the probes are not entirely due to environmental factors as demonstrated, for example, by the emission polarization differences observed in the isotropic solvent paraffin oil. In summary, while these findings point out some of the complexities in the 9-anthroyloxy-fatty acids as membrane probes, they also indicate how these complexities might be used as a sensitive measure of lipid-probe interaction. PMID:7260317

  8. Insights into 6‐Methylsalicylic Acid Bio‐assembly by Using Chemical Probes

    PubMed Central

    Parascandolo, James S.; Havemann, Judith; Potter, Helen K.; Huang, Fanglu; Riva, Elena; Connolly, Jack; Wilkening, Ina; Song, Lijiang; Leadlay, Peter F.

    2016-01-01

    Abstract Chemical probes capable of reacting with KS (ketosynthase)‐bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6‐methylsalicylic acid synthase (6‐MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6‐MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6‐MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.

  9. Molecular imaging of biothiols and in vitro diagnostics based on an organic chromophore bearing a terbium hybrid probe.

    PubMed

    Zhou, Zhan; Wang, Qianming; Zhang, Cheng Cheng; Gao, Jinwei

    2016-04-25

    In this research, a novel terbium-based luminescent hybrid inorganic/organic probe was designed and synthesized. Mesoporous silica nanospheres dispersed in water were used as the appropriate host for the covalently linked lanthanide-containing organic structures. The lanthanide structure was linked to a sulfonate ester unit, which, in the presence of biothiols, was cleaved to result in terbium emission. The hybrid probe exhibited the capabilities of quantitative determination and detection limits for biothiols were presented (36.8 nM for Cys, 32.5 nM for GSH, and 34.7 nM for Hcy). Evaluation of luminescence changes in cell culture demonstrated that this smart probe is cell membrane permeable and selectively lights up in the presence of cysteine and glutathione in human embryonic kidney cells and human lung adenocarcinoma cells. This variation in the presence of biothiols can be controlled by the treatment with N-methylmaleimide. The narrow line-like bands and long-lived excited states of this terbium luminescent sensor allows the discrimination of scattering signals and interfering fluorescence derived from biological tissues. PMID:27041001

  10. A method for preventing artifactual binding of cRNA probes to neurons caused by in situ hybridization.

    PubMed

    Blödorn, B; Brück, W; Rieckmann, P; Felgenhauer, K; Mäder, M

    1998-01-01

    When in situ hybridization was used for the detection of mRNA for the beta-trace protein (beta-trace; prostaglandin-D-synthase) in sections of rat and porcine brains, unspecific binding reactions of sense and antisense probes to neurons were observed. The beta-trace fragment which served as a template for the synthesis of cRNA probes was blunt end-cloned in the vector pCR-Script SK (+). It was demonstrated that the unspecific signals were caused by artifactual binding of two portions of the cRNA which correspond to sequences of the multicloning site of this vector. These sequences are localized between the SrfI restriction site (or the insert) and the promoter for the T7 RNA polymerase. Thus, artifactual binding could be prevented using riboprobes synthesized by T3 RNA polymerase instead of T7 RNA polymerase. Because of the relatively weak transcription efficiency of T3 RNA polymerase, as compared with T7 RNA polymerase, a blocking procedure was established which allowed successful in situ hybridization with T7 RNA polymerase-synthesized probes. Blocking was performed using synthetic oligonucleotides deduced from the two sequences of the multicloning site which were found to be responsible for artifactual binding. PMID:9448846

  11. Producing a trimethylpentanoic acid using hybrid polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2014-10-07

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

  12. Resonant uv pump-probe spectroscopy of dipicolinic acid via impulsive excitation

    SciTech Connect

    Murawski, Robert K.; Rostovtsev, Yuri V.; Sariyanni, Zoe-Elizabeth; Sautenkov, Vladimir A.; Backus, Sterling; Raymondson, Daisy; Kapteyn, Henry C.; Murnane, Margaret M.; Scully, Marlan O.

    2008-02-15

    We present experimental evidence of coherent wave packet motion in dipicolinic acid (C{sub 7}H{sub 5}NO{sub 4}) which is an important marker molecule for bacterial spores. Resonant impulsive excitation is achieved by applying a uv pump pulse (267 nm, 16 fs) which has a duration that is shorter than the vibrational period of the molecules. The resulting dynamics is then probed with a weaker pulse of the same width and frequency. Evidence of the important 'fingerprint' region for this molecule (between 1000 cm{sup -1} and 1500 cm{sup -1}) is found in the transient absorption of the probe. We present simulations of the pump-probe experiment, based on the Liouville equation for the density matrix, and predict the optimal pulse width and detuning.

  13. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    NASA Astrophysics Data System (ADS)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  14. Nucleic acid hybridization with RNA immobilized on filter paper.

    NASA Technical Reports Server (NTRS)

    Saxinger, W. C.; Ponnamperuma, C.; Gillespie, D.

    1972-01-01

    RNA has been immobilized in a manner suitable for use in molecular hybridization experiments with dissolved RNA or DNA by a nonaqueous solid-phase reaction with carbonyldiimidazole and RNA 'dry coated' on cellulose or, preferably, on previously activated phosphocellulose filters. Immobilization of RNA does not appear to alter its chemical character or cause it to acquire affinity for unspecific RNA or DNA. The versatility and efficiency of this method make it potentially attractive for use in routine analytical or preparative hybridization experiments, among other applications.

  15. Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization.

    PubMed

    Yang, G; Matocha, M F; Rapoport, S I

    1988-08-01

    An in situ hybridization procedure was applied to quantify glucocorticoid receptor (GR) mRNAs in the hippocampus of rat brain. Hybridization was carried out using a radiolabeled antisense probe complementary to the rat liver GR gene. The specificity of the method was validated by showing: 1) a high cellular grain density in sections hybridized with an antisense but not a sense probe; 2) agreement between the experimental and theoretical temperature at which 50% of the hybrids melted, and 3) a high signal distribution of GR mRNA in the hippocampus, a region of brain known to preferentially concentrate steroid hormones. Within the hippocampus, however, subregional differences in hybridization densities were observed. Quantitative autoradiography indicated that the average neuronal silver grain number was highest in the pyramidal cell layers of CA2 and CA4 and lowest in those of CA1 and CA3. Also, there was a significant difference in the average grain number between all of the cell fields except for that between CA2 and CA4. These results show that contiguous but neuroanatomically distinct cell fields of the hippocampus express different levels of GR transcripts, and indicate that differential regulation of GR expression occurs in subpopulations of hippocampal neurons. PMID:3211154

  16. Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization

    SciTech Connect

    Yang, G.; Matocha, M.F.; Rapoport, S.I.

    1988-08-01

    An in situ hybridization procedure was applied to quantify glucocorticoid receptor (GR) mRNAs in the hippocampus of rat brain. Hybridization was carried out using a radiolabeled antisense probe complementary to the rat liver GR gene. The specificity of the method was validated by showing: 1) a high cellular grain density in sections hybridized with an antisense but not a sense probe; 2) agreement between the experimental and theoretical temperature at which 50% of the hybrids melted, and 3) a high signal distribution of GR mRNA in the hippocampus, a region of brain known to preferentially concentrate steroid hormones. Within the hippocampus, however, subregional differences in hybridization densities were observed. Quantitative autoradiography indicated that the average neuronal silver grain number was highest in the pyramidal cell layers of CA2 and CA4 and lowest in those of CA1 and CA3. Also, there was a significant difference in the average grain number between all of the cell fields except for that between CA2 and CA4. These results show that contiguous but neuroanatomically distinct cell fields of the hippocampus express different levels of GR transcripts, and indicate that differential regulation of GR expression occurs in subpopulations of hippocampal neurons.

  17. Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system

    PubMed Central

    Liew, Michael; Rowe, Leslie; Clement, Parker W.; Miles, Rodney R.; Salama, Mohamed E.

    2016-01-01

    Introduction: Detection of MYC translocations using fluorescence in situ hybridization (FISH) is important in the evaluation of lymphomas, in particular, Burkitt lymphoma and diffuse large B-cell lymphoma. Our aim was to validate a digital FISH capture and imaging system for the detection of MYC 8q24 translocations using LSI-MYC (a break-apart probe) and MYC 8;14 translocation using IGH-MYC (a fusion probe). Materials and Methods: LSI-MYC probe was evaluated using tissue sections from 35 patients. IGH-MYC probe was evaluated using tissue sections from forty patients. Sections were processed for FISH and analyzed using traditional methods. FISH slides were then analyzed using the GenASIs capture and analysis system. Results: Results for LSI-MYC had a high degree of correlation between traditional method of FISH analysis and digital FISH analysis. Results for IGH-MYC had a 100% concordance between traditional method of FISH analysis and digital FISH analysis. Conclusion: Annotated whole slide images of H and E and FISH sections can be digitally aligned, so that areas of tumor within a section can be matched and evaluated with a greater degree of accuracy. Images can be archived permanently, providing a means for examining the results retrospectively. Digital FISH imaging of the MYC translocations provides a better diagnostic tool compared to traditional methods for evaluating lymphomas. PMID:27217970

  18. 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.

  19. Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe.

    PubMed Central

    Tevere, V J; Hewitt, P L; Dare, A; Hocknell, P; Keen, A; Spadoro, J P; Young, K K

    1996-01-01

    Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections. PMID:8815108

  20. Design and Synthesis of Novel Isoxazole Tethered Quinone-Amino Acid Hybrids

    PubMed Central

    Ravi Kumar, P.; Sambaiah, M.; Kandula, Venu; Payili, Nagaraju; Jaya Shree, A.; Yennam, Satyanarayana

    2014-01-01

    A new series of isoxazole tethered quinone-amino acid hybrids has been designed and synthesized involving 1,3-dipolar cycloaddition reaction followed by an oxidation reaction using cerium ammonium nitrate (CAN). Using this method, for the first time various isoxazole tethered quinone-phenyl alanine and quinone-alanine hybrids were synthesized from simple commercially available 4-bromobenzyl bromide, propargyl bromide, and 2,5-dimethoxybenzaldehyde in good yield. PMID:25709839

  1. Reactivity, Selectivity, and Stability in Sulfenic Acid Detection: A Comparative Study of Nucleophilic and Electrophilic Probes.

    PubMed

    Gupta, Vinayak; Paritala, Hanumantharao; Carroll, Kate S

    2016-05-18

    The comparative reaction efficiencies of currently used nucleophilic and electrophilic probes toward cysteine sulfenic acid have been thoroughly evaluated in two different settings-(i) a small molecule dipeptide based model and (ii) a recombinant protein model. We further evaluated the stability of corresponding thioether and sulfoxide adducts under reducing conditions which are commonly encountered during proteomic protocols and in cell analysis. Powered by the development of new cyclic and linear C-nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under competitive conditions is achieved and thus holds great promise as highly potent tools for activity-based sulfenome profiling. PMID:27123991

  2. The chromatin remodeler DDM1 promotes hybrid vigor by regulating salicylic acid metabolism.

    PubMed

    Zhang, Qingzhu; Li, Yanqiang; Xu, Tao; Srivastava, Ashish Kumar; Wang, Dong; Zeng, Liang; Yang, Lan; He, Li; Zhang, Heng; Zheng, Zhimin; Yang, Dong-Lei; Zhao, Cheng; Dong, Juan; Gong, Zhizhong; Liu, Renyi; Zhu, Jian-Kang

    2016-01-01

    In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism. Interestingly, our data suggest that salicylic acid is a hormetic regulator of seedling growth heterosis, and that hybrid vigor arises from crosses that produce optimal salicylic acid levels. Although DNA methylation failed to correlate with differential non-additively expressed gene expression, we uncovered DDM1 as an epigenetic link between salicylic acid metabolism and heterosis, and propose that the endogenous salicylic acid levels of parental plants can be used to predict the heterotic outcome. Salicylic acid protects plants from pathogens and abiotic stress. Thus, our findings suggest that stress-induced hormesis, which has been associated with increased longevity in other organisms, may underlie specific hybrid vigor traits. PMID:27551435

  3. The chromatin remodeler DDM1 promotes hybrid vigor by regulating salicylic acid metabolism

    PubMed Central

    Zhang, Qingzhu; Li, Yanqiang; Xu, Tao; Srivastava, Ashish Kumar; Wang, Dong; Zeng, Liang; Yang, Lan; He, Li; Zhang, Heng; Zheng, Zhimin; Yang, Dong-Lei; Zhao, Cheng; Dong, Juan; Gong, Zhizhong; Liu, Renyi; Zhu, Jian-Kang

    2016-01-01

    In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism. Interestingly, our data suggest that salicylic acid is a hormetic regulator of seedling growth heterosis, and that hybrid vigor arises from crosses that produce optimal salicylic acid levels. Although DNA methylation failed to correlate with differential non-additively expressed gene expression, we uncovered DDM1 as an epigenetic link between salicylic acid metabolism and heterosis, and propose that the endogenous salicylic acid levels of parental plants can be used to predict the heterotic outcome. Salicylic acid protects plants from pathogens and abiotic stress. Thus, our findings suggest that stress-induced hormesis, which has been associated with increased longevity in other organisms, may underlie specific hybrid vigor traits. PMID:27551435

  4. Nanofiltration, bipolar electrodialysis and reactive extraction hybrid system for separation of fumaric acid from fermentation broth.

    PubMed

    Prochaska, Krystyna; Staszak, Katarzyna; Woźniak-Budych, Marta Joanna; Regel-Rosocka, Magdalena; Adamczak, Michalina; Wiśniewski, Maciej; Staniewski, Jacek

    2014-09-01

    A novel approach based on a hybrid system allowing nanofiltration, bipolar electrodialysis and reactive extraction, was proposed to remove fumaric acid from fermentation broth left after bioconversion of glycerol. The fumaric salts can be concentrated in the nanofiltration process to a high yield (80-95% depending on pressure), fumaric acid can be selectively separated from other fermentation components, as well as sodium fumarate can be conversed into the acid form in bipolar electrodialysis process (stack consists of bipolar and anion-exchange membranes). Reactive extraction with quaternary ammonium chloride (Aliquat 336) or alkylphosphine oxides (Cyanex 923) solutions (yield between 60% and 98%) was applied as the final step for fumaric acid recovery from aqueous streams after the membrane techniques. The hybrid system permitting nanofiltration, bipolar electrodialysis and reactive extraction was found effective for recovery of fumaric acid from the fermentation broth. PMID:24983693

  5. Evaluation of hybridization efficiencies of centromeric probes for chromosomes X, Y, 18 and 13/21 on uncultured amniocytes by a `rapid one-step` fluorescence in situ hybridization

    SciTech Connect

    Prashad, N.; Cubbage, M.; Weber, W.

    1994-09-01

    Aprogenex has developed a rapid one-step FISH assay, KaryoSite{trademark}, for the detection of chromosomes X, Y, 13, 18 and 21. The chromosome probes used are synthetic oligonucleotides with covalently attached chromosomes. In combination with a unique hybridization cocktail, these chromosomes are detected in a one-step hybridization. This method allows the detection of aneuploidy for these five chromosomes in uncultured amniocytes as well as metaphase chromosome preparations. Hybridization efficiency of dual probe combinations, X-Rhodamine plus Y-FITC and 18-FITC plus 13/21-Rhodamine, was determined on 50 amniocyte samples. Probes were added to the cells on slides and target cellular DNA was denatured at 90{degrees}C for 5 minutes followed by hybridization at 42{degrees}C for 30 minutes. Hybridization efficiency for SY probes was greater than 95% and for 18-13/21 probes was greater than 90%. Aneuploidy data from amniocyte samples will be presented. Aprogenex`s FISH technology is a reliable and rapid one-step method for prenatal diagnosis of aneuploidy for chromosome X, Y, 13, 18 and 21 in uncultured amniocytes.

  6. A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

    PubMed Central

    2012-01-01

    Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These

  7. Formation of a hybrid plasmonic waveguide mode probed by dispersion measurement

    SciTech Connect

    Saito, H.; Kurata, H.

    2015-04-07

    Hybrid waveguides, i.e., dielectric waveguides combined with plasmonic waveguides, have great potential for concomitantly exhibiting subwavelength confinement and long range propagation, enabling a highly integrated photonic circuit. We report the characterization of hybrid waveguide modes excited in Si/SiO{sub 2}/Al films, by dispersion measurement using angle-resolved electron energy-loss spectroscopy. This experiment directly verifies the formation of the hybrid waveguide mode with a strongly localized electromagnetic field in a 6-nm-thick SiO{sub 2} layer. The results clearly describe the characteristic behavior of the hybrid waveguide mode, which depends on the effective index of the constituent dielectric waveguide and the surface plasmon-polariton modes.

  8. Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

    PubMed Central

    Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2015-01-01

    Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

  9. Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

    PubMed

    Ho, M S; Barr, B C; Tarantal, A F; Lai, L T; Hendrickx, A G; Marsh, A E; Sverlow, K W; Packham, A E; Conrad, P A

    1997-07-01

    Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora. PMID:9196184

  10. Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid Agent Development

    PubMed Central

    Seibold, Uwe; Wängler, Björn; Schirrmacher, Ralf; Wängler, Carmen

    2014-01-01

    Molecular imaging—and especially positron emission tomography (PET)—has gained increasing importance for diagnosis of various diseases and thus experiences an increasing dissemination. Therefore, there is also a growing demand for highly affine PET tracers specifically accumulating and visualizing target structures in the human body. Beyond the development of agents suitable for PET alone, recent tendencies aim at the synthesis of bimodal imaging probes applicable in PET as well as optical imaging (OI), as this combination of modalities can provide clinical advantages. PET, due to the high tissue penetration of the γ-radiation emitted by PET nuclides, allows a quantitative imaging able to identify and visualize tumors and metastases in the whole body. OI on the contrary visualizes photons exhibiting only a limited tissue penetration but enables the identification of tumor margins and infected lymph nodes during surgery without bearing a radiation burden for the surgeon. Thus, there is an emerging interest in bimodal agents for PET and OI in order to exploit the potential of both imaging techniques for the imaging and treatment of tumor diseases. This short review summarizes the available hybrid probes developed for dual PET and OI and discusses future directions for hybrid agent development. PMID:24822177

  11. Bimodal imaging probes for combined PET and OI: recent developments and future directions for hybrid agent development.

    PubMed

    Seibold, Uwe; Wängler, Björn; Schirrmacher, Ralf; Wängler, Carmen

    2014-01-01

    Molecular imaging--and especially positron emission tomography (PET)--has gained increasing importance for diagnosis of various diseases and thus experiences an increasing dissemination. Therefore, there is also a growing demand for highly affine PET tracers specifically accumulating and visualizing target structures in the human body. Beyond the development of agents suitable for PET alone, recent tendencies aim at the synthesis of bimodal imaging probes applicable in PET as well as optical imaging (OI), as this combination of modalities can provide clinical advantages. PET, due to the high tissue penetration of the γ-radiation emitted by PET nuclides, allows a quantitative imaging able to identify and visualize tumors and metastases in the whole body. OI on the contrary visualizes photons exhibiting only a limited tissue penetration but enables the identification of tumor margins and infected lymph nodes during surgery without bearing a radiation burden for the surgeon. Thus, there is an emerging interest in bimodal agents for PET and OI in order to exploit the potential of both imaging techniques for the imaging and treatment of tumor diseases. This short review summarizes the available hybrid probes developed for dual PET and OI and discusses future directions for hybrid agent development. PMID:24822177

  12. Development of a new colorimetric and red-emitting fluorescent dual probe for G-quadruplex nucleic acids.

    PubMed

    Yan, Jin-Wu; Chen, Shuo-Bin; Liu, Hui-Yun; Ye, Wen-Jie; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

    2014-07-01

    A tailor-made colorimetric and red-emitting fluorescent dual probe for G-quadruplex nucleic acids was developed by incorporating a coumarin-hemicyanine fluorophore into an isaindigotone framework. The significant and distinct changes in both the color and fluorescence of this probe enable the label-free and visual detection of G-quadruplex structures. PMID:24841696

  13. Quantitative determination of uric acid using CdTe nanoparticles as fluorescence probes.

    PubMed

    Jin, Dongri; Seo, Min-Ho; Huy, Bui The; Pham, Quoc-Thai; Conte, Maxwell L; Thangadurai, Daniel; Lee, Yong-Ill

    2016-03-15

    A convenient enzymatic optical method for uric acid detection was developed based on the fluorescence quenching of ligand-capped CdTe nanoparticles by H2O2 which was generated from the enzymatic reaction of uric acid. The interactions between the CdTe nanoparticles capped with different ligands (glutathione, 3-mercaptopropionic acid, and thioglycerol) and H2O2 were investigated. The fluorescence quenching studies of GSH-capped CdTe nanoparticles demonstrated an excellent sensitivity to H2O2. The effects of uric acid, uricase and H2O2 on the fluorescence intensity of CdTe nanoparticles were also explored. The detection conditions, reaction time, pH value, incubation period and the concentration of uricase and uric acid were optimized. The detection limit of uric acid was found to be 0.10 µM and the linear range was 0.22-6 µM under the optimized experimental conditions. These results typify that CdTe nanoparticles could be used as a fluorescent probe for uric acid detection. PMID:26433069

  14. Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus

    SciTech Connect

    Uhl, G.R.; Zingg, H.H.; Habener, J.F.

    1985-08-01

    Hybridizable vasopressin mRNA may be quantitatively localized in situ in sections from rat hypothalamus. Radiolabeled oligonucleotide cDNA probes, synthesized by chemical and enzymatic means, provide strong hybridization in zones known to contain vasopressin cell bodies. Multiple single-stranded /sup 32/P-, /sup 35/S-, or /sup 3/H-labeled oligonucleotides demonstrate localized hybridization that increases as probes are lengthened from 8 to 75 bases. Competition studies, RNase experiments, anatomic localization, and use of multiple probes all support hybridization specificity. An approximate doubling of hybridizable mRNA in both supraoptic and paraventricular nuclei can be detected with dehydration of the animals. Hybridizable mRNA densities are virtually normal in hypothalamic nuclei of Brattleboro rats given free access to water. These methods can provide insight into regional mRNA dynamics and may reflect functional activity of peptidergic neurons.

  15. Zebrafish Whole-Mount In Situ Hybridization Followed by Sectioning.

    PubMed

    Doganli, Canan; Nyengaard, Jens Randel; Lykke-Hartmann, Karin

    2016-01-01

    In situ hybridization is a powerful technique used for locating specific nucleic acid targets within morphologically preserved tissues and cell preparations. A labeled RNA or DNA probe hybridizes to its complementary mRNA or DNA sequence within a sample. Here, we describe RNA in situ hybridization protocol for whole-mount zebrafish embryos. PMID:26695046

  16. Indole-3-acetic acid biosensor based on G-rich DNA labeled AuNPs as chemiluminescence probe coupling the DNA signal amplification

    NASA Astrophysics Data System (ADS)

    Hun, Xu; Mei, Zhenghua; Wang, Zhouping; He, Yunhua

    2012-09-01

    A highly sensitive chemiluminescence (CL) method for detection of phytohormone indole-3-acetic acid (IAA) was developed by using G-rich DNA labeled gold nanoparticles (AuNPs) as CL probe coupling the DNA signal amplification technology. The IAA antibody was immobilized on carboxyl terminated magnetic beads (MBs). In the presence of IAA, antibody labeled AuNPs were captured by antibody functionalized MBs. The DNA on AuNPs is released by a ligand exchange process induced by the addition of DTT. The released DNA is then acted as the linker and hybridized with the capture DNA on MBs and probe DNA on AuNPs CL probe. The CL signal is obtained via the instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG), and the G-rich DNA on AuNPs CL probe. IAA can be detected in the concentration range from 0.02 ng/mL to 30 ng/mL, and the limit of detection is 0.01 ng/mL.

  17. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    PubMed

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions. PMID:24310436

  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. 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.

  20. 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

  1. 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.

  2. Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

    NASA Astrophysics Data System (ADS)

    Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

    2016-05-01

    A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

  3. Bioactivity in silica/poly(γ-glutamic acid) sol-gel hybrids through calcium chelation.

    PubMed

    Valliant, Esther M; Romer, Frederik; Wang, Daming; McPhail, David S; Smith, Mark E; Hanna, John V; Jones, Julian R

    2013-08-01

    Bioactive glasses and inorganic/organic hybrids have great potential as biomedical implant materials. Sol-gel hybrids with interpenetrating networks of silica and biodegradable polymers can combine the bioactive properties of a glass with the toughness of a polymer. However, traditional calcium sources such as calcium nitrate and calcium chloride are unsuitable for hybrids. In this study calcium was incorporated by chelation to the polymer component. The calcium salt form of poly(γ-glutamic acid) (γCaPGA) was synthesized for use as both a calcium source and as the biodegradable toughening component of the hybrids. Hybrids of 40wt.% γCaPGA were successfully formed and had fine scale integration of Ca and Si ions, according to secondary ion mass spectrometry imaging, indicating a homogeneous distribution of organic and inorganic components. (29)Si magic angle spinning nuclear magnetic resonance data demonstrated that the network connectivity was unaltered with changing polymer molecular weight, as there was no perturbation to the overall Si speciation and silica network formation. Upon immersion in simulated body fluid a hydroxycarbonate apatite surface layer formed on the hybrids within 1week. The polymer molecular weight (Mw 30-120kDa) affected the mechanical properties of the resulting hybrids, but all hybrids had large strains to failure, >26%, and compressive strengths, in excess of 300MPa. The large strain to failure values showed that γCaPGA hybrids exhibited non-brittle behaviour whilst also incorporating calcium. Thus calcium incorporation by chelation to the polymer component is justified as a novel approach in hybrids for biomedical materials. PMID:23632373

  4. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    SciTech Connect

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. )

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  5. 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.

  6. Insights into 6‐Methylsalicylic Acid Bio‐assembly by Using Chemical Probes

    PubMed Central

    Parascandolo, James S.; Havemann, Judith; Potter, Helen K.; Huang, Fanglu; Riva, Elena; Connolly, Jack; Wilkening, Ina; Song, Lijiang; Leadlay, Peter F.

    2016-01-01

    Abstract Chemical probes capable of reacting with KS (ketosynthase)‐bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6‐methylsalicylic acid synthase (6‐MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6‐MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6‐MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities. PMID:26833898

  7. Use of enrichments and nucleic acid probes in monitoring bioremediation of a deep trichloroethylene plume

    SciTech Connect

    Brockman, F.; Payne, W.; Workman, D.; Soong, A.; Manley, S.; Sun, W.; Ogram, A.

    1994-12-31

    The field site was manipulated with injection of air (control experiment), 1% methane (in air), pulsing of air only and 4% methane, and pulsing of 4% methane supplemented with gaseous forms of nitrogen and phosphorus. Gases were injected through a horizontal well into the aquifer and a vacuum was established in a second horizontal well in the vadose zone. Following each injection regime, sediment samples from the contaminated region were analyzed. Analyses included most-probable-number enrichments for physiological groups known or suspected to degrade trichloroethylene (TCE) and per-chloroethylene (PCE), TCE and PCE removal from enrichments, and DNA extraction and hybridization with various gene probes corresponding to enzymes known to degrade TCE or TCE metabolites.

  8. Photoluminescence and quantum yields of organic/inorganic hybrids prepared through formic acid solvolysis

    NASA Astrophysics Data System (ADS)

    Fu, Lianshe; Sá Ferreira, R. A.; Fernandes, M.; Nunes, S. C.; de Zea Bermudez, V.; Hungerford, Graham; Rocha, J.; Carlos, L. D.

    2008-03-01

    Three undoped di-urea cross-linked poly(oxyethylene) (POE)/siloxane hybrid matrices, classed as di-ureasils, incorporating POE segments with different lengths were prepared through the carboxylic acid solvolysis sol-gel method using formic acid. The resulting hybrids were characterized by X-ray diffraction, Fourier transform mid-infrared spectroscopy, 29Si and cross-polarization 13C magic-angle spinning nuclear magnetic resonance and photoluminescence spectroscopy. The hybrids' structure is essentially independent of the polymer chain length and the materials are room temperature white-light emitters with emission quantum yields of ˜10 ± 1% and lifetime average values between 2 and 4 ns. For the di-ureasil host with short polymer chains the solvolysis method favours the increase of the PL quantum yields relatively to conventional sol-gel route.

  9. Fluorescence Probe Based on Hybrid Mesoporous Silica/Quantum Dot/Molecularly Imprinted Polymer for Detection of Tetracycline.

    PubMed

    Zhang, Liang; Chen, Ligang

    2016-06-29

    A newly designed fluorescence probe made from a hybrid quantum dot/mesoporous silica/molecularly imprinted polymer (QD/MS/MIP) was successfully created, and the probe was used for the detection of tetracycline (TC) in serum sample. QD/MS/MIP was characterized by transmission electron microscope, Fourier transform infrared spectroscopy, UV spectroscopy, X-ray powder diffraction, nitrogen adsorption-desorption experiment and fluorescence spectroscopy. Tetracycline, which is a type of broad-spectrum antibiotic, was selected as the template. The monomer and the template were combined by covalent bonds. After the template was removed to form a binding site, a hydrogen bonding interaction formed between the hole and the target molecule. Moreover, when rebinding TC, a new complex was produced between the amino group of QD/MS/MIP and the hydroxyl group of TC. After that, the energy of the QDs could transfer to the complex, which explains the fluorescence quenching phenomenon. The fluorescent intensity of QD/MS/MIP decreased in 10 min, and an excellent linearity from 50 to 1000 ng mL(-1) was correspondingly obtained. This composite material has a high selectivity with an imprinting factor of 6.71. In addition, the confirmed probe strategy was successfully applied to serum sample analyses, and the recoveries were 90.2%-97.2% with relative standard deviations of 2.2%-5.7%. This current work offers a novel and suitable method to synthesize QD/MS/MIP with a highly selective recognition ability. This composite material will be valuable for use in fluorescence probe applications. PMID:27280785

  10. Nucleic Acid Hybridization Studies within the Genus Cucurbita

    PubMed Central

    Goldberg, Robert B.; Bemis, William P.; Siegel, Albert

    1972-01-01

    The DNAs of Cucurbita species were examined by several methods. All Cucurbita DNAs have a similar CsCl isopycnic banding pattern consisting of a major band at 1.695 g/cc and a well separated satellite band at 1.707 g/cc. Compared to other plant and animal genera, Cucurbita species have a large genomic proportion of rDNA; this value ranging from 1.4% to 3.1%. The genomic proportion of rDNA was found not to be useful as a characteristic indicating degree of relatedness of the various Cucurbita species. However, Cucurbita DNAs can be distinguished by the extent to which their repetitive sequences cross-hybridize to each other and an assessment of species relationships can be made on this basis. PMID:17248582

  11. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    PubMed Central

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  12. Using NV centers to probe magnetization dynamics in normal metal/magnetic insulator hybrid system at the nanoscale

    NASA Astrophysics Data System (ADS)

    Zhang, Huiliang; Ku, Mark J. H.; Han, Minyong; Casola, Francesco; van der Sar, Toeno; Yacoby, Amir; Walsworth, Ronald L.

    2016-05-01

    Understanding magnetization dynamics induced by electric current is of great interest for both fundamental and practical reasons. Great endeavor has been dedicated to spin-orbit torques (SOT) in metallic structures, while quantitative study of analogous phenomena in magnetic insulators remains challenging where transport measurements are not feasible. Recently we have developed techniques using nitrogen vacancy (NV) centers in diamond to probe few-nanometre-scale correlated-electron magnetic excitations (i.e., spin waves). Here we demonstrate how this powerful tool can be implemented to study magnetization dynamics inside ferromagnetic insulator, Yttrium iron garnet (YIG) with spin injection from electrical current through normal metal (Platinum in our case). Particularly our work will focus on NV magnetic detection, imaging, and spectroscopy of coherent auto-oscillations in Pt/YIG microdisc. Magnetic fluctuations and local temperature measurements, both with nearby NV centers, will also be interesting topics relevant to SOT physics in Pt/YIG hybrid system.

  13. Method and apparatus for staining immobilized nucleic acids

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

    2000-01-01

    A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

  14. Interactions of ibuprofen with hybrid lipid bilayers probed by complementary surface-enhanced vibrational spectroscopies

    PubMed Central

    Levin, Carly S.; Kundu, Janardan; Janesko, Benjamin G.; Scuseria, Gustavo E.; Raphael, Robert M.; Halas, Naomi J.

    2016-01-01

    The incorporation of small molecules into lipid bilayers is a process of biological importance and clinical relevance that can change the material properties of cell membranes and cause deleterious side effects for certain drugs. Here we report the direct observation, using surface enhanced Raman and IR spectroscopies (SERS, SEIRA), of the insertion of ibuprofen molecules into hybrid lipid bilayers. The alkanethiol-phospholipid hybrid bilayers were formed onto gold nanoshells by self-assembly, where the underlying nanoshell substrates provided the necessary enhancements for SERS and SEIRA. The spectroscopic data reveal specific interactions between ibuprofen and phospholipid moieties and indicate that the overall hydrophobicity of ibuprofen plays an important role in its intercalation in these membrane mimics. PMID:18942873

  15. Hydroxyapatite-phosphonoformic acid hybrid compounds prepared by hydrothermal method

    NASA Astrophysics Data System (ADS)

    Turki, Thouraya; Othmani, Masseoud; Bantignies, Jean-Louis; Bouzouita, Khaled

    2014-01-01

    Hydroxyapatites were prepared in the presence of different amounts of phosphonoformic acid (PFA) via the hydrothermal method. The obtained powders were characterized through chemical analysis, XRD, IR, 31P MAS-NMR, TEM, and TG-TDA. The XRD showed that the PFA did not affect the apatite composition. Indeed, only a reduction of the crystallite size was noted. After grafting of PFA, the IR spectroscopy revealed the appearance of new bands belonging to HPO42- and carboxylate groups of the apatite and organic moiety, respectively. Moreover, the 31P MAS-NMR spectra exhibited a peak with a low intensity assigned to the terminal phosphonate group of the organic moiety in addition to that of the apatite. Based on these results, a reaction mechanism involving the surface hydroxyl groups (tbnd Casbnd OH) of the apatite and the carboxyl group of the acid was proposed.

  16. Quantitative approaches to monitor protein–nucleic acid interactions using fluorescent probes

    PubMed Central

    Pagano, John M.; Clingman, Carina C.; Ryder, Sean P.

    2011-01-01

    Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods. PMID:21098142

  17. 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. PMID:25602505

  18. Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond

    PubMed Central

    Hadiwikarta, W. W.; Walter, J.-C.; Hooyberghs, J.; Carlon, E.

    2012-01-01

    In this article, it is shown how optimized and dedicated microarray experiments can be used to study the thermodynamics of DNA hybridization for a large number of different conformations in a highly parallel fashion. In particular, free energy penalties for mismatches are obtained in two independent ways and are shown to be correlated with values from melting experiments in solution reported in the literature. The additivity principle, which is at the basis of the nearest-neighbor model, and according to which the penalty for two isolated mismatches is equal to the sum of the independent penalties, is thoroughly tested. Additivity is shown to break down for a mismatch distance below 5 nt. The behavior of mismatches in the vicinity of the helix edges, and the behavior of tandem mismatches are also investigated. Finally, some thermodynamic outlying sequences are observed and highlighted. These sequences contain combinations of GA mismatches. The analysis of the microarray data reported in this article provides new insights on the DNA hybridization parameters and can help to increase the accuracy of hybridization-based technologies. PMID:22661582

  19. Advanced bipolar lead-acid battery for hybrid electric vehicles

    NASA Astrophysics Data System (ADS)

    Saakes, Michel; Kleijnen, Christian; Schmal, Dick; ten Have, Peter

    A large size 80 V bipolar lead acid battery was constructed and tested successfully with a drive cycle especially developed for a HEV. The bipolar battery was made using the bipolar plate developed at TNO and an optimised paste developed by Centurion. An empirical model was derived for calculating the Ragone plot from the results from a small size 12 V bipolar lead-acid battery. This resulted in a specific power of 340 W/kg for the 80 V module. The Ragone plot was calculated at t=5 and t=10 s after the discharge started for current densities varying from 0.02 to 1.2 A/cm 2. A further development of the bipolar lead-acid battery will result in a specific power of 500 W/kg or more. From the economic analysis we estimate that the price of this high power battery will be in the order of 500 US$/kWh. This price is substantially lower than for comparable high power battery systems. This makes it an acceptable candidate future for HEV.

  20. 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

  1. A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Wu, Changfeng; Barnhill, Hannah; Liang, Xiaoping; Wang, Qian; Jiang, Huabei

    2005-11-01

    A fluorescent probe based on bionanoparticle cowpea mosaic virus has been developed for near-infrared fluorescence tomography. A unique advantage of this probe is that over 30 dye molecules can be loaded onto each viral nanoparticle with an average diameter of 30 nm, making high local dye concentration (∼1.8 mM) possible without significant fluorescence quenching. This ability of high loading of local dye concentration would increase the signal-to-noise ratio considerably, thus sensitivity for detection. We demonstrate successful tomographic fluorescence imaging of a target containing the virus-dye nanoparticles embedded in a tissue-like phantom. Tomographic fluorescence data were obtained through a multi-channel frequency-domain system and the spatial maps of fluorescence quantum yield were recovered with a finite-element-based reconstruction algorithm.

  2. A new microcolumn-type microchip for examining the expression of chimeric fusion genes using a nucleic acid sandwich hybridization technique.

    PubMed

    Ohnishi, Michihiro; Sasaki, Naoyuki; Kishimoto, Takuya; Watanabe, Hidetoshi; Takagi, Masatoshi; Mizutani, Shuki; Kishii, Noriyuki; Yasuda, Akio

    2014-11-01

    We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification. PMID:25240923

  3. Comparative study of colony hybridization with synthetic oligonucleotide probes and enzyme-linked immunosorbent assay for identification of enterotoxigenic Escherichia coli.

    PubMed Central

    Sommerfelt, H; Svennerholm, A M; Kalland, K H; Haukanes, B I; Bjorvatn, B

    1988-01-01

    On the basis of the published nucleotide sequences of the genes that code for the heat-labile toxin LTh and the heat-stable toxins STaI and STaII of human enterotoxigenic Escherichia coli, a 34-mer and two 33-mer oligonucleotide probes were synthesized. To compare their relative efficacies in the detection and differentiation of enterotoxigenic E. coli, a colony hybridization technique using these probes and a GM1 ganglioside enzyme-linked immunosorbent assay using monoclonal anti-LT and anti-ST antibodies were used with 76 strains of E. coli with known enterotoxin profiles. For further evaluation of probe specificity, the enterotoxigenic bacteria Vibrio cholerae O1 and non-O1 and Yersinia enterocolitica were examined with the colony hybridization technique. The sensitivity of colony hybridization compared favorably with that of GM1 ganglioside enzyme-linked immunosorbent assay, and the two assays showed a high level of concordance in specific detection and differentiation of E. coli with various enterotoxin profiles (kappa = 0.906, P less than 0.00001). The probes did not hybridize with DNAs from strains of V. cholerae O1 or non-O1 or Y. enterocolitica. PMID:3281978

  4. Specific identification of human papillomavirus type in cervical smears and paraffin sections by in situ hybridization with radioactive probes: a preliminary communication

    SciTech Connect

    Gupta, J.; Gendelman, H.E.; Naghashfar, Z.; Gupta, P.; Rosenshein, N.; Sawada, E.; Woodruff, J.D.; Shah, K.

    1985-01-01

    Cervical Papanicolaou smears and paraffin sections of biopsy specimens obtained from women attending dysplasia clinics were examined for viral DNA sequences by in situ hybridization technique using TVS-labeled cloned recombinant DNA probes of human papillomavirus (HPV) types 6, 11, and 16. These and one unrelated DNA probe complementary to measles virus RNA were labeled by nick translation using either one or two TVS-labeled nucleotides. Paraffin sections and cervical smears were collected on pretreated slides, hybridized with the probes under stringent or nonstringent conditions for 50 h, and autoradiographed. Additional cervical specimens from the same women were examined for the presence of genus-specific papillomavirus capsid antigen by the immunoperoxidase technique. Preliminary results may be summarized as follows. The infecting virus could be identified in smears as well as in sections. Viral DNA sequences were detected only when there were condylomatous cells in the specimen and in only a proportion of the condylomatous cells. Even under stringent conditions, some specimens reacted with both HPV-6 and HPV-11. In some instances, the cells did not hybridize with any of the three probes even when duplicate specimens contained frankly condylomatous, capsid antigen-positive cells. In situ hybridization of Papanicolaou smears or of tissue sections is a practical method for diagnosis and follow-up of specific papillomavirus infection using routinely collected material.

  5. Designed diblock hairpin probes for the nonenzymatic and label-free detection of nucleic acid.

    PubMed

    Wen, Junlin; Chen, Junhua; Zhuang, Li; Zhou, Shungui

    2016-05-15

    The detection of nucleic acid sequences is of great importance in a variety of fields. An ultrasensitive DNA sensing platform is constructed using elaborately designed diblock hairpin probes (DHPs) that are composed of hairpin and poly-adenine blocks. The introduction of an initiator DNA target triggers the catalytic assembly of probes DHP1, DHP2 and DHP3 to fabricate numerous poly-adenine-tailed branched DNA junctions, which significantly amplify the signal of the target-DNA-recognizing event without any enzyme. Coupled to a gold nanoparticle-based colorimetric assay, the amplified recognition signal can be quantitatively detected or visually read with the naked eye. The combination of the high-efficiency target-catalyzed DHP assembly and sensitive gold-based colorimetric assay offers an ultrasensitive detection of DNA with a detection limit of 0.1 pM and a dynamic range from 0.01 to 5 pM. The proposed sensing platform can discriminate even single-base mutations. Moreover, the sensing platform can be expanded to detect pollutant-degrading-bacteria-specific DNA sequences. The proposed sensing system should offer an alternative approach for the detection of nucleic acids in the fields of microbiology, biogeochemistry, and environmental sciences. PMID:26765529

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

    PubMed

    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

  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. 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

  9. Characterization of serovars of the genus Leptospira by DNA hybridization with hardjobovis and icterohaemorrhagiae recombinant probes with special attention to serogroup sejroe.

    PubMed Central

    Van Eys, G J; Gerritsen, M J; Korver, H; Schoone, G J; Kroon, C C; Terpstra, W J

    1991-01-01

    Recombinant DNA probes derived from genomic libraries of serovars hardjobovis and icterohaemorrhagiae were applied for the characterization of leptospires. Differences in hybridization signals in combination with the banding pattern appear to provide good characteristics for strain typing. The banding patterns were easy to distinguish, since the recombinant DNA probes hybridized with a limited number of fragments. They were also indicative of genomic relationships between serovars. The probes suggested the existence of four subgroups with extensive genomic homology within the serogroup Sejroe. A number of serovars outside the serogroup Sejroe showed genomic homology with these subgroups. Amplification with the polymerase chain reaction showed a correlation with the genomic homologies demonstrated by Southern analysis. Knowledge about genomic relationships between leptospiral strains, as revealed by Southern analysis, may lead to a more rational approach for primer selection for polymerase chain reaction or cloning of particular genes. Images PMID:2056039

  10. Hybrid femtosecond/picosecond rotational coherent anti-Stokes Raman scattering at flame temperatures using a second-harmonic bandwidth-compressed probe.

    PubMed

    Kearney, Sean P; Scoglietti, Daniel J

    2013-03-15

    We demonstrate an approach for picosecond probe-beam generation that enables hybrid femtosecond/picosecond pure-rotational coherent anti-Stokes Raman scattering (CARS) measurements in flames. Sum-frequency generation of bandwidth-compressed picosecond radiation from femtosecond pumps with phase-conjugate chirps provides probe pulses with energies in excess of 1 mJ that are temporally locked to the femtosecond pump/Stokes preparation. This method overcomes previous limitations on hybrid femtosecond/picosecond rotational CARS techniques, which have relied upon less efficient bandwidth-reduction processes that have generally resulted in prohibitively low probe energy for flame measurements. We provide the details of the second-harmonic approach and demonstrate the technique in near-adiabatic hydrogen/air flames. PMID:23503231

  11. Probing photocurrent generation mechanisms in hybrid IR-senstive quantum dot/conjugated polymer solar cells

    NASA Astrophysics Data System (ADS)

    Strein, Elisabeth

    The work in this dissertation aims to improve the ability of hybrid polymer/quantum dot solar cells to harvest and utilize sunlight by contributing mechanistic insights into photocurrent generation. The mechanisms of charge transfer and energy transfer are explored spectroscopically in chapter three and both are found to contribute to photocurrent. Chapter four looks at excitation energy in excess of the bandgap and finds a rise in polaron yield which correlates with excess photon energy. Chapter two discusses details of the experimental techniques used to access the data discussed in the chapters that follow.

  12. Heteropoly Acid/Nitrogen Functionalized Onion-like Carbon Hybrid Catalyst for Ester Hydrolysis Reactions.

    PubMed

    Liu, Wei; Qi, Wei; Guo, Xiaoling; Su, Dangsheng

    2016-02-18

    A novel heteropoly acid (HPA)/nitrogen functionalized onion-like carbon (NOLC) hybrid catalyst was synthesized through supramolecular (electrostatic and hydrogen bond) interactions between the two components. The chemical structure and acid strength of the HPA/NOLC hybrid have been fully characterized by thermogravimetric analysis, IR spectroscopy, X-ray photoelectron spectroscopy, NH3 temperature-programmed desorption and acid-base titration measurements. The proposed method for the fabrication of the HPA/NOLC hybrid catalyst is a universal strategy for different types of HPAs to meet various requirements of acidic or redox catalysis. The hydrophobic environment of NOLC effectively prevents the deactivation of HPA in an aqueous system, and the combination of uniformly dispersed HPA clusters and the synergistic effect between NOLC and HPA significantly promotes its activity in ester hydrolysis reactions, which is higher than that of bare PWA as homogeneous catalyst. The kinetics of the hydrolysis reactions indicate that the aggregation status of the catalyst particles has great influence on the apparent activity. PMID:26606266

  13. A novel biocompatible hyaluronic acid-chitosan hybrid hydrogel for osteoarthrosis therapy.

    PubMed

    Kaderli, S; Boulocher, C; Pillet, E; Watrelot-Virieux, D; Rougemont, A L; Roger, T; Viguier, E; Gurny, R; Scapozza, L; Jordan, O

    2015-04-10

    A conventional therapy for the treatment of osteoarthrosis is intra-articular injection of hyaluronic acid, which requires repeated, frequent injections. To extend the viscosupplementation effect of hyaluronic acid, we propose to associate it with another biopolymer in the form of a hybrid hydrogel. Chitosan was chosen because of its structural similarity to synovial glycosaminoglycans, its anti-inflammatory effects and its ability to promote cartilage growth. To avoid polyelectrolyte aggregation and obtain transparent, homogeneous gels, chitosan was reacetylated to a 50% degree, and different salts and formulation buffers were investigated. The biocompatibility of the hybrid gels was tested in vitro on human arthrosic synoviocytes, and in vivo assessments were made 1 week after subcutaneous injection in rats and 1 month after intra-articular injection in rabbits. Hyaluronic acid-chitosan polyelectrolyte complexes were prevented by cationic complexation of the negative charges of hyaluronic acid. The different salts tested were found to alter the viscosity and thermal degradation of the gels. Good biocompatibility was observed in rats, although the calcium-containing formulation induced calcium deposits after 1 week. The sodium chloride formulation was further tested in rabbits and did not show acute clinical signs of pain or inflammation. Hybrid HA-Cs hydrogels may be a valuable alternative viscosupplementation agent. PMID:25666331

  14. 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. PMID:26802748

  15. Radiolytic Modification and Reactivity of Amino Acid Residues Serving as Structural Probes for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectible products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by -rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectible. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

  16. Community Analysis of Biofilters Using Fluorescence In Situ Hybridization Including a New Probe for the Xanthomonas Branch of the Class Proteobacteria

    PubMed Central

    Friedrich, Udo; Naismith, Michèle M.; Altendorf, Karlheinz; Lipski, André

    1999-01-01

    Domain-, class-, and subclass-specific rRNA-targeted probes were applied to investigate the microbial communities of three industrial and three laboratory-scale biofilters. The set of probes also included a new probe (named XAN818) specific for the Xanthomonas branch of the class Proteobacteria; this probe is described in this study. The members of the Xanthomonas branch do not hybridize with previously developed rRNA-targeted oligonucleotide probes for the α-, β-, and γ-Proteobacteria. Bacteria of the Xanthomonas branch accounted for up to 4.5% of total direct counts obtained with 4′,6-diamidino-2-phenylindole. In biofilter samples, the relative abundance of these bacteria was similar to that of the γ-Proteobacteria. Actinobacteria (gram-positive bacteria with a high G+C DNA content) and α-Proteobacteria were the most dominant groups. Detection rates obtained with probe EUB338 varied between about 40 and 70%. For samples with high contents of gram-positive bacteria, these percentages were substantially improved when the calculations were corrected for the reduced permeability of gram-positive bacteria when formaldehyde was used as a fixative. The set of applied bacterial class- and subclass-specific probes yielded, on average, 58.5% (± a standard deviation of 23.0%) of the corrected eubacterial detection rates, thus indicating the necessity of additional probes for studies of biofilter communities. The Xanthomonas-specific probe presented here may serve as an efficient tool for identifying potential phytopathogens. In situ hybridization proved to be a practical tool for microbiological studies of biofiltration systems. PMID:10427047

  17. Effects of novel hybrids of caffeic acid phenethyl ester and NSAIDs on experimental ocular inflammation.

    PubMed

    Pittalà, Valeria; Salerno, Loredana; Romeo, Giuseppe; Siracusa, Maria Angela; Modica, Maria Nunziata; Romano, Giovanni Luca; Salomone, Salvatore; Drago, Filippo; Bucolo, Claudio

    2015-04-01

    In this study, we report the design and synthesis of novel hybrids of caffeic acid phenetyl ester (CAPE) and non-steroidal anti-inflammatory drugs (NSAIDs). We assessed their effects on an experimental ocular inflammation in New Zealand rabbits. The formulations of CAPE-aspirin and CAPE-indomethacin hybrids were topical instilled in the rabbit׳s eye. Afterwards, the anti-inflammatory activity was evaluated by grading the clinical signs and by assessing the inflammatory cell count, protein, PGE2 and TNFα levels in the aqueous humor. Furthermore, ocular tolerability of hybrids formulations was evaluated in a separate set of animals by using a modified Draize test. The ocular inflammation in the control group was significantly higher than in both the hybrid-treated groups, as indicated by clinical grading and biomarkers assessment. However, only the CAPE-aspirin hybrid reduced, in a significant dose-dependent manner, the ocular inflammation elicited by paracentesis. CAPE-indomethacin hybrid was able to significantly attenuate the clinical grading and the PGE2 aqueous levels only at the highest dose (0.1%). CAPE-aspirin significantly reduced PGE2 and TNFα levels in the aqueous humor as well as proteins and PMNs. Finally, all formulations showed no ocular irritation compared with vehicle-treated group. In conclusion, CAPE-aspirin shows full anti-inflammatory efficacy in experimental model of ocular inflammation demonstrating an optimal pharmacological and safety profile. Taken together these data indicate that CAPE-aspirin hybrid represents a valid and safe new chemical entity potentially useful for the treatment of ocular inflammation. PMID:25704612

  18. 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.

  19. Hall probe measurements of the poloidal magnetic field in Compact Toroidal Hybrid plasmas

    SciTech Connect

    Stevenson, B. A.; Knowlton, S. F.; Hartwell, G. J. Hanson, J. D.; Maurer, D. A.

    2014-09-15

    A linear array of 16 Hall effect sensors has been developed to directly measure the poloidal magnetic field inside the boundary of a non-axisymmetric hybrid torsatron/tokamak plasma. The array consists of miniature gallium arsenide Hall sensor elements mounted 8 mm apart on a narrow, rotatable printed circuit board inserted into a re-entrant stainless steel tube sheathed in boron nitride. The sensors are calibrated on the bench and in situ to provide accurate local measurements of the magnetic field to aid in reconstructing the equilibrium plasma current density profiles in fully three-dimensional plasmas. Calibrations show that the sensor sensitivities agree with the nominal manufacturers specifications of 1.46 V/T. Poloidal fields measured with the Hall sensor array are found to be within 5% of poloidal fields modeled with a Biot-Savart code.

  20. Growth characteristics of Ti-based fumaric acid hybrid thin films by molecular layer deposition.

    PubMed

    Cao, Yan-Qiang; Zhu, Lin; Li, Xin; Cao, Zheng-Yi; Wu, Di; Li, Ai-Dong

    2015-09-01

    Ti-based fumaric acid hybrid thin films were successfully prepared using inorganic TiCl4 and organic fumaric acid as precursors by molecular layer deposition (MLD). The effect of deposition temperature from 180 °C to 350 °C on the growth rate, composition, chemical state, and topology of hybrid films has been investigated systematically by means of a series of analytical tools such as spectroscopic ellipsometry, atomic force microscopy (AFM), high resolution X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The MLD process of the Ti-fumaric acid shows self-limiting surface reaction with a reasonable growth rate of ∼0.93 Å per cycle and small surface roughness of ∼0.59 nm in root-mean-square value at 200 °C. A temperature-dependent growth characteristic has been observed in the hybrid films. On increasing the temperature from 180 °C to 300 °C, the growth rate decreases from 1.10 to 0.49 Å per cycle and the XPS composition of the film's C : O : Ti ratio changes from 8.35 : 7.49 : 1.00 to 4.66 : 4.80 : 1.00. FTIR spectra indicate that the hybrid films show bridging bonding mode at a low deposition temperature of 200 °C and bridging/bidentate mixed bonding mode at elevated deposition temperatures of 250 and 300 °C. The higher C and O amounts deviating from the ideal composition may be ascribed to increased organic incorporation into the hybrid films at lower deposition temperature and temperature-dependent density of reactive sites (-OH). The composition of hybrid films grown at 350 °C shows a dramatic decrease in C and O elemental composition (C : O : Ti = 1.97 : 2.76 : 1.00) due to the thermal decomposition of the fumaric acid precursor. The produced by-product H2O changes the structure of the hybrid films, resulting in the formation of more Ti-O bonds at high temperatures. The stability of the hybrid films against chemical and thermal treatment, and long-term storage by

  1. Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid.

    PubMed Central

    Takayama, Kuni; Hayes, Barry; Vestling, Matha M; Massey, Randall J

    2003-01-01

    Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery. PMID:12879902

  2. Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

    PubMed Central

    Muangchuen, Ajima; Chaumpluk, Piyasak; Suriyasomboon, Annop; Ekgasit, Sanong

    2014-01-01

    Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease. PMID:25111239

  3. In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes.

    PubMed Central

    Manz, W; Szewzyk, U; Ericsson, P; Amann, R; Schleifer, K H; Stenström, T A

    1993-01-01

    Free-water-phase and surface-associated microorganisms from drinking water were detected and roughly identified by hybridization with fluorescence-labeled oligonucleotide probes complementary to regions of 16S and 23S rRNA characteristic for the domains Bacteria, Archaea, and Eucarya and the beta and gamma subclasses of Proteobacteria. Samples of glass-attached biofilms and plankton were taken from a Robbins device installed in a water distribution system. More than 70% of the surface-associated cells and less than 40% of the planktonic cells visualized by 4',6-diamidino-2-phenylindole staining bound detectable amounts of rRNA-targeted probes. These findings are an indication for higher average rRNA content and consequently higher physiological activity of the attached microbial cells compared with the free-living cells. All detectable cells hybridized with the bacterial probe, whereas no Archaea and no Eucarya cells could be detected. Simultaneous hybridization with probes specific for the beta and gamma subclasses of Proteobacteria revealed that microcolonies already consisted of mixed populations in early stages with fewer than 50 cells. These observations provide further evidence that the coexistence and interaction of bacteria in drinking water biofilms may be an integral part of their growth and survival strategies. Images PMID:8357261

  4. Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans.

    PubMed Central

    Pujol, C; Joly, S; Lockhart, S R; Noel, S; Tibayrenc, M; Soll, D R

    1997-01-01

    Randomly amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Southern blot hybridization with moderately repetitive DNA probes have emerged as effective fingerprinting methods for the infectious fungus Candida albicans. The three methods have been compared for their capacities to identify identical or highly related isolates, to cluster weakly related isolates, to discriminate between unrelated isolates, and to assess microevolution within a strain. By computing similarity coefficients between 29 isolates from three cities within the continental United States, strong concordance of the results is demonstrated for RAPD analysis, MLEE, and Southern blot hybridization with the moderately repetitive probe Ca3, and weaker concordance of the results is demonstrated for these three fingerprinting methods and Southern blot hybridization with the moderately repetitive probe CARE2. All methods were also demonstrated to be able to resolve microevolution within a strain, with the Ca3 probe exhibiting the greatest resolving power. The strong correlations demonstrated between polymorphic markers assessed by the four independent fingerprinting methods and the nonrandom association between loci demonstrated by RAPD analysis and MLEE provide evidence for strong linkage disequilibrium and a clonal population structure for C. albicans. In addition, a synapomorphic allele, Pep-3A, was found to be present in all members of one of the three clusters discriminated by RAPD analysis, MLEE, and Ca3 fingerprinting, supporting the concordance of the clustering capacities of the three methods, the robustness of the clusters, and the clonal nature of the clusters. PMID:9276415

  5. 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

  6. 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.

  7. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  8. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds

    PubMed Central

    Feng, Zi-ming; Zhan, Zhi-lai; Yang, Ya-nan; Jiang, Jian-shuang; Zhang, Pei-cheng

    2016-01-01

    The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of γ-amino acids and sugars from the roots of Ranunculus ternatus, which possess potential tail to tail ether-connected (6,6-ether-connected) modes in the sugar moiety. The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method. PMID:27166276

  9. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds.

    PubMed

    Feng, Zi-Ming; Zhan, Zhi-Lai; Yang, Ya-Nan; Jiang, Jian-Shuang; Zhang, Pei-Cheng

    2016-01-01

    The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of γ-amino acids and sugars from the roots of Ranunculus ternatus, which possess potential tail to tail ether-connected (6,6-ether-connected) modes in the sugar moiety. The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method. PMID:27166276

  10. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds

    NASA Astrophysics Data System (ADS)

    Feng, Zi-Ming; Zhan, Zhi-Lai; Yang, Ya-Nan; Jiang, Jian-Shuang; Zhang, Pei-Cheng

    2016-05-01

    The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of γ-amino acids and sugars from the roots of Ranunculus ternatus, which possess potential tail to tail ether-connected (6,6-ether-connected) modes in the sugar moiety. The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method.

  11. Using nitrile-derivatized amino acids as infrared probes of local environment.

    PubMed

    Getahun, Zelleka; Huang, Cheng-Yen; Wang, Ting; De León, Brenda; DeGrado, William F; Gai, Feng

    2003-01-15

    It is well-known that the C=N stretching vibration in acetonitrile is sensitive to solvent. Therefore, we proposed in this contribution to use this vibrational mode to report local environment of a particular amino acid in proteins or local environmental changes upon binding or folding. We have studied the solvent-induced frequency shift of two nitrile-derivatized amino acids, which are, AlaCN and PheCN, in H(2)O and tetrahydrofuran (THF), respectively. Here, THF was used to approximate a protein's hydrophobic interior because of its low dielectric constant. As expected, the C=N stretching vibrations of both AlaCN and PheCN shift as much as approximately 10 cm(-1) toward higher frequency when THF was replaced with H2O, indicative of the sensitivity of this vibration to solvation. To further test the utility of nitrile-derivatized amino acids as probes of the environment within a peptide, we have studied the binding between calmodulin (CaM) and a peptide from the CaM binding domain of skeletal muscle myosin light chain kinase (MLCK(579-595)), which contains a single PheCN. MLCK(579-595) binds to CaM in a helical conformation. When the PheCN was substituted on the polar side of the helix, which was partially exposed to water, the C=N stretching vibration is similar to that of PheCN in water. In constrast, when PheCN is introduced at a site that becomes buried in the interior of the protein, the C=N stretch is similar to that of PheCN in THF. Together, these results suggest that the C=N stretching vibration of nitrile-derivatized amino acids can indeed be used as local internal environmental markers, especially for protein conformational studies. PMID:12517152

  12. Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.

    PubMed

    Schramm, Andreas; Fuchs, Bernhard M; Nielsen, Jeppe L; Tonolla, Mauro; Stahl, David A

    2002-11-01

    A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. PMID:12460279

  13. Validation of a new catalysed reporter deposition-fluorescence in situ hybridization probe for the accurate quantification of marine Bacteroidetes populations.

    PubMed

    Acinas, Silvia G; Ferrera, Isabel; Sarmento, Hugo; Díez-Vives, Cristina; Forn, Irene; Ruiz-González, Clara; Cornejo-Castillo, Francisco M; Salazar, Guillem; Gasol, Josep M

    2015-10-01

    Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) is a powerful approach to quantify bacterial taxa. In this study, we compare the performance of the widely used Bacteroidetes CF319a probe with the new CF968 probe. In silico analyses and tests with isolates demonstrate that CF319a hybridizes with non-Bacteroidetes sequences from the Rhodobacteraceae and Alteromonadaceae families. We test the probes' accuracy in 37 globally distributed marine samples and over two consecutive years at the Blanes Bay Microbial Observatory (NW Mediterranean). We also compared the CARD-FISH data with the Bacteroidetes 16S rRNA gene sequences retrieved from 27 marine metagenomes from the TARA Oceans expedition. We find no significant differences in abundances between both approaches, although CF319a targeted some unspecific sequences and both probes displayed different abundances of specific Bacteroidetes phylotypes. Our results demonstrate that quantitative estimations by using both probes are significantly different in certain oceanographic regions (Mediterranean Sea, Red Sea and Arabian Sea) and that CF968 shows seasonality within marine Bacteroidetes, notably large differences between summer and winter that is overlooked by CF319a. We propose CF968 as an alternative to CF319a for targeting the whole Bacteroidetes phylum since it has better coverage, greater specificity and overall better quantifies marine Bacteroidetes. PMID:24890225

  14. Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

    PubMed

    Kamstra, S A; Ramanna, M S; de Jeu, M J; Kuipers, A G; Jacobsen, E

    1999-01-01

    A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed. PMID:10087627

  15. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

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

    1998-01-01

    A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

  16. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

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

    1998-07-21

    A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

  17. A versatile solid support system for oligodeoxynucleotide probe-based hybridization assays.

    PubMed Central

    Van Ness, J; Kalbfleisch, S; Petrie, C R; Reed, M W; Tabone, J C; Vermeulen, N M

    1991-01-01

    A procedure for immobilization of well-defined quantities of oligodeoxyribonucleotides (ODNs) to a versatile nylon support is described. The solid support, a nylon-6/6 bead, is covalently coated with poly(ethyleneimine) to provide a reactive spacer-arm for attachment of ODNs. 5'-Aminohexyl-tailed ODNs are selectively activated using 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) and then covalently attached to the bead via the triazine moiety. The modified nylon support has a low level of binding of nonspecific nucleic acid and efficiently captures both RNA and DNA targets. PMID:2062652

  18. Fluorescence in situ hybridization (FISH) using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes

    PubMed Central

    Halder, Ashutosh; Jain, Manish; Chaudhary, Isha; Gupta, Neerja; Kabra, Madhulika

    2013-01-01

    Background & objectives: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. Methods: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. Results: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis. PMID:24056568

  19. A hybrid analog-digital phase-locked loop for frequency mode non-contact scanning probe microscopy.

    PubMed

    Mehta, M M; Chandrasekhar, V

    2014-01-01

    Non-contact scanning probe microscopy (SPM) has developed into a powerful technique to image many different properties of samples. The conventional method involves monitoring the amplitude, phase, or frequency of a cantilever oscillating at or near its resonant frequency as it is scanned across the surface of a sample. For high Q factor cantilevers, monitoring the resonant frequency is the preferred method in order to obtain reasonable scan times. This can be done by using a phase-locked-loop (PLL). PLLs can be obtained as commercial integrated circuits, but these do not have the frequency resolution required for SPM. To increase the resolution, all-digital PLLs requiring sophisticated digital signal processors or field programmable gate arrays have also been implemented. We describe here a hybrid analog/digital PLL where most of the components are implemented using discrete analog integrated circuits, but the frequency resolution is provided by a direct digital synthesis chip controlled by a simple peripheral interface controller (PIC) microcontroller. The PLL has excellent frequency resolution and noise, and can be controlled and read by a computer via a universal serial bus connection. PMID:24517775

  20. A hybrid analog-digital phase-locked loop for frequency mode non-contact scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Mehta, M. M.; Chandrasekhar, V.

    2014-01-01

    Non-contact scanning probe microscopy (SPM) has developed into a powerful technique to image many different properties of samples. The conventional method involves monitoring the amplitude, phase, or frequency of a cantilever oscillating at or near its resonant frequency as it is scanned across the surface of a sample. For high Q factor cantilevers, monitoring the resonant frequency is the preferred method in order to obtain reasonable scan times. This can be done by using a phase-locked-loop (PLL). PLLs can be obtained as commercial integrated circuits, but these do not have the frequency resolution required for SPM. To increase the resolution, all-digital PLLs requiring sophisticated digital signal processors or field programmable gate arrays have also been implemented. We describe here a hybrid analog/digital PLL where most of the components are implemented using discrete analog integrated circuits, but the frequency resolution is provided by a direct digital synthesis chip controlled by a simple peripheral interface controller (PIC) microcontroller. The PLL has excellent frequency resolution and noise, and can be controlled and read by a computer via a universal serial bus connection.

  1. Multiplex Ligation-Dependent Probe Amplification Versus Multiprobe Fluorescence in Situ Hybridization To Detect Genomic Aberrations in Chronic Lymphocytic Leukemia

    PubMed Central

    Al Zaabi, Eiman A.; Fernandez, Louis A.; Sadek, Irene A.; Riddell, D. Christie; Greer, Wenda L.

    2010-01-01

    Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH, genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%), trisomy 12 (7%), ATM deletion (6%), 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes, the most common being 19p13 (LDLR and CDKN2D). Moreover, the cost for MLPA analysis, including technical time and reagents, is 86% less than FISH. In conclusion, cytogenetic abnormalities are a common finding in CLL patients, and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis. PMID:20093390

  2. Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

    SciTech Connect

    Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

    2003-08-01

    Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

  3. A highly selective turn-on fluorescent probe for hypochlorous acid based on hypochlorous acid-induced oxidative intramolecular cyclization of boron dipyrromethene-hydrazone.

    PubMed

    Chen, Wei-Chieh; Venkatesan, Parthiban; Wu, Shu-Pao

    2015-07-01

    A BODIPY-based fluorescent probe, HBP, was developed for the detection of hypochlorous acid based on the specific hypochlorous acid-promoted oxidative intramolecular cyclization of heterocyclic hydrazone in response to the amount of HOCl. The reaction is accompanied by a 41-fold increase in the fluorescent quantum yield (from 0.004 to 0.164). The fluorescence intensity of the reaction between HOCl and HBP is linear in the HOCl concentration range of 1-8 μM with a detection limit of 2.4 nM (S/N=3). Confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe HBP could be used as an effective fluorescent probe for detecting HOCl in living cells. PMID:26043093

  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. Hybrid Compounds Strategy in the Synthesis of Oleanolic Acid Skeleton-NSAID Derivatives.

    PubMed

    Pawełczyk, Anna; Olender, Dorota; Sowa-Kasprzak, Katarzyna; Zaprutko, Lucjusz

    2016-01-01

    The current study focuses on the synthesis of several hybrid individuals combining a natural oleanolic acid skeleton and synthetic nonsteroidal anti-inflammatory drug moieties (NSAIDs). It studied structural modifications of the oleanolic acid structure by use of the direct reactivity of hydroxyl or hydroxyimino groups at position C-3 of the triterpenoid skeleton with the carboxylic function of anti-inflammatory drugs leading to new perspective compounds with high potential pharmacological activities. Novel ester- and iminoester-type derivatives of oleanolic unit with the different NSAIDs, such as ibuprofen, aspirin, naproxen, and ketoprofen, were obtained and characterized. Moreover, preliminary research of compounds obtaining structure stability under acidic conditions was examined and the PASS method of prediction of activity spectra for substances was used to estimate the potential biological activity of these compounds. PMID:27077841

  6. Porous Zirconium-Phytic Acid Hybrid: a Highly Efficient Catalyst for Meerwein-Ponndorf-Verley Reductions.

    PubMed

    Song, Jinliang; Zhou, Baowen; Zhou, Huacong; Wu, Lingqiao; Meng, Qinglei; Liu, Zhimin; Han, Buxing

    2015-08-01

    The utilization of compounds from natural sources to prepare functional materials is of great importance. Herein, we describe for the first time the preparation of organic-inorganic hybrid catalysts by using natural phytic acid as building block. Zirconium phosphonate (Zr-PhyA) was synthesized by reaction of phytic acid and ZrCl4 and was obtained as a mesoporous material with pore sizes centered around 8.5 nm. Zr-PhyA was used to catalyze the mild and selective Meerwein-Ponndorf-Verley (MPV) reduction of various carbonyl compounds, e.g., of levulinic acid and its esters into γ-valerolactone. Further studies indicated that both Zr and phosphate groups contribute significantly to the excellent performance of Zr-PhyA. PMID:26177726

  7. Effect of acidic solutions on the surface degradation of a micro-hybrid composite resin.

    PubMed

    Münchow, Eliseu A; Ferreira, Ana Cláudia A; Machado, Raissa M M; Ramos, Tatiana S; Rodrigues-Junior, Sinval A; Zanchi, Cesar H

    2014-01-01

    Composite resins may undergo wear by the action of chemical substances (e.g., saliva, alcohol, bacterial acids) of the oral environment, which may affect the material's structure and surface properties. This study evaluated the effect of acidic substances on the surface properties of a micro-hybrid composite resin (Filtek Z-250). Eighty specimens were prepared, and baseline hardness and surface roughness (KMN0 and Ra0, respectively) were measured. The specimens were subjected to sorption (SO) and solubility (SL) tests according to ISO 4049:2009, but using different storage solutions: deionized water; 75/25 vol% ethanol/water solution; lactic acid; propionic acid; and acetic acid. The acids were used in two concentrations: PA and 0.02 N. pH was measured for all solutions and final hardness (KMN1) and surface roughness (Ra1) were measured. Data were analyzed with paired t-tests and one-way ANOVA and Tukey's test (a=5%). All solutions decreased hardness and increased the Ra values, except for the specimens stored in water and 0.02 N lactic acid, which maintained the hardness. All solutions produced similar SO and SL phenomena, except for the 0.02 N lactic acid, which caused lower solubility than the other solutions. Ethanol showed the highest pH (6.6) and the 0.02 N lactic acid the lowest one (2.5). The solutions affected negatively the surface properties of the composite resin; in addition, an acidic pH did not seem to be a significant factor that intensifies the surface degradation phenomena. PMID:25250496

  8. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    PubMed Central

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-01-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera. PMID:9097423

  9. Deoxyribonucleic acid-based hybrid thin films for potential application as high energy density capacitors

    NASA Astrophysics Data System (ADS)

    Joyce, Donna M.; Venkat, Narayanan; Ouchen, Fahima; Singh, Kristi M.; Smith, Steven R.; Grabowski, Christopher A.; Terry Murray, P.; Grote, James G.

    2014-03-01

    Deoxyribonucleic acid (DNA) based hybrid films incorporating sol-gel-derived ceramics have shown strong promise as insulating dielectrics for high voltage capacitor applications. Our studies of DNA-CTMA (cetyltrimethylammonium) complex/sol-gel ceramic hybrid thin film devices have demonstrated reproducibility and stability in temperature- and frequency-dependent dielectric properties with dielectric constant k ˜ 5.0 (1 kHz), as well as reliability in DC voltage breakdown measurements, attaining values consistently in the range of 300-350 V/μm. The electrical/dielectric characteristics of DNA-CTMA films with sol-gel-derived ceramics were examined to determine the critical energy storage parameters such as voltage breakdown and dielectric constant.

  10. Testing and evaluation of EV-1300 lead-acid modules for the hybrid vehicle application

    SciTech Connect

    Gay, E.C.; Webster, C.E.; Hornstra, F.; Yao, N.P.

    1984-01-01

    This paper presents the results of testing and evaluation of GE/Globe EV-1300 lead-acid modules developed by Globe Battery Division of Johnson Controls, Inc. for the hybrid vehicle, HTV-I, developed by General Electric (GE) for the Department of Energy. The design of this battery was derived from that of the Globe Improved State of the Art (ISOA) battery under development for the ETV-1 all-electric vehicle. Key differences in the battery performance requirements for the HTV-1 hybrid vehicle, as opposed to the ETV-1, are higher specific power (137 W/kg versus 104 W/kg sustained for 15 seconds at 50% depth of discharge (DOD)) and less specific energy (36.1 Wh/kg versus 37.5 Wh/kg at a 3h discharge rate).

  11. Porous polylactic acid-silica hybrids: preparation, characterization, and study of mesenchymal stem cell osteogenic differentiation.

    PubMed

    Pandis, Christos; Trujillo, Sara; Matos, Joana; Madeira, Sara; Ródenas-Rochina, Joaquín; Kripotou, Sotiria; Kyritsis, Apostolos; Mano, João F; Gómez Ribelles, José Luis

    2015-02-01

    A novel approach to reinforce polymer porous membranes is presented. In the prepared hybrid materials, the inorganic phase of silica is synthesized in-situ and inside the pores of aminolyzed polylactic acid (PLA) membranes by sol-gel reactions using tetraethylorthosilicate (TEOS) and glycidoxypropyltrimethoxysilane (GPTMS) as precursors. The hybrid materials present a porous structure with a silica layer covering the walls of the pores while GPTMS serves also as coupling agent between the organic and inorganic phase. The adjustment of silica precursors ratio allows the modulation of the thermomechanical properties. Culture of mesenchymal stem cells on these supports in osteogenic medium shows the expression of characteristic osteoblastic markers and the mineralization of the extracellular matrix. PMID:25303745

  12. Heavy metal removal from acid mine drainage by calcined eggshell and microalgae hybrid system.

    PubMed

    Choi, Hee-Jeong; Lee, Seung-Mok

    2015-09-01

    This study investigates the use of calcined eggshells and microalgae for the removal of heavy metals from acid mine drainage (AMD) and the simultaneous enhancement of biomass productivity. The experiment was conducted over a period of 6 days in a hybrid system containing calcined eggshells and the microalgae Chlorella vulgaris. The results show that the biomass productivity increased to ~8.04 times its initial concentration of 0.367 g/L as measured by an optical panel photobioreactor (OPPBR) and had a light transmittance of 95 % at a depth of 305 mm. On the other hand, the simultaneous percent removal of Fe, Cu, Zn, Mn, As, and Cd from the AMD effluent was found to be 99.47 to 100 %. These results indicate that the hybrid system with calcined eggshells and microalgae was highly effective for heavy metal removal in the AMD. PMID:25940497

  13. Host selection and probing behavior of the poplar aphid Chaitophorus leucomelas (Sternorrhyncha: Aphididae) on two poplar hybrids with contrasting susceptibility to aphids.

    PubMed

    Barrios-San Martín, Joceline; Quiroz, Andrés; Verdugo, Jaime A; Parra, Leonardo; Hormazabal, Emilio; Astudillo, Luis A; Rojas-Herrera, Marcelo; Ramírez, Claudio C

    2014-02-01

    Poplars are frequently attacked by aphids. The differential susceptibility of poplar hybrids to the aphid Chaitophorus leucomelas Koch (Sternorrhyncha: Aphididae) has been described, but the mechanism underlying this pattern is unknown. This work tested the hypothesis that poplar resistance to this aphid is associated with the presence of volatiles and secondary plant compounds that affect host selection and feeding behavior. This hypothesis was tested by studying the host choice and feeding behavior of C. leucomelas on two poplar hybrids with contrasting susceptibilities to this aphid ([Populus trichocarpa Torrey & Gray x Populus deltoides Bartram ex Marshall] x P. deltoides [TD x D], and [P. trichocarpa x Populus maximowiczii Henry] x [P. trichocarpa x P. maximowiczii] [TM x TM]). The results showed that C. leucomelas rejected leaves of the TM x TM hybrid and did not prefer odors from either hybrid. Electronic monitoring of the probing behavior of C. leucomelas suggested the involvement of antifeedant factors in the TM x TM hybrid. In addition, the chemical characterization of volatiles, epicuticular waxes, and internal phenols of leaves from both poplar hybrids revealed that TM x TM had a higher abundance of monoterpenes, sesquiterpenes, n-alkanes, and phenols. These results are discussed in terms of their contribution to poplar breeding programs aimed at enhancing insect resistance. PMID:24665710

  14. Rapid Quantification of Drug Resistance Gene Expression in Candida albicans by Reverse Transcriptase LightCycler PCR and Fluorescent Probe Hybridization

    PubMed Central

    Frade, Joao P.; Warnock, David W.; Arthington-Skaggs, Beth A.

    2004-01-01

    We developed a rapid, sensitive, and reproducible assay to quantify Candida albicans ACT1, CDR1, CDR2, ERG11, and MDR1 mRNA using a two-step reverse transcription and LightCycler real-time PCR (RT-LightCycler PCR) method with sequence-specific hybridization probes. We compared RT-LightCycler PCR with Northern hybridization for quantitative analysis of gene expression in isolates with various fluconazole susceptibilities. Specificity of each LightCycler PCR was verified by LightCycler melting curve analysis and agarose gel electrophoresis of amplified products. Correlation of quantification results between RT-LightCycler PCR and Northern hybridization yielded correlation coefficients of ≥0.91 for all genes except MDR1 (0.74). In this case, reduced correlation was due to the inability of Northern hybridization to accurately quantify the high MDR1 expression in a susceptible dose-dependent isolate which was shown by RT-LightCycler PCR to overexpress MDR1 >200-fold relative to the other isolates tested. In four isolates, low levels of CDR2 mRNA were detected by RT-LightCycler PCR but were undetectable by Northern hybridization. mRNA quantification by RT-LightCycler PCR correlates with Northern hybridization and offers additional advantages, including increased sensitivity and speed of analysis, along with lower RNA concentration requirements and an increased dynamic range of signal detection. PMID:15131174

  15. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  16. 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

  17. Quest for a reliable, valid, and sensitive in situ hybridization procedure to detect viral nucleic acids in the central nervous system.

    PubMed

    Tourtellotte, W W; Schmid, P; Pick, P; Verity, N; Martinez, S; Shapshak, P

    1987-06-01

    In situ hybridization (ISH) to detect and to quantitate viral nucleic acid sequences in cryopreserved central nervous system (CNS) tissue is a reliable, valid and sensitive molecular technique. On the other hand, utilization of formaldehyde fixed paraffin embedded (FFPE) tissue to improve cytomorphology requires fundamental changes in the procedure since it is necessary to cleave the elaborate protein network cross-linked by formaldehyde using elevated concentrations of proteinases in order to permit diffusion of complementary DNA probes to the targets (genomic viral nucleic acid sequences and/or viral mRNA). Adversely, this procedure hydrolyzed the proteinaceous glues generally used to fix tissue to glass slides resulting in loss of tissue sections during the ISH protocol. This report describes the application of a novel procedure utilizing a silano-organic compound to covalently bond to glass slides FFPE sections as well as cryopreserved tissue sections and cultured cells with and without virus infections. This covalent bonding procedure has permitted optimization of the ISH procedure for virus detection and quantification, especially for exploratory studies of specificity and wash stringency in relation to the Tm of the hybridized product. Progressive multifocal leucoencephalopathy (PML) caused by an opportunistic papovavirus (JC) was chosen because of the ready availability of tissue, stability of papovavirus nucleic acids, and specificity of 3H- and 35S-radiolabeled JC cloned DNA probes. Further, this laboratory is utilizing the optimized sensitive procedure to search for several virus etiologies in human diseases such as multiple sclerosis, temporal lobe epilepsy, Alzheimer's disease, schizophrenia, and Parkinson's disease, as well as normal aging.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3299127

  18. Hybrid femtosecond/picosecond rotational coherent anti-Stokes Raman scattering temperature and concentration measurements using two different picosecond-duration probes.

    PubMed

    Kearney, Sean P; Scoglietti, Daniel J; Kliewer, Christopher J

    2013-05-20

    A hybrid fs/ps pure-rotational CARS scheme is characterized in furnace-heated air at temperatures from 290 to 800 K. Impulsive femtosecond excitation is used to prepare a rotational Raman coherence that is probed with a ps-duration beam generated from an initially broadband fs pulse that is bandwidth limited using air-spaced Fabry-Perot etalons. CARS spectra are generated using 1.5- and 7.0-ps duration probe beams with corresponding coarse and narrow spectral widths. The spectra are fitted using a simple phenomenological model for both shot-averaged and single-shot measurements of temperature and oxygen mole fraction. Our single-shot temperature measurements exhibit high levels of precision and accuracy when the spectrally coarse 1.5-ps probe beam is used, demonstrating that high spectral resolution is not required for thermometry. An initial assessment of concentration measurements in air is also provided, with best results obtained using the higher resolution 7.0-ps probe. This systematic assessment of the hybrid CARS technique demonstrates its utility for practical application in low-temperature gas-phase systems. PMID:23736451

  19. Measurement of mRNA by solution hybridization with /sup 32/P-labelled single stranded cRNA probe (SP6 test). Comparison with a /sup 32/P-labelled single stranded cDNA as hybridization probe (S1 test) for measurement of AVP mRNA

    SciTech Connect

    Ludwig, G.; Haenze, J.L.; Lehmann, E.; Lang, R.E.; Burbach, J.H.; Ganten, D.

    1988-01-01

    Radioactively labelled cRNA for the rat AVP gene exon C was synthetized from a pSP64-vector and used for solution hybridization measurement of AVP mRNA (SP6 test). For comparison hybridization was carried out with a gel-purified radioactively labelled cDNA probe synthetized by primer extension of AVP gene exon C cloned into an M13mp9 phage vector DNA (S1 test). Both tests had a comparable sensitivity of up to 0.2 pg AVP mRNA. Under optimal hybridization conditions kinetics were similar in both tests. The fast and easy preparation of large amounts of labelled cDNA probe and simple determination of absolute amounts of mRNA by saturation kinetics without need of a mRNA standard makes the SP6 test an attractive alternative to the known S1 test. The SP6 test should be applicable for a wide variety of genes.

  20. Design and Evaluation of a Lactobacillus manihotivorans Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation

    PubMed Central

    Ampe, Frédéric

    2000-01-01

    Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria. PMID:10788405

  1. The dietary branched chain amino acid requirements of hybrid striped bass(Morone chrysops x M. saxatilis)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The requirements for branched chain amino acids (BCAAs) are unknown in hybrid striped bass and necessary for formulating efficient and nutritious diets. Moreover, the dietary balance among these three amino acids can substantially influence the performance of meat animals fed those diets. The diet...

  2. Internalization of Locked Nucleic Acids/DNA Hybrid Oligomers into Escherichia coli.

    PubMed

    Traglia, German M; Sala, Carol Davies; Fuxman Bass, Juan I; Soler-Bistué, Alfonso J C; Zorreguieta, Angeles; Ramírez, María Soledad; Tolmasky, Marcelo E

    2012-10-01

    Delivery inside the cells is essential for practical application of antisense technologies. The hybrid locked nucleic acid (LNA)/DNA CAAGTACTGTTCCACCA (LNA residues are underlined) was labeled by conjugation to Alexa Fluor 488 (fLNA/DNA) and tested to determine its ability to penetrate Escherichia coli cells and reach the cytoplasm. Flow cytometry analysis showed that the fLNA/DNA was associated with 14% of cells from a stationary phase culture, while association with a labeled isosequential oligodeoxynucleotide was negligible. Laser scanning confocal microscopy confirmed that the fLNA/DNA was located inside the cytoplasm. PMID:23515318

  3. A novel chaotic based image encryption using a hybrid model of deoxyribonucleic acid and cellular automata

    NASA Astrophysics Data System (ADS)

    Enayatifar, Rasul; Sadaei, Hossein Javedani; Abdullah, Abdul Hanan; Lee, Malrey; Isnin, Ismail Fauzi

    2015-08-01

    Currently, there are many studies have conducted on developing security of the digital image in order to protect such data while they are sending on the internet. This work aims to propose a new approach based on a hybrid model of the Tinkerbell chaotic map, deoxyribonucleic acid (DNA) and cellular automata (CA). DNA rules, DNA sequence XOR operator and CA rules are used simultaneously to encrypt the plain-image pixels. To determine rule number in DNA sequence and also CA, a 2-dimension Tinkerbell chaotic map is employed. Experimental results and computer simulations, both confirm that the proposed scheme not only demonstrates outstanding encryption, but also resists various typical attacks.

  4. Electrochemical sensor for dopamine based on imprinted silica matrix-poly(aniline boronic acid) hybrid as recognition element.

    PubMed

    Li, Jian; Zhang, Ning; Sun, Qingqing; Bai, Zhanming; Zheng, Jianbin

    2016-10-01

    A novel imprinted silica matrix-poly(aniline boronic acid) hybrid for electrochemical detection of dopamine (DA) was developed. Boronic acid functionalized conducting polymer was electrochemically prepared on Au electrode. The number of covalent binding sites toward DA templates was controlled by potential cycles. A precursory sol solution of ammonium fluorosilicate (as cross-linking monomer) containing DA was spin-coated on the polymer modified electrode. Under NH3 atmosphere, the hydroxyl ions were generated in the solution and catalyzed the hydrolysis of fluorosilicate to form silica matrix. After this aqueous sol-gel process, an inorganic framework around the DA template was formed and the imprinted hybrid for DA was also produced. As revealed by scanning electron microscopy, UV-vis spectroscopy and cyclic voltammetry characterization, DA was embedded in the imprinted hybrid successfully. The affinity and selectivity of the imprinted hybrid were also characterized by cyclic voltammetry. The imprinted hybrid showed higher affinity for DA than that for epinephrine, and little or no affinity for ascorbic acid and uric acid due to the combined effects of covalent interaction, cavities matching and electrostatic repulsion. The imprinted hybrid sensor exhibited a quick response (within 5min) to DA in the concentration range from 0.05 to 500μmolL(-1) with a detection limit of 0.018μmolL(-1). The prepared sensor was also applied to detect DA in real samples with a satisfactory result. PMID:27474321

  5. Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

    PubMed Central

    Minnigan, H; Moyer, R W

    1985-01-01

    The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses. Images PMID:2991586

  6. Tartaric Acid-Assisted Self-Assembly of Hybrid Block Copolymer Composites

    NASA Astrophysics Data System (ADS)

    Yao, Li; Lin, Ying; Watkins, James

    2014-03-01

    Enantiopure tartaric acid was used as an additive to increase the segregation strength of poly(ethylene oxide-block-tert-butyl acrylate) (PEO-b-PtBA) copolymers through strong, selective interactions with one of the polymer chain segments. Addition of tartaric acid to PEO-b-PtBA exhibiting cylindrical morphologies resulted in the formation of helical superstructures as observed by transmission electron microscopy. It was also found that this small acid additive can also enable phase-selective ultra-high loading of nanoparticles (NPs) into target domains of the block copolymer composites. The loading of tartaric acid can increase enthalpically favorable interactions between the nanoparticle ligands and the host domain and mitigate entropic penalties associated with NP incorporation into the target domain. A metal content of over 40 weight percent by mass of the resulting well ordered composites was achieved as measured by thermal gravimetric analysis in PEO-b-PtBA/tartaric acid/4-hydroxythiophenol functionalized Au NP hybrid system. Funding from Center for Hierarchical Manufacturing (CHM); Facility support from Materials Research Science and Engineering Center at UMass Amherst.

  7. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  8. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.

    PubMed

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS-PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS-PKS hybrid enzyme. PMID:26503170

  9. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

    PubMed Central

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

  10. Fibrillar networks of glycyrrhizic acid for hybrid nanomaterials with catalytic features.

    PubMed

    Saha, Abhijit; Adamcik, Jozef; Bolisetty, Sreenath; Handschin, Stephan; Mezzenga, Raffaele

    2015-04-27

    Self-assembly of the naturally occurring sweetening agent, glycyrrhizic acid (GA) in water is studied by small-angle X-ray scattering and microscopic techniques. Statistical analysis on atomic force microscopy images reveals the formation of ultralong GA fibrils with uniform thickness of 2.5 nm and right-handed twist with a pitch of 9 nm, independently of GA concentration. Transparent nematic GA hydrogels are exploited to create functional hybrid materials. Two-fold and three-fold hybrids are developed by introducing graphene oxide (GO) and in situ-synthesized gold nanoparticles (Au NPs) in the hydrogel matrix for catalysis applications. In the presence of GO, the catalytic efficiency of Au NPs in the reduction of p-nitrophenol to p-aminophenol is enhanced by 2.5 times. Gold microplate single crystals are further synthesized in the GA hydrogel, expanding the scope of these hybrids and demonstrating their versatility in materials design. PMID:25759108

  11. Multicolor fluorescence in situ hybridization with centromeric DNA probes as a new approach to distinguish chromosome breakage from aneuploidy in interphase cells and micronuclei

    SciTech Connect

    Eastmond, D.A.; Rupa, D.S.; Chen, H.W.; Hasegawa, L.

    1993-12-31

    Chromosomal abnormalities are believed to contribute significantly to human reproductive failure, carcinogenesis and other pathophysiological conditions. For example, approximately 15% of recognized pregnancies terminate in spontaneous abortion, and of these approximately 30% have been shown to be chromosomally abnormal. The contribution of chromosomal abnormalities to early embryonic and fetal death appears to decrease with gestational age, suggesting that as many as 67% of the aborted embryos in early embryonic deaths are chromosomally abnormal. Furthermore, clinically significant chromosomal abnormalities can also be found to be present in approximately 0.58 to 0.67% of live births. These figures indicate that within a given year, hundreds of thousands of chromosomally abnormal babies will be born throughout the world and additional millions of chromosomally abnormal embryos will have been spontaneously aborted. For the past several years, our research has focused on utilizing new molecular cytogenetic techniques to develop assays for detecting aneuploidy-inducing agents in mammalian cells. One approach that we have sucessfully employed involves the use of fluorescence in situ hybridization with chromosome-specific DNA probes to determine the number of copies of a representative chromosome present within the nucleus following chemical exposure. DNA sequences (probes) which hybridize to blocks of repetitive centromeric DNA on specific chromosomes have been developed for most of the human chromosomes. In situ hybridization with these probes results in the staining of a compact chromosomal region which can be easily detected in interphase nuclei. The presence of 3 (or more) hybridization domains in an interphase nucleus indicates the presence of three centromeric regions and has been presumed to indicate that three copies of the entire chromosome were present in the nucleus.

  12. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1991-01-01

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

  13. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1991-07-02

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

  14. Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

    PubMed

    Xu, Yao; Zheng, Zhi

    2016-05-15

    We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. PMID:26761615

  15. p-Aminophenylacetic acid-mediated synthesis of monodispersed titanium oxide hybrid microspheres in ethanol solution.

    PubMed

    Zhang, Hongye; Xie, Yun; Liu, Zhimin; Tao, Ranting; Sun, Zhenyu; Ding, Kunlun; An, Guimin

    2009-10-15

    Monodispersed TiO2 hybrid microspheres were prepared via the hydrolysis of titanium isopropoxide (TTIP) in ethanol solution containing p-aminophenylacetic acid (APA). The effects of the APA:TTIP molar ratio, water content, reaction time and reaction temperature on the morphology of the resultant spheres were investigated. The products were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction. It was demonstrated that the diameters of the resultant TiO2 spheres could be tuned in the range of 380-800 nm by changing the APA:TTIP molar ratio (1:3 to 3:1) and water content (1-3 v/v%) in the reaction medium, and that increasing the APA:TTIP molar ratio led to larger TiO2 hybrid spheres while increasing the water content decreased their size. The loading content of APA in the hybrid spheres could reach 20 wt.% as they were prepared with the APA:TTIP ratio of 3:1. The possible formation mechanism of the hybrid spheres was also investigated. It was found that APA slowed down the hydrolysis rate of the titanium precursor so that resulted in the formation of the TiO2 spheres. In addition, the APA present in TiO2 spheres acted as a reducing agent to in situ convert HAuCl4 into metallic Au on the surface of the TiO2 spheres. The catalytic activity of the resultant Au/APA-TiO2 composite was examined using transfer hydrogenation of phenylacetone with 2-propanol, and it was indicated that the catalyst displayed high efficiency for this reaction. PMID:19616218

  16. Hybrid Processes Combining Photocatalysis and Ceramic Membrane Filtration for Degradation of Humic Acids in Saline Water

    PubMed Central

    Song, Lili; Zhu, Bo; Gray, Stephen; Duke, Mikel; Muthukumaran, Shobha

    2016-01-01

    This study explored the combined effects of photocatalysis with ceramic membrane filtration for the removal of humic acid in the presence of salt; to simulate saline wastewater conditions. The effects of operating parameters, such as salinity and TiO2 concentration on permeate fluxes, total organic carbon (TOC), and UV absorbance removal, were investigated. The interaction between the humic acids and TiO2 photocatalyst played an important role in the observed flux change during ceramic membrane filtration. The results for this hybrid system showed that the TOC removal was more than 70% for both without NaCl and with the 500 ppm NaCl concentration, and 62% and 66% for 1000 and 2000 ppm NaCl concentrations. The reduction in UV absorbance was more complete in the absence of NaCl compared to the presence of NaCl. The operation of the integrated photoreactor-ceramic membrane filter over five repeat cycles is described. It can be concluded that the overall removal performance of the hybrid system was influenced by the presence of salts, as salt leads to agglomeration of TiO2 particles by suppressing the stabilising effects of electrostatic repulsion and thereby reduces the effective surface contact between the pollutant and the photocatalyst. PMID:26938568

  17. Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model

    NASA Astrophysics Data System (ADS)

    Dora Tang, T.-Y.; Rohaida Che Hak, C.; Thompson, Alexander J.; Kuimova, Marina K.; Williams, D. S.; Perriman, Adam W.; Mann, Stephen

    2014-06-01

    Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes.

  18. Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model.

    PubMed

    Dora Tang, T-Y; Rohaida Che Hak, C; Thompson, Alexander J; Kuimova, Marina K; Williams, D S; Perriman, Adam W; Mann, Stephen

    2014-06-01

    Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes. PMID:24848239

  19. Novel method for high throughput DNA methylation marker evaluation using PNA-probe library hybridization and MALDI-TOF detection.

    PubMed

    Schatz, Philipp; Distler, Jürgen; Berlin, Kurt; Schuster, Matthias

    2006-01-01

    The methylation of CpG dinucleotides has become a topic of great interest in cancer research, and the methylation of promoter regions of several tumor suppressor genes has been identified as a marker of tumorigenesis. Evaluation of DNA methylation markers in tumor tissue requires hundreds of samples, which must be analyzed quantitatively due to the heterogeneous composition of biological material. Therefore novel, fast and inexpensive methods for high throughput analysis are needed. Here we introduce a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. This method is multiplexable, allows the use of standard 384 well automated pipetting, and is more specific and flexible than established methods, such as microarrays and MS-SNuPE. The approach was used to evaluate three candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Ade-nomatous polyposis coli (APC), glycogen synthase kinase-beta-3 (GSK3beta) and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. APC and EYA4 were confirmed as being differentially methylated in colon cancer patients whereas GSK3beta did not show differential methylation. PMID:16670426

  20. Novel method for high throughput DNA methylation marker evaluation using PNA-probe library hybridization and MALDI-TOF detection

    PubMed Central

    Schatz, Philipp; Distler, Jürgen; Berlin, Kurt; Schuster, Matthias

    2006-01-01

    The methylation of CpG dinucleotides has become a topic of great interest in cancer research, and the methylation of promoter regions of several tumor suppressor genes has been identified as a marker of tumorigenesis. Evaluation of DNA methylation markers in tumor tissue requires hundreds of samples, which must be analyzed quantitatively due to the heterogeneous composition of biological material. Therefore novel, fast and inexpensive methods for high throughput analysis are needed. Here we introduce a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. This method is multiplexable, allows the use of standard 384 well automated pipetting, and is more specific and flexible than established methods, such as microarrays and MS-SNuPE. The approach was used to evaluate three candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Ade-nomatous polyposis coli (APC), glycogen synthase kinase-β-3 (GSK3β) and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. APC and EYA4 were confirmed as being differentially methylated in colon cancer patients whereas GSK3β did not show differential methylation. PMID:16670426

  1. Effect of Backbone Design on Hybridization Thermodynamics of Oligo-nucleic Acids: A Coarse-Grained Molecular Dynamics Simulation Study

    NASA Astrophysics Data System (ADS)

    Ghobadi, Ahmadreza F.; Jayaraman, Arthi

    DNA hybridization is the basis of various bio-nano technologies, such as DNA origami and assembly of DNA-functionalized nanoparticles. A hybridized double stranded (ds) DNA is formed when complementary nucleobases on hybridizing strands exhibit specific and directional hydrogen bonds through canonical Watson-Crick base-pairing interactions. In recent years, the need for cheaper alternatives and significant synthetic advances have driven design of DNA mimics with new backbone chemistries. However, a fundamental understanding of how these backbone modifications in the oligo-nucleic acids impact the hybridization and melting behavior of the duplex is still lacking. In this talk, we present our recent findings on impact of varying backbone chemistry on hybridization of oligo-nucleic acid duplexes. We use coarse-grained molecular dynamics simulations to isolate the effect of strand flexibility, electrostatic interactions and nucleobase spacing on the melting curves for duplexes with various strand sequences and concentrations. Since conjugation of oligo-nucleic acids with polymers serve as building blocks for thermo-responsive polymer networks and gels, we also present the effect of such conjugation on hybridization thermodynamics and polymer conformation.

  2. Salinomycin Hydroxamic Acids: Synthesis, Structure, and Biological Activity of Polyether Ionophore Hybrids.

    PubMed

    Borgström, Björn; Huang, Xiaoli; Chygorin, Eduard; Oredsson, Stina; Strand, Daniel

    2016-06-01

    The polyether ionophore salinomycin has recently gained attention due to its exceptional ability to selectively reduce the proportion of cancer stem cells within a number of cancer cell lines. Efficient single step strategies for the preparation of hydroxamic acid hybrids of this compound varying in N- and O-alkylation are presented. The parent hydroxamic acid, salinomycin-NHOH, forms both inclusion complexes and well-defined electroneutral complexes with potassium and sodium cations via 1,3-coordination by the hydroxamic acid moiety to the metal ion. A crystal structure of an cationic sodium complex with a noncoordinating anion corroborates this finding and, moreover, reveals a novel type of hydrogen bond network that stabilizes the head-to-tail conformation that encapsulates the cation analogously to the native structure. The hydroxamic acid derivatives display down to single digit micromolar activity against cancer cells but unlike salinomycin selective reduction of ALDH(+) cells, a phenotype associated with cancer stem cells was not observed. Mechanistic implications are discussed. PMID:27326340

  3. Near-infrared fluorescence probe for the determination of acid phosphatase and imaging of prostate cancer cells.

    PubMed

    Lin, Zihan; Liu, Ziping; Zhang, Hao; Su, Xingguang

    2015-03-01

    In this paper, we developed a near-infrared mercaptopropionic acid (MPA)-capped CuInS2 quantum dot (QD) fluorescence probe for the detection of acid phosphatases (ACP), which is an important biomarker and indicator of prostate cancer. The fluorescence of CuInS2 QDs could be quenched by Cu(2+), and then the addition of adenosine-5'-triphosphate (ATP) could effectively turn on the quenched fluorescence due to the strong interaction between Cu(2+) and ATP. The ACP could catalyze the hydrolysis of ATP, which would disassemble the complex of Cu(2+)-ATP. Therefore, the recovered fluorescence could be quenched again by the addition of ACP. In our method, the limit of detection (LOD) is considerably low for ACP detection in solution. Using the CuInS2 QDs fluorescence probe, we successfully performed in vitro imaging of human prostate cancer cells. PMID:25632410

  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. PMID:18494480

  5. Development of SSR Markers Linked to Low Hydrocyanic Acid Content in Sorghum-Sudan Grass Hybrid Based on BSA Method.

    PubMed

    Xiao-Xia, Yu; Zhi-Hua, Liu; Zhuo, Yu; Yue, Shi; Xiao-Yu, Li

    2016-01-01

    Sorghum-Sudan grass hybrid containing high hydrocyanic acid content can cause hydrocyanic acid poisoning to the livestock and limit the popularization of this forage crop. Molecular markers associated with low hydrocyanic acid content can speed up the process of identification of genotypes with low hydrocyanic acid content. In the present study, 11 polymorphic SSR primers were screened and used for bulked segregant analysis and single marker analysis. Three SSR markers Xtxp7230, Xtxp7375 and Bnlg667960 associated with low hydrocyanic acid content were rapidly identified by BSA. In single marker analysis, six markers Xtxp7230, Xtxp7375, Bnlg667960, Xtxp67-11, Xtxp295-7 and Xtxp12-9 were linked to low hydrocyanic acid content, which explained the proportion of phenotypic variation from 7.6 % to 41.2 %. The markers identified by BSA were also verified by single marker analysis. The three SSR marker bands were then cloned and sequenced for sequence homology analysis in NCBI. It is the first report on the development of molecular markers associated with low hydrocyanic acid content in sorghum- Sudan grass hybrid. These markers will be useful for genetic improvement of low hydrocyanic acid sorghum-Sudan grass hybrid by marker-assisted breeding. PMID:27001403

  6. One-pot transformation of cellobiose to formic acid and levulinic acid over ionic-liquid-based polyoxometalate hybrids.

    PubMed

    Li, Kaixin; Bai, Linlu; Amaniampong, Prince Nana; Jia, Xinli; Lee, Jong-Min; Yang, Yanhui

    2014-09-01

    Currently, levulinic acid (LA) and formic acid (FA) are considered as important carbohydrates for the production of value-added chemicals. Their direct production from biomass will open up a new opportunity for the transformation of biomass resource to valuable chemicals. In this study, one-pot transformation of cellobiose into LA and FA was demonstrated, using a series of multiple-functional ionic liquid-based polyoxometalate (IL-POM) hybrids as catalytic materials. These IL-POMs not only markedly promoted the production of valuable chemicals including LA, FA and monosaccharides with high selectivities, but also provided great convenience of the recovery and the reuse of the catalytic materials in an environmentally friendly manner. Cellobiose conversion of 100%, LA selectivity of 46.3%, and FA selectivity of 26.1% were obtained at 423 K and 3 MPa for 3 h in presence of oxygen. A detailed catalytic mechanism for the one-pot transformation of cellobiose was also presented. PMID:25110998

  7. Theoretical study of the NLO responses of some natural and unnatural amino acids used as probe molecules.

    PubMed

    Derrar, S N; Sekkal-Rahal, M; Derreumaux, P; Springborg, M

    2014-08-01

    The first hyperpolarizabilities β of the natural aromatic amino acids tryptophan and tyrosine have been investigated using several methods and basis sets. Some of the theoretical results obtained were compared to the only experimental hyper-Rayleigh scattering data available. The sensitivity of tryptophan to its local environment was analyzed by constructing two-dimensional potential energy plots around the dipeptide tryptophan-lysine. Static hyperpolarizabilities β(0) of the found minima were calculated by a second-order Møller-Plesset (MP2) method in combination with the 6-31+G(d) basis set. Moreover, the efficiency of tryptophan and those of a series of unnatural amino acids as endogenous probe molecules were tested by calculating the nonlinear responses of some peptides. Impressive results were obtained for the amino acid ALADAN, which shows significantly improved nonlinear performance compared to other amino acids with weak nonlinear responses. PMID:25092242

  8. 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.

  9. A carbon dot-based "off-on" fluorescent probe for highly selective and sensitive detection of phytic acid.

    PubMed

    Gao, Zhao; Wang, Libing; Su, Rongxin; Huang, Renliang; Qi, Wei; He, Zhimin

    2015-08-15

    We herein report a facile, one-step pyrolysis synthesis of photoluminescent carbon dots (CDs) using citric acid as the carbon source and lysine as the surface passivation reagent. The as-prepared CDs show narrow size distribution, excellent blue fluorescence and good photo-stability and water dispersivity. The fluorescence of the CDs was found to be effectively quenched by ferric (Fe(III)) ions with high selectivity via a photo-induced electron transfer (PET) process. Upon addition of phytic acid (PA) to the CDs/Fe(III) complex dispersion, the fluorescence of the CDs was significantly recovered, arising from the release of Fe(III) ions from the CDs/Fe(III) complex because PA has a higher affinity for Fe(III) ions compared to CDs. Furthermore, we developed an "off-on" fluorescence assay method for the detection of phytic acid using CDs/Fe(III) as a fluorescent probe. This probe enables the selective detection of PA with a linear range of 0.68-18.69 μM and a limit of detection (signal-to-noise ratio is 3) of 0.36 μM. The assay method demonstrates high selectivity, repeatability, stability and recovery ratio in the detection of the standard and real PA samples. We believe that the facile operation, low-cost, high sensitivity and selectivity render this CD-based "off-on" fluorescent probe an ideal sensing platform for the detection of PA. PMID:25829220

  10. 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

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

    PubMed

    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 (CC) 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 (210nm) and the two well-resolved emission peaks separated by 140nm. 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 50s), 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. PMID:27236136

  12. 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

  13. A universal strategy for visual chiral recognition of α-amino acids with l-tartaric acid-capped gold nanoparticles as colorimetric probes.

    PubMed

    Song, Guoxin; Zhou, Fulin; Xu, Chunli; Li, Baoxin

    2016-02-01

    The ability to recognize and quantify the chirality of alpha-amino acids constitutes the basis of many critical areas for specific targeting in drug development and metabolite probing. It is still challenging to conveniently distinguish the enantiomer of amino acids largely due to the lack of a universal and simple strategy. In this work, we report a strategy for the visual recognition of α-amino acids. It is based on the chirality of l-tartaric acid-capped gold nanoparticles (l-TA-capped AuNPs, ca. 13 nm in diameter). All of 19 right-handed α-amino acids can induce a red-to-blue color change of l-TA-capped AuNP solution, whereas all of the left-handed amino acids (except cysteine) cannot. The chiral recognition can be achieved by the naked eye and a simple spectrophotometer. This method does not require complicated chiral modification, and excels through its low-cost, good availability of materials and its simplicity. Another notable feature of this method is its high generality, and this method can discriminate almost all native α-amino acid enantiomers. This versatile method could be potentially used for high-throughput chiral recognition of amino acids. PMID:26759834

  14. Automated DNA electrophoresis, hybridization and detection

    SciTech Connect

    Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

    1986-05-01

    A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; /sup 32/P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing.

  15. Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization

    SciTech Connect

    Arnold, N.; Wienberg, J.; Ermert, K.

    1995-03-01

    Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.

  16. A hybrid Li-air battery with buckypaper air cathode and sulfuric acid electrolyte

    SciTech Connect

    Li, YF; Huang, K; Xing, YC

    2012-10-30

    We demonstrate a type of carbon nanotube based buckypaper cathode in a hybrid electrolyte Li-air battery (HyLAB) that showed outstanding discharging performances. The HyLAB has sulfuric acid as the catholyte and a large active electrode area (10 cm(2)). The active cathode layer was made from a buckypaper with 5 wt.% Pt supported on carbon nanotubes (Pt/CNTs) for oxygen reduction and evolution. A similar cathode was constructed with a catalyst of 5 wt.% Pt supported on carbon black (Pt/CB). It is demonstrated that sulfuric acid can achieve high discharging current densities while maintaining relatively high cell potentials. The cell with Pt/CNTs showed a much better performance than with Pt/CB at high current densities. The HyLAB with Pt/CNTs achieved a discharging capacity of 306 mAh/g and a cell voltage of 3.15 V at 0.2 mA/cm(2). The corresponding specific energy is 1067 Wh/kg based on the total weight of the sulfuric acid. Slow decrease in performance was observed, but it can be recovered by refilling the cell with new electrolyte after continuous discharging of more than 75 h. A charge-discharge experiment at 0.2 mA/cm(2) showed that the cell was rechargeable with a capacity of more than 300 mAh/g. (c) 2012 Elsevier Ltd. All rights reserved.

  17. Development and operation of a hybrid acid-alkaline advanced water electrolysis cell

    NASA Astrophysics Data System (ADS)

    Teschke, O.; Zwanziger, M.

    A hybrid acid-alkaline water electrolysis cell has been developed for hydrogen production. The cell is based on the use of an acidic solution at the cathode and a basic solution at the anode to reduce the minimum theoretical voltage for water decomposition from the thermoneutral potential of 1.47 V to close to 1.4 V at 25 C and 1 atm. The pH differential is maintained by the removal of OH ions from the cathode section and water removal from the anode section, which can be driven by heat energy. A practical cell has been built using a solid polymer electrolyte in which, however, the cathodic compartment is not acidic but neutral. Tests with a platinum black cathode catalyst and a platinum-iridium anode catalyst have resulted in steady-state water hydrolysis at an applied voltage of 0.9 V, and a V-I diagram with a considerably lower slope than that of a conventional cell has been obtained at 90 C.

  18. Split-probe hybrid femtosecond/picosecond rotational CARS for time-domain measurement of S-branch Raman linewidths within a single laser shot.

    PubMed

    Patterson, Brian D; Gao, Yi; Seeger, Thomas; Kliewer, Christopher J

    2013-11-15

    We introduce a multiplex technique for the single-laser-shot determination of S-branch Raman linewidths with high accuracy and precision by implementing hybrid femtosecond (fs)/picosecond (ps) rotational coherent anti-Stokes Raman spectroscopy (CARS) with multiple spatially and temporally separated probe beams derived from a single laser pulse. The probe beams scatter from the rotational coherence driven by the fs pump and Stokes pulses at four different probe pulse delay times spanning 360 ps, thereby mapping collisional coherence dephasing in time for the populated rotational levels. The probe beams scatter at different folded BOXCARS angles, yielding spatially separated CARS signals which are collected simultaneously on the charge coupled device camera. The technique yields a single-shot standard deviation (1σ) of less than 3.5% in the determination of Raman linewidths and the average linewidth values obtained for N(2) are within 1% of those previously reported. The presented technique opens the possibility for correcting CARS spectra for time-varying collisional environments in operando. PMID:24322075

  19. Application of Sequence-Specific Labeled 16S rRNA Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial Abundance and Diversity by Array Hybridization

    PubMed Central

    Rudi, Knut; Skulberg, Olav M.; Skulberg, Randi; Jakobsen, Kjetill S.

    2000-01-01

    DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats. PMID:10966421

  20. Lead-acid batteries in micro-hybrid applications. Part II. Test proposal

    NASA Astrophysics Data System (ADS)

    Schaeck, S.; Stoermer, A. O.; Albers, J.; Weirather-Koestner, D.; Kabza, H.

    In the first part of this work [1] selected key parameters for applying lead-acid (LA) batteries in micro-hybrid power systems (MHPS) were investigated. Main results are integrated in an accelerated, comprehensive test proposal presented here. The test proposal aims at a realistic representation of the pSoC operation regime, which is described in Refs. [1,6]. The test is designed to be sensitive with respect to dynamic charge acceptance (DCA) at partially discharged state (critical for regenerative braking) and the internal resistance at high-rate discharge (critical for idling stop applications). First results are presented for up-to-date valve-regulated LA batteries with absorbent glass mat (AGM) separators. The batteries are close to the limits of the first proposal of pass/fail-criteria. Also flooded batteries were tested; the first out of ten units failed already.

  1. Advanced design of valve-regulated lead-acid battery for hybrid electric vehicles

    NASA Astrophysics Data System (ADS)

    Lam, L. T.; Newnham, R. H.; Ozgun, H.; Fleming, F. A.

    A novel design of lead-acid battery has been developed for use in hybrid electric vehicles (HEVs). The battery has current take-offs at both ends of each of the positive and negative plates. This feature markedly reduces battery operating temperatures, improves battery capacity, and extends cycle-life under HEV duty. The battery also performs well under partial-state-of-charge (PSoC)/fast-charge, electric-vehicle operation. The improvements in performance are attributed to more uniform utilization of the plate active-materials. The battery, combined with an internal-combustion engine and a new type of supercapacitor, will be used to power an HEV, which is being designed and constructed by an Australian industry-government consortium.

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

    SciTech Connect

    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.

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

    SciTech Connect

    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.

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

    SciTech Connect

    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.

  5. 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.

  6. 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.

  7. Lead-acid batteries in micro-hybrid applications. Part I. Selected key parameters

    NASA Astrophysics Data System (ADS)

    Schaeck, S.; Stoermer, A. O.; Kaiser, F.; Koehler, L.; Albers, J.; Kabza, H.

    Micro-hybrid electric vehicles were launched by BMW in March 2007. These are equipped with brake energy regeneration (BER) and the automatic start and stop function (ASSF) of the internal combustion engine. These functions are based on common 14 V series components and lead-acid (LA) batteries. The novelty is given by the intelligent onboard energy management, which upgrades the conventional electric system to the micro-hybrid power system (MHPS). In part I of this publication the key factors for the operation of LA batteries in the MHPS are discussed. Especially for BER one is high dynamic charge acceptance (DCA) for effective boost charging. Vehicle rest time is identified as a particular negative parameter for DCA. It can be refreshed by regular fully charging at elevated charge voltage. Thus, the batteries have to be outstandingly robust against overcharge and water loss. This can be accomplished for valve-regulated lead-acid (VRLA) batteries at least if they are mounted in the trunk. ASSF goes along with frequent high-rate loads for warm cranking. The internal resistance determines the drop of the power net voltage during cranking and is preferably low for reasons of power net stability even after years of operation. Investigations have to be done with aged 90 Ah VRLA-absorbent glass mat (AGM) batteries. Battery operation at partial state-of-charge gives a higher risk of deep discharging (overdischarging). Subsequent re-charging then is likely to lead to the formation of micro-short circuits in the absorbent glass mat separator.

  8. Ferulic acid-carbazole hybrid compounds: Combination of cholinesterase inhibition, antioxidant and neuroprotection as multifunctional anti-Alzheimer agents.

    PubMed

    Fang, Lei; Chen, Mohao; Liu, Zhikun; Fang, Xubin; Gou, Shaohua; Chen, Li

    2016-02-15

    In order to search for novel multifunctional anti-Alzheimer agents, a series of ferulic acid-carbazole hybrid compounds were designed and synthesized. Ellman's assay revealed that the hybrid compounds showed moderate to potent inhibitory activity against the cholinesterases. Particularly, the AChE inhibition potency of compound 5k (IC50 1.9μM) was even 5-fold higher than that of galantamine. In addition, the target compounds showed pronounced antioxidant ability and neuroprotective property, especially against the ROS-induced toxicity. Notably, the neuroprotective effect of 5k was obviously superior to that of the mixture of ferulic acid and carbazole, indicating the therapeutic effect of the hybrid compound is better than the combination administration of the corresponding mixture. PMID:26795115

  9. Nitrilotriacetic acid-coated magnetic nanoparticles as affinity probes for enrichment of histidine-tagged proteins and phosphorylated peptides.

    PubMed

    Li, Yi-Cheng; Lin, Ya-Shiuan; Tsai, Pei-Jane; Chen, Cheng-Tai; Chen, Wei-Yu; Chen, Yu-Chie

    2007-10-01

    We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA) that can bind with different immobilized metal ions are capable of probing diverse target species. Immobilized Ni(II) on the surfaces of the NTA-magnetic nanoparticles have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with 6x His, i.e., C192S (MW approximately 42 kDa), from cell lysates. Enrichment was achieved by vigorously mixing the sample solution and the nanoparticles by pipetting in and out of a sample vial for only 30 s. After enrichment, the probe-target species could be readily isolated by magnetic separation. We also characterized the proteins enriched on the affinity probes using on-probe tryptic digestion under microwave irradiation for only 2 min, followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. Using this enrichment and tryptic digestion, the target species can be rapidly enriched and characterized, reducing the time required for carrying out the complete analysis to less than 10 min. Furthermore, when either Zr(IV) or Ga (III) ions are immobilized on the surfaces of the NTA-magnetic nanoparticles, the nanoparticles have the capability of selectively enriching phosphorylated peptides from tryptic digests of alpha-, beta-caseins, and diluted milk. The detection limit for the tryptic digests of alpha- and beta-caseins is approximately 50 fmol. PMID:17784733

  10. Nanoparticle Formation from Hybrid, Multiblock Copolymers of Poly(Acrylic Acid) and VPGVG Peptide

    PubMed Central

    Grieshaber, Sarah E.; Paik, Bradford A.; Bai, Shi; Kiick, Kristi L.; Jia, Xinqiao

    2012-01-01

    Elastin-mimetic hybrid copolymers with an alternating molecular architecture were synthesized via the step growth polymerization of azide-functionalized, telechelic poly(tert-butyl acrylate) (PtBA) and an alkyne-terminated, valine and glycine-rich peptide with a sequence of (VPGVG)2 (VG2). The resultant hybrid copolymer, [PtBA-VG2]n, contains up to six constituent building blocks and has a polydispersity index (PDI) of ~1.9. Trifluoroacetic acid (TFA) treatment of [PtBA-VG2]n gave rise to an alternating copolymer of poly(acrylic acid) (PAA) and VG2 ([PAA-VG2]n). The modular design permits facile adjustment of the copolymer composition by varying the molecular weight of PAA (22 and 63 repeat units). Characterization by dynamic light scattering indicated that the multiblock copolymers formed discrete nanoparticles at room temperature in aqueous solution at pH 3.8, with an average diameter of 250-270 nm and a particle size distribution of 0.34 for multiblock copolymers containing PAA22 and 0.17 for those containing PAA63. Upon increasing the pH to 7.4, both types of particles were able to swell without being disintegrated, reaching an average diameter of 285-300 nm for [PAA22-VG2]n and 330-350 nm for [PAA63-VG2]n, respectively. The nanoparticles were not dissociated upon the addition of urea, further confirming their unusual stability. The nanoparticles were capable of sequestering a hydrophobic fluorescent dye (pyrene), and the critical aggregation concentration (CAC) was determined to be 1.09 × 10-2 or 1.05 × 10-2 mg/mL for [PAA22-VG2]n and [PAA63-VG2]n, respectively. We suggest that the multiblock copolymers form through collective H-bonding and hydrophobic interactions between the PAA and VG2 peptide units, and that the unusual stability of the multiblock nanoparticles is conferred by the multiblock architecture. These hybrid multiblock copolymers are potentially useful as pH-responsive drug delivery vehicles, with the possibility of drug loading through

  11. DNA and RNA "traffic lights": synthetic wavelength-shifting fluorescent probes based on nucleic acid base substitutes for molecular imaging.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2013-08-01

    The DNA base substitute approach by the (S)-3-amino-1,2-propanediol linker allows placing two fluorophores in a precise way inside a given DNA framework. The double helical architecture around the fluorophores, especially the DNA-induced twist, is crucial for the desired photophysical interactions. Excitonic, excimer, and energy transfer interactions yield fluorescent DNA and RNA probes with dual emission color readout. Especially, our DNA and RNA "traffic light" that combines the green emission of TO with the red emission of TR represents an important tool for molecular imaging and can be applied as aptasensors and as probes to monitor the siRNA delivery into cells. The concept can be extended to the synthetically easier to access postsynthetic 2'-modifications and the NIR range. Thereby, the pool of tailor-made fluorescent nucleic acid conjugates can be extended. PMID:23796243

  12. 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

  13. Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

    PubMed Central

    Diez, Cristina; Lorenz, Virginia; Ortiz, Silvia; Gonzalez, Verónica; Racca, Andrea; Bontempi, Iván; Manattini, Silvia; Solari, Aldo; Marcipar, Iván

    2010-01-01

    We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe. PMID:20064998

  14. The Interaction of the Solar Wind with Solar Probe Plus - 3D Hybrid Simulation. Report 1; The Study for the Distance 4.5Rs

    NASA Technical Reports Server (NTRS)

    Lipatov, Alexander S.; Sittler, Edward C.; Hartle, Richard E.; Cooper, John F.

    2010-01-01

    Our report devotes a 3D numerical hybrid model of the interaction of the solar wind with the Solar Probe spacecraft. The Solar Probe Plus (SPP) model includes 3 main parts, namely, a non-conducting heat shield, a support system, and cylindrical section or spacecraft bus that contains the particle analysis devices and antenna. One observes an excitation of the low frequency Alfven and whistler type wave directed by the magnetic field with an amplitude of about (0.06-0.6) V/m. The compression waves and the jumps in an electric field with an amplitude of about (0.15-0.7) V/m were also observed. The wave amplitudes are comparable to or greater than previously estimated max wave amplitudes that SPP is expected to measure. The results of our hybrid simulation will be useful for understanding the plasma environment near the SPP spacecraft at the distance 4.5 Rs. Future simulation will take into account the charging of the spacecraft, the charge separation effects, an outgassing from heat shield, a photoionization and an electron impact ionization effects near the spacecraft.

  15. Development of a lead-acid battery for a hybrid electric vehicle

    NASA Astrophysics Data System (ADS)

    Cooper, A.

    In September 2000, a project reliable, highly optimized lead-acid battery (RHOLAB) started under the UK Foresight Vehicle Programme with the objective of developing an optimized lead-acid battery solution for hybrid electric vehicles. The work is based on a novel, individual, spirally-wound valve-regulated lead-acid 2 V cell optimized for HEV use and low variability. This cell is being used as a building block for the development of a complete battery pack that is managed at the cell level. Following bench testing, this battery pack is to be thoroughly evaluated by substituting it for the Ni-MH pack in a Honda Insight. The RHOLAB cell is based on the 8 Ah Hawker Cyclon cell which has been modified to have current take-off at both ends—the dual-tab design. In addition, a variant has been produced with modified cell chemistry to help deal with problems that can occur when these valve-regulated lead-acid battery (VRLA) cells operate in a partial-state-of-charge condition. The cells have been cycled to a specially formulated test cycle based on real vehicle data derived from testing the Honda Insight on the various test tracks at the Millbrook Proving Grounds in the UK. These cycling tests have shown that the lead-acid pack can be successfully cycled when subjected to the high current demands from the vehicle, which have been measured at up to 15 C on discharge and 8 C during regenerative recharging, and cycle life is looking very promising under this arduous test regime. Concurrent with this work, battery development has been taking place. It was decided early on to develop the 144 V battery as four 36 V modules. Data collection and control has been built-in and special steps taken to minimize the problems of interconnect in this complex system. Development of the battery modules is now at an advanced stage. The project plan then allows for extensive testing of the vehicle with its lead-acid battery at Millbrook so it can be compared with the benchmark tests which

  16. A ratiometric fluorescent probe based on boron dipyrromethene and rhodamine Förster resonance energy transfer platform for hypochlorous acid and its application in living cells.

    PubMed

    Liu, Ying; Zhao, Zhi-Min; Miao, Jun-Ying; Zhao, Bao-Xiang

    2016-05-19

    We have developed a ratiometric fluorescent probe BRT based on boron dipyrromethene (BODIPY) and rhodamine-thiohydrazide Förster resonance energy transfer (FRET) platform for sensing hypochlorous acid (HOCl) with high selectivity and sensitivity. The probe can detect HOCl in 15 s with the detection limit of 38 nM. Upon mixing with HOCl the fluorescence colour of probe BRT changed from green to orange. Moreover, probe BRT was applied to successfully monitor HOCl in living RAW 264.7 cells. PMID:27126792

  17. Preparation and Electrochemical Performance of Hybrid Materials Containing Heteropoly Acid with Dawson Structure and Polymers

    NASA Astrophysics Data System (ADS)

    Tong, Xia; Wu, Wen; Zhou, Shengming; Wu, Qingyin; Cao, Fahe; Yaroslavtsev, A. B.

    2012-11-01

    Highly proton-conducting hybrid materials (P2W17V/PEG and P2W17V/PEG/SiO2) were prepared by heptadecatungstovanadodiphosphoric heteropoly acid with Dawson structure (P2W17V, 90 wt.%), polyethylene glycol (PEG, 10 wt.% and 5 wt.%) and silica gel (SiO2, 0 wt.% and 5 wt.%). The products were characterized by the infrared (IR) spectrum, X-ray powder diffraction (XRD) analysis and electrochemical impedance spectrum (EIS). The result reveals that their conductivity values are 1.02 × 10-2 and 2.58 × 10-2S ṡ cm-1 at room temperature (26°C) and 75% relative humidity (RH), respectively. Their conductivities increase with higher temperature and these activation energies of proton conduction are 9.51 and 14.95 kJṡmol-1, which are lower than that of pure heteropoly acid (32.23 kJṡmol-1). These mechanisms of proton conduction for these two materials are Grotthuss mechanism.

  18. THE EFFECT OF ANOLYTE PRODUCT ACID CONCENTRATION ON HYBRID SULFUR CYCLE PERFORMANCE

    SciTech Connect

    Gorensek, M.; Summers, W.

    2010-03-24

    The Hybrid Sulfur (HyS) cycle (Fig. 1) is one of the simplest, all-fluids thermochemical cycles that has been devised for splitting water with a high-temperature nuclear or solar heat source. It was originally patented by Brecher and Wu in 1975 and extensively developed by Westinghouse in the late 1970s and early 1980s. As its name suggests, the only element used besides hydrogen and oxygen is sulfur, which is cycled between the +4 and +6 oxidation states. HyS comprises two steps. One is the thermochemical (>800 C) decomposition of sulfuric acid (H{sub 2}SO{sub 4}) to sulfur dioxide (SO{sub 2}), oxygen (O{sub 2}), and water. H{sub 2}SO{sub 4} = SO{sub 2} + 1/2 O{sub 2} + H{sub 2}O. The other is the SO{sub 2}-depolarized electrolysis of water to H{sub 2}SO{sub 4} and hydrogen (H{sub 2}), SO{sub 2} + 2 H{sub 2}O = H{sub 2}SO{sub 4} + H{sub 2}, E{sup o} = -0.156 V, explaining the 'hybrid' designation. These two steps taken together split water into H{sub 2} and O{sub 2} using heat and electricity. Researchers at the Savannah River National Laboratory (SRNL) and at the University of South Carolina (USC) have successfully demonstrated the use of proton exchange membrane (PEM) electrolyzers (Fig. 2) for the SO{sub 2}-depolarized electrolysis (sulfur oxidation) step, while Sandia National Laboratories (SNL) successfully demonstrated the high-temperature sulfuric acid decomposition (sulfur reduction) step using a bayonet-type reactor (Fig. 3). This latter work was performed as part of the Sulfur-Iodine (SI) cycle Integrated Laboratory Scale demonstration at General Atomics (GA). The combination of these two operations results in a simple process that will be more efficient and cost-effective for the massive production of hydrogen than alkaline electrolysis. Recent developments suggest that the use of PEMs other than Nafion will allow sulfuric acid to be produced at higher concentrations (>60 wt%), offering the possibility of net thermal efficiencies around 50% (HHV basis

  19. Real-Time PCR Genotyping using Taqman Probes to Detect High Oleic Acid Peanuts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oleic acid, a monounsaturated, omega-9 fatty acid is an important agronomic trait in peanut cultivars because it provides increased shelf life, improved flavor, enhanced fatty acid composition, and a beneficial effect on human health. Currently, most high oleic peanuts confer limited resistance to ...

  20. Synthesis and utilization of chitin humic acid hybrid as sorbent for Cr(III)

    NASA Astrophysics Data System (ADS)

    Santosa, Sri Juari; Siswanta, Dwi; Sudiono, Sri; Sehol, Muhamad

    2007-11-01

    New types of hybrid material have been synthesized by using four different methods of immobilization of humic acid (HA) on chitin. The most stable hybrid material toward the change of medium acidity was then utilized as sorbent for Cr(III). The HA was extracted from peat soil of Gambut District, South Kalimantan, Indonesia, using the recommended procedure of International Humic Substances Society (IHSS), while the chitin was isolated from crab shell waste through deproteination using 3.5% (w/v) NaOH and followed by removal of inorganic impurities using 1 M HCl. The four methods of immobilization of HA on chitin were (i) Method A: chitin powder (4 g) was gently poured into the stirred solution of 0.4 g HA in 40 mL of 0.01 M NaOH. After overnight stirring, the solid was separated, washed with water, and dried in oven at 70 °C. (ii) Method B: gelatinous chitin (40 g) in 250 mL of 0.5 M HCl was reacted with HA (4 g) in 500 mL of 0.5 M NaOH and aged for 24 h. The product was washed with water and dried. (iii) Method C: HA powder (0.5 g) was mixed with the stirred gel of chitin (2.5 g) in 60 mL of CaCl 2 saturated methanol and the mixture was then washed with the mixed solution of 25 mL of 2 M sodium citrate and ethylene glycol 1:1. The solid was separated, washed with water, and dried. (iv) Method D: the solution of HA (0.056 g) in 10 mL of 0.01 M NaOH was reacted with the gel of chitin (0.2 g) in 10 mL of CaCl 2 saturated methanol. After 24 h stirring, the solid was separated from the reaction medium, washed with the mixed solution of 2 M sodium citrate and ethylene glycol 1:1, and followed by washing with water and drying. Parameters investigated in this study consisted of the stability test of the immobilized HA, as well as the rate constant ( k1), capacity ( b), and energy ( E) of sorption as well as the rate constant of desorption ( k-1). The k1 and k-1 were determined according to a kinetic model of first order sorption reaching equilibrium, while the b and E

  1. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

    PubMed Central

    Seal, S E; Jackson, L A; Daniels, M J

    1992-01-01

    A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers. Images PMID:1482193

  2. Interfacial micropore defect formation in PEDOT:PSS-Si hybrid solar cells probed by TOF-SIMS 3D chemical imaging.

    PubMed

    Thomas, Joseph P; Zhao, Liyan; Abd-Ellah, Marwa; Heinig, Nina F; Leung, K T

    2013-07-16

    Conducting p-type polymer layers on n-type Si have been widely studied for the fabrication of cost-effective hybrid solar cells. In this work, time-of-flight secondary ion mass spectrometry (TOF-SIMS) is used to provide three-dimensional chemical imaging of the interface between poly(3,4-ethylene-dioxythiophene):polystyrenesulfonate (PEDOT:PSS) and SiOx/Si in a hybrid solar cell. To minimize structural damage to the polymer layer, an Ar cluster sputtering source is used for depth profiling. The present result shows the formation of micropore defects in the interface region of the PEDOT:PSS layer on the SiOx/Si substrate. This interfacial micropore defect formation becomes more prominent with increasing thickness of the native oxide layer, which is a key device parameter that greatly affects the hybrid solar cell performance. Three-dimensional chemical imaging coupled with Ar cluster ion sputtering has therefore been demonstrated as an emerging technique for probing the interface of this and other polymer-inorganic systems. PMID:23745755

  3. Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans.

    PubMed Central

    Meyer, W; Mitchell, T G; Freedman, E Z; Vilgalys, R

    1993-01-01

    In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms. Images PMID:8408543

  4. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

    PubMed Central

    Rijpens, N P; Jannes, G; Van Asbroeck, M; Rossau, R; Herman, L M

    1996-01-01

    The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods. PMID:8633866

  5. Probe-pulse optimization for nonresonant suppression in hybrid fs/ps coherent anti-Stokes Raman scattering at high temperature.

    PubMed

    Miller, Joseph D; Slipchenko, Mikhail N; Meyer, Terrence R

    2011-07-01

    Hybrid femtosecond/picosecond coherent anti-Stokes Raman scattering (fs/ps CARS) offers accurate thermometry at kHz rates for combustion diagnostics. In high-temperature flames, selection of probe-pulse characteristics is key to simultaneously optimizing signal-to-nonresonant-background ratio, signal strength, and spectral resolution. We demonstrate a simple method for enhancing signal-to-nonresonant-background ratio by using a narrowband Lorentzian filter to generate a time-asymmetric probe pulse with full-width-half-maximum (FWHM) pulse width of only 240 fs. This allows detection within just 310 fs after the Raman excitation for eliminating nonresonant background while retaining 45% of the resonant signal at 2000 K. The narrow linewidth is comparable to that of a time-symmetric sinc2 probe pulse with a pulse width of ~2.4 ps generated with a conventional 4-f pulse shaper. This allows nonresonant-background-free, frequency-domain vibrational spectroscopy at high temperature, as verified using comparisons to a time-dependent theoretical fs/ps CARS model. PMID:21747487

  6. Organic-inorganic hybrid proton exchange membranes based on silicon-containing polyacrylate nanoparticles with phosphotungstic acid

    NASA Astrophysics Data System (ADS)

    Cui, Xuejun; Zhong, Shuangling; Wang, Hongyan

    A series of silicon-containing polyacrylate nanoparticles (SiPANPs) were successfully synthesized by simple emulsifier-free emulsion polymerization technique. The resulting latex particles were characterized by Fourier transform infrared (FTIR) spectrometry, dynamic light scattering (DLS) analysis, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The SiPANP membranes and SiPANP/phosphotungstic acid (SiPANP/PWA) hybrid membranes were also prepared and characterized to evaluate their potential as proton exchange membranes in proton exchange membrane fuel cell (PEMFC). Compared with the pure SiPANP membrane, the hybrid membranes displayed lower thermal stability. However, the degradation temperatures were still above 190 °C, satisfying the requirement of thermal stability for PEMFC operation. In addition, the hybrid membranes showed lower water uptake but higher proton conductivity than the SiPANP precursor. The proton conductivity of the hybrid membranes was in the range of 10 -3 to 10 -2 S cm -1 and increased gradually with PWA content and temperature. The excellent hydrolytic stability was also observed in the hybrid membranes because of the existence of crosslinked silica network. The good thermal stability, reasonable water uptake, excellent hydrolytic stability, suitable proton conductivity and cost effectiveness make these hybrids quite attractive as proton exchange membranes for PEMFC applications.

  7. Camera-based ratiometric fluorescence transduction of nucleic acid hybridization with reagentless signal amplification on a paper-based platform using immobilized quantum dots as donors.

    PubMed

    Noor, M Omair; Krull, Ulrich J

    2014-10-21

    Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an

  8. Cationic liposome-hyaluronic acid hybrid nanoparticles for intranasal vaccination with subunit antigens

    PubMed Central

    Ochyl, Lukasz J.; Akerberg, Jonathan; Moon, James J.

    2015-01-01

    Here we report the development of a new cationic liposome-hyaluronic acid (HA) hybrid nanoparticle (NP) system and present our characterization of these NPs as an intranasal vaccine platform using a model antigen and F1-V, a candidate recombinant antigen for Yersinia pestis, the causative agent of plague. Incubation of cationic liposomes composed of DOTAP and DOPE with anionic HA biopolymer led to efficient ionic complexation and formation of homogenous liposome-polymer hybrid NPs, as evidenced by fluorescence resonance energy transfer, dynamic light scattering, and nanoparticle tracking analyses. Incorporation of cationic liposomes with thiolated HA allowed for facile surface decoration of NPs with thiol-PEG, resulting in the formation of DOTAP/HA core-PEG shell nanostructures. These NPs, termed DOTAP-HA NPs, exhibited improved colloidal stability and prolonged antigen release. In addition, cytotoxicity associated with DOTAP liposomes (LC50 ~0.2 mg/ml) was significantly reduced by at least 20-fold with DOTAP-HA NPs (LC50 > 4 mg/ml), as measured with bone marrow dendritic cells (BMDCs). Furthermore, NPs co-loaded with ovalbumin (OVA) and a molecular adjuvant, monophosphoryl lipid A (MPLA) promoted BMDC maturation and upregulation of co-stimulatory markers, including CD40, CD86, and MHC-II, and C57BL/6 mice vaccinated with NPs via intranasal route generated robust OVA-specific CD8+ T cell and antibody responses. Importantly, intranasal vaccination with NPs co-loaded with F1-V and MPLA induced potent humoral immune responses with 11-, 23-, and 15-fold increases in F1-V-specific total IgG, IgG1, and IgG2c titers in immune sera by day 77, respectively, and induced balanced Th1/Th2 humoral immune responses, compared with the lack of sero-conversion in mice immunized with the equivalent doses of soluble F1-V vaccine. Overall, these results suggest that liposome-polymer hybrid NPs may serve as a promising vaccine delivery platform for intranasal vaccination against Y

  9. Cationic liposome-hyaluronic acid hybrid nanoparticles for intranasal vaccination with subunit antigens.

    PubMed

    Fan, Yuchen; Sahdev, Preety; Ochyl, Lukasz J; J Akerberg, Jonathan; Moon, James J

    2015-06-28

    Here we report the development of a new cationic liposome-hyaluronic acid (HA) hybrid nanoparticle (NP) system and present our characterization of these NPs as an intranasal vaccine platform using a model antigen and F1-V, a candidate recombinant antigen for Yersinia pestis, the causative agent of plague. Incubation of cationic liposomes composed of DOTAP and DOPE with anionic HA biopolymer led to efficient ionic complexation and formation of homogenous liposome-polymer hybrid NPs, as evidenced by fluorescence resonance energy transfer, dynamic light scattering, and nanoparticle tracking analyses. Incorporation of cationic liposomes with thiolated HA allowed for facile surface decoration of NPs with thiol-PEG, resulting in the formation of DOTAP/HA core-PEG shell nanostructures. These NPs, termed DOTAP-HA NPs, exhibited improved colloidal stability and prolonged antigen release. In addition, cytotoxicity associated with DOTAP liposomes (LC50~0.2mg/ml) was significantly reduced by at least 20-fold with DOTAP-HA NPs (LC50>4mg/ml), as measured with bone marrow derived dendritic cells (BMDCs). Furthermore, NPs co-loaded with ovalbumin (OVA) and a molecular adjuvant, monophosphoryl lipid A (MPLA) promoted BMDC maturation and upregulation of co-stimulatory markers, including CD40, CD86, and MHC-II, and C57BL/6 mice vaccinated with NPs via intranasal route generated robust OVA-specific CD8(+) T cell and antibody responses. Importantly, intranasal vaccination with NPs co-loaded with F1-V and MPLA induced potent humoral immune responses with 11-, 23-, and 15-fold increases in F1-V-specific total IgG, IgG1, and IgG2c titers in immune sera by day 77, respectively, and induced balanced Th1/Th2 humoral immune responses, whereas mice immunized with the equivalent doses of soluble F1-V vaccine failed to achieve sero-conversion. Overall, these results suggest that liposome-polymer hybrid NPs may serve as a promising vaccine delivery platform for intranasal vaccination against Y

  10. The Use of p-Aminobenzoic Acid as a Probe Substance for the Targeted Profiling of Glycine Conjugation.

    PubMed

    Nortje, Carla; van der Sluis, Rencia; van Dijk, Alberdina Aike; Erasmus, Elardus

    2016-03-01

    Glycine conjugation facilitates the metabolism of toxic aromatic acids, capable of disrupting mitochondrial integrity. Owing to the high exposure to toxic substrates, characterization of individual glycine conjugation capacity, and its regulatory factors has become increasingly important. Aspirin and benzoate have been employed for this purpose; however, adverse reactions, aspirin intolerance, and Reye's syndrome in children are substantial drawbacks. The goal of this study was to investigate p-aminobenzoic acid (PABA) as an alternative glycine conjugation probe. Ten human volunteers participated in a PABA challenge test, and p-aminohippuric acid (PAHA), p-acetamidobenzoic acid, and p-acetamidohippuric acid were quantified in urine. The glycine N-acyltransferase gene of the volunteers was also screened for two polymorphisms associated with normal and increased enzyme activity. All of the individuals were homozygous for increased enzyme activity, but excretion of PAHA varied significantly (16-56%, hippurate ratio). The intricacies of PABA metabolism revealed possible limiting factors and the potential of PABA as an indicator of Phase 0 biotransformation. PMID:26484797

  11. 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.

  12. Probing the active center of benzaldehyde lyase with substitutions and the pseudo-substrate analog benzoylphosphonic acid methyl ester

    PubMed Central

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

    2009-01-01

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg2+ 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 type of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analog 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 Å (PDB ID: 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. PMID:18570438

  13. 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. PMID:25683730

  14. Hybrid hyaluronic acid hydrogel/poly(ɛ-caprolactone) scaffold provides mechanically favorable platform for cartilage tissue engineering studies.

    PubMed

    Mintz, Benjamin R; Cooper, James A

    2014-09-01

    Hybrid scaffolds for cartilage tissue engineering provide the potential for high stiffness properties in tension and compression while exhibiting the viscoelastic response found in hydrogels and native cartilage tissue. We investigate the impact of a hybrid scaffold fabricated from a hyaluronic acid (HA)-based hydrogel combined with porous poly(ε-caprolactone) (PCL) material formed by a particulate leaching method to study dedifferentiated chondrocyte response. The material properties of the hybrid scaffold showed mean Young's moduli in tension which were similar to human articular cartilage but not statistically different between the hybrid and porous PCL scaffolds at 2.02 and 2.07 MPa, respectively. For both the hybrid and porous PCL control scaffolds in compression at low loading frequencies (<1 Hz) and 10% strain peak amplitude the Young's moduli are not statistically distinct. However, at frequencies in the range of normal human gait from 1 to2 Hz, hybrid scaffolds exhibit significantly (p < 0.01) increased loss moduli indicating additional contribution of the viscous phase to stiffness. Dedifferentiated chondrocytes seeded onto the scaffolds exhibited a rounded morphology in hybrid scaffolds however ECM protein expression levels of collagen type I, collagen type II, and aggrecan are not different from the PCL control scaffolds. These results provide a model platform to investigate cell response to mechanical and chemical cues in a hybrid scaffold system with mechanical properties similar to human cartilage that does not contribute to differentiation in order to identify the appropriate design and development parameters to promote formation of extracellular matrix and investigate chondrocyte scaffold interactions. PMID:24115629

  15. Enhanced photocatalytic performance of ZnO-reduced graphene oxide hybrid synthesized via ultrasonic probe-assisted study

    NASA Astrophysics Data System (ADS)

    Prakash, A.; Sahu, N. K.; Bahadur, D.

    2013-02-01

    A facile ultrasonic horn-assisted reaction has been used to synthesize zinc oxide-reduced graphene oxide (ZnO-RGO) hybrids in dimethylformamide. The incorporation of graphene oxide prevents the cluster formation of ZnO nanoparticles. The photocatalytic performance in degradation of methylene blue has been investigated with ZnO and ZnO-RGO hybrids and the results show that the RGO plays an important role in the enhancement of photocatalytic performance of ZnO-RGO. A direct evidence of electron exchange between ZnO and RGO is confirmed by zeta potentials measurements, which is an established reason for photocatalytic degradation of organic dyes.

  16. Monitoring of ppm level humic acid in surface water using ZnO-chitosan nano-composite as fluorescence probe

    NASA Astrophysics Data System (ADS)

    Basumallick, Srijita; Santra, Swadeshmukul

    2015-05-01

    Surface water contains natural pollutants humic acid (HA) and fulvic acid at ppm level which form carcinogenic chloro-compounds during chlorination in water treatment plants. We report here synthesis of ZnO-chitosan (CS) nano-composites by simple hydrothermal technique and examined their application potential as fluorescent probe for monitoring ppm level HA. These ZnO-CS composites have been characterized by HRTEM, EDX, FTIR, AFM and Fluorescence Spectra. HRTEM images show the formation of ZnO-CS nano-composites of average diameter of 50-250 nm. Aqueous dispersions of these nano-composites show fluorescence emission at 395 nm when excited at 300 nm which is strongly quenched by ppm level HA indicating their possible use in monitoring ppm level HA present in surface water.

  17. Hybrid of chitin and humic acid as high performance sorbent for Ni(II)

    NASA Astrophysics Data System (ADS)

    Santosa, Sri Juari; Siswanta, Dwi; Kurniawan, Agusta; Rahmanto, Wasino H.

    2007-11-01

    Hybrid of humic acid (HA) and chitin has been synthesized and the hybrid material (chitin-HA) was then applied as sorbent to adsorb Ni(II). The HA was extracted from peat soil of Gambut District, South Kalimantan, Indonesia, according to the procedure recommended by IHSS (International Humic Substances Society). The chitin was isolated from crab shell waste of sea food restaurants through deproteination using NaOH 3.5% (w/v) and followed by removal of inorganic impurities using HCl 1 M. The synthesis of chitin-HA was performed by reacting gelatinous chitin solution in HCl 0.5 M and HA solution in NaOH 0.5 M. Parameters investigated in this work consists of effect of medium acidity on the sorption, sorption rate ( ks) and desorption rate ( kd) constants, Langmuir (monolayer) and Freundlich (multilayer) sorption capacities, and energy ( E) of sorption. The ks and kd were determined according to a kinetic model of first order sorption reaching equilibrium, monolayer sorption capacity ( b) and energy ( E) were determined according to the Langmuir isotherm model, and multilayer sorption capacity ( B) was determined based on the Freundlich isotherm model. Sorption of Ni(II) on both chitin and chitin-HA was maximum at pH 8.0. The kinetic expression resulted from the proposed kinetic model has been shown to be more applicable than the commonly known Lagergren equation obtained from the pseudo-first order sorption model. The application of the proposed model revealed that the presence of HA increased the ks from 0.018 min -1 for chitin to 0.031 min -1 for chitin-HA. As for ks, the value of b was also bigger in the presence of HA, i.e. 7.42 × 10 -5 mol/g for chitin and 9.93 × 10 -5 mol/g for the chitin-HA. Unlike ks and b, the value of E slightly decreased from 23.23 to 21.51 kJ/mol for the absence and presence of HA, respectively. It can also be deduced that the presence of HA on chitin contributed more to the additional layer of Ni(II) sorbed on sorbent. Without HA, B

  18. Silylated melamine and cyanuric acid as precursors for imprinted and hybrid silica materials with molecular recognition properties.

    PubMed

    Arrachart, Guilhem; Carcel, Carole; Trens, Philippe; Moreau, Jöel J E; Wong Chi Man, Michel

    2009-06-15

    Two monotrialkoxysilylated compounds that consist of complementary fragments of melamine (M) and cyanuric acid (CA) have been synthesised. The molecular recognition properties of the M and CA fragments through complementary hydrogen bonds (DAD and ADA; D=donor, A=acceptor) are the key factor used to direct the formation of hybrid silica materials by using a sol-gel process. These materials were synthesised following two methods: First, an organo-bridged silsesquioxane was obtained by the hydrolysis of the two complementary monotrialkoxysilylated melamine and cyanuric acid derivatives, with fluoride ions as a catalyst. The hydrogen-bonding interactions between the two organic fragments are responsible for the formation of the bridging unit. The transcription of the assembly into the hybrid material was characterised and evidenced by solid-state NMR (29Si, 13C) and FTIR spectroscopic experiments. Second, the molecular recognition was exploited to synthesise an imprinted hybrid silica. This material was prepared by co-condensation of tetraethyl orthosilicate (TEOS) with the monosilylated cyanuric acid derivative (CA) templated by nonsilylated melamine. The melamine template was completely removed by treating the solid material with hydrochloric acid. The reintroduction of the template was performed by treating the resulting material with an aqueous suspension of melamine. These steps were monitored and analysed by several techniques, such as solid-state NMR (29Si, 13C) and FTIR spectroscopic analysis and nitrogen adsorption-desorption isotherms. PMID:19440996

  19. 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

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

    PubMed

    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

  1. Probing the local environment of hybrid materials designed from ionic liquids and synthetic clay by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Siqueira, Leonardo J. A.; Constantino, Vera R. L.; Camilo, Fernanda F.; Torresi, Roberto M.; Temperini, Marcia L. A.; Ribeiro, Mauro C. C.; Izumi, Celly M. S.

    2014-03-01

    Hybrid organic-inorganic material containing Laponite clay and ionic liquids forming cations have been prepared and characterized by FT-Raman spectroscopy, X-ray diffraction, and thermal analysis. The effect of varying the length of the alkyl side chain and conformations of cations has been investigated by using different ionic liquids based on piperidinium and imidazolium cations. The structure of the N,N-butyl-methyl-piperidinium cation and the assignment of its vibrational spectrum have been further elucidated by quantum chemistry calculations. The X-ray data indicate that the organic cations are intercalated parallel to the layers of the clay. Comparison of Raman spectra of pure ionic liquids with different anions and the resulting solid hybrid materials in which the organic cations have been intercalated into the clay characterizes the local environment experienced by the cations in the hybrid materials. The Raman spectra of hybrid materials suggest that the local environment of all confined cations, in spite of this diversity in properties, resembles the liquid state of ionic liquids with a relatively disordered structure.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. Detection of supercoiled hepatitis B virus DNA and related forms by means of molecular hybridization to an oligonucleotide probe

    SciTech Connect

    Lin, H.J.; Chung, H.T.; Lai, C.L.; Leong, S.; Tam, O.S. )

    1989-12-01

    A novel assay for supercoiled and other fully double-stranded forms of hepatitis B virus (HBV) DNA in blood is presented that utilizes molecular hybridisation to a radiophosphorous-labeled oligonucleotide probe. The probe (5'-d(ACGTGCAGAGGTGAAGCGA)) is complementary to the S(+)-strand sequence furthest downstream, at the end of the gap. We examined blood specimens from 137 healthy HBsAg-positive individuals, applying the probe to dots representing 2-3.5 ml serum or plasma. We found that supercoiled HBV is present in many HBV DNA-positive blood specimens albeit in small quantities. Of the 104 specimens that were positive for HBV DNA of any form, 53 annealed to the probe. Serial specimens from the same subject taken over a period of months showed that the proportion of supercoil to other HBV DNA forms was variable. The presence of supercoil HBV DNA was not closely correlated with the level of serum HBV DNA polymerase. The supercoil is an HBV DNA form that can persist in the liver in the presence or absence of other replicative intermediates. This assay may enable further characterization of the status of HBV infection.

  7. Photochemical properties in flag leaves of a super-high-yielding hybrid rice and a traditional hybrid rice (Oryza sativa L.) probed by chlorophyll a fluorescence transient.

    PubMed

    Zhang, Meiping; Shan, YongJie; Kochian, Leon; Strasser, Reto J; Chen, GuoXiang

    2015-12-01

    Chlorophyll a fluorescence of flag leaves in a super-high-yielding hybrid rice (Oryza sativa L.) LYPJ, and a traditional hybrid rice SY63 cultivar with lower grain yield, which were grown in the field, were investigated from emergence through senescence of flag leaves. As the flag leaf matured, there was an increasing trend in photosynthetic parameters such as quantum efficiency of primary photochemistry ([Formula: see text] Po) and efficiency of electron transport from PS II to PS I (Ψ Eo). The overall photosynthetic performance index (PIABS) was significantly higher in the high-yielding LYPJ compared to SY63 during the entire reproductive stage of the plant, the same to MDA content. However, [Formula: see text] Po(=F V/F M), an indicator of the primary photochemistry of the flag leaf, did not display significant changes with leaf age and was not significantly different between the two cultivars, suggesting that PIABS is a more sensitive parameter than [Formula: see text] Po (=F V/F M) during leaf age for distinguishing between cultivars differing in yield. PMID:25972274

  8. 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

  9. Material balance studies with continuous SSF for ethanol production using dilute-acid pretreated hybrid popular

    SciTech Connect

    Kadam, K.L.; Hayward, T.K.; Philippidis, G.P.

    1995-10-01

    Simultaneous saccharification and fermentation (SSF) is a leading process option for converting lignocellulosic biomass to ethanol. Economic industrial production of ethanol by SSF would likely require a continuous mode of operation, and development of a database on continuous SSF using industrial substrates is essential. To this end, a single-stage continuous SSF system was designed and used for studying ethanol production by Saccharomyces cerevisiae strain D{sub 5}A with dilute-acid preheated hybrid poplar as substrate. Material balance studies were conducted using the bench-scale system; the objectives of these studies were to gain insight into how the cells allocate carbon and to account for all the substrate flowing through the system. It was possible to close the total mass and carbon balances with {+-}3% accuracy, which is within the limits of experimental error. In a typical continuous SSF run, about 57% of the consumed carbon was used to produce ethanol and less than 1% to synthesize cell mass; thus the majority of carbon flow was to ethanol. The ethanol yields based on consumed substrate and volumetric productivities were in the range of 85% and 0.2 g ethanol/l{times}h, respectively; both values are within the expectations for a nearterm biomass-to-ethanol process. In the single-stage system used, continuous inoculation was not needed even for whole-slurry fermentations; this has significant economic implications since the inoculum cost can be significant.

  10. Mapping the regioisomeric distribution of fatty acids in triacylglycerols by hybrid mass spectrometry[S

    PubMed Central

    Nagy, Kornél; Sandoz, Laurence; Destaillats, Frédéric; Schafer, Olivier

    2013-01-01

    This study describes the use of hybrid mass spectrometry for the mapping, identification, and semi-quantitation of triacylglycerol regioisomers in fats and oils. The identification was performed based on the accurate mass and fragmentation pattern obtained by data-dependent fragmentation. Quantitation was based on the high-resolution ion chromatograms, and relative proportion of sn-1(3)/sn-2 regioisomers was calculated based on generalized fragmentation models and the relative intensities observed in the product ion spectra. The key performance features of the developed method are inter-batch mass accuracy < 1 ppm (n = 10); lower limit of detection (triggering threshold) 0.1 μg/ml (equivalent to 0.2 weight % in oil); lower limit of quantitation 0.2 μg/ml (equivalent to 0.4 weight % in oil); peak area precision 6.5% at 2 μg/ml concentration and 15% at 0.2 μM concentration; inter-batch precision of fragment intensities < 1% (n = 10) independent of the investigated concentration; and averaged accuracy using the generic calibration 3.8% in the 1–10 μg/ml range and varies between 1–23% depending on analytes. Inter-esterified fat, beef tallow, pork lard, and butter fat samples were used to show how well regioisomeric distribution of palmitic acid can be captured by this method. PMID:23093552

  11. 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.

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

    PubMed

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

    2005-03-01

    The molecular recognition interaction of alpha-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 alpha-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine approximately DL-beta-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L-iso-leucine > L-leucine approximately L-methionine approximately DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, delta G, delta H, delta 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 alpha-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K. PMID:15683802

  13. Synthesis and biological evaluation of boswellic acid-NSAID hybrid molecules as anti-inflammatory and anti-arthritic agents.

    PubMed

    Shenvi, Suvarna; Kiran, K R; Kumar, Krishna; Diwakar, Latha; Reddy, G Chandrasekara

    2015-06-15

    Methyl esters of the β-boswellic acid (BA) and 11-keto-β-boswellic acid (KBA) obtained from Boswellia serrata resin were subjected to Steglich esterification with the different non-steroidal anti-inflammatory drugs (NSAID) viz., ibuprofen, naproxen, diclophenac and indomethacin. The novel hybrids of methyl boswellate (5-8) and that of methyl 11-keto boswellate (9-12) were evaluated for anti-inflammatory activity by carrageenan-induced rat hind paw edema model and anti-arthritic activity by Complete Freund's Adjuvant (CFA) induced arthritis in Wister albino rat. Significant inhibition on carrageenan-induced paw edema has been observed with 5, 6 and 10 where as in CFA induced rats, hybrids 5, 8, 9 and 12 exhibited pronounced antiarthritic activity. Hybrid molecules 5 and 9 have been found to be more effective in inhibiting in-vivo COX-2 than ibuprofen by itself, thus showing the synergistic effect. Hybrid 5 and 9 tested for in-vitro lipoxygenase and cyclooxygenase-2 (LOX/COX-2) inhibitory activity. The studies revealed that both 5 and 9 inhibited COX-2 relatively better than LOX enzyme. PMID:26010018

  14. Maximum power output and load matching of a phosphoric acid fuel cell-thermoelectric generator hybrid system

    NASA Astrophysics Data System (ADS)

    Chen, Xiaohang; Wang, Yuan; Cai, Ling; Zhou, Yinghui

    2015-10-01

    Based on the current models of phosphoric acid fuel cells (PAFCs) and thermoelectric generators (TGs), a new hybrid system is proposed, in which the effects of multi-irreversibilities resulting from the activation, concentration, and ohmic overpotentials in the PAFC, Joule heat and heat leak in the TG, finite-rate heat transfer between the TG and the heat reservoirs, and heat leak from the PAFC to the environment are taken into account. Expressions for the power output and efficiency of the PAFC, TG, and hybrid system are analytically derived and directly used to discuss the performance characteristics of the hybrid system. The optimal relationship between the electric currents in the PAFC and TG is obtained. The maximum power output is numerically calculated. It is found that the maximum power output density of the hybrid system will increase about 150 Wm-2, compared with that of a single PAFC. The problem how to optimally match the load resistances of two subsystems is discussed. Some significant results for practical hybrid systems are obtained.

  15. Use of the Verrucomicrobia-Specific Probe EUB338-III and Fluorescent In Situ Hybridization for Detection of “Candidatus Xiphinematobacter” Cells in Nematode Hosts

    PubMed Central

    Vandekerckhove, Tom T. M.; Coomans, August; Cornelis, Karen; Baert, Philippe; Gillis, Monique

    2002-01-01

    Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium “Candidatus Xiphinematobacter” in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria. PMID:12039775

  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. Molecular cytogenetics using fluorescence in situ hybridization

    SciTech Connect

    Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

    1990-12-07

    Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

  18. Visual detection of telomerase activity with a tunable dynamic range by using a gold nanoparticle probe-based hybridization protection strategy

    NASA Astrophysics Data System (ADS)

    Wang, Jiasi; Wu, Li; Ren, Jinsong; Qu, Xiaogang

    2014-01-01

    We developed a novel telomere complementary (TC) oligonucleotide modified AuNP probe (TC-AuNPs) for colorimetric analysis of telomerase activity. The mechanism of this method is that the telomerase reaction products (TRP), which can hybridize with the TC-AuNPs, are able to protect the AuNPs from the aggregation induced by salt. It is demonstrated that the colorimetric method enabled the analysis of the telomerase activity in 1000 HeLa cells with the naked eye, and down to 100 HeLa cells with the aid of UV-Vis spectroscopy. This strategy is not only convenient and sensitive, but also has a tunable dynamic range. The platform is also applicable for the initial screening of a telomerase inhibitor to discover new anticancer drugs.We developed a novel telomere complementary (TC) oligonucleotide modified AuNP probe (TC-AuNPs) for colorimetric analysis of telomerase activity. The mechanism of this method is that the telomerase reaction products (TRP), which can hybridize with the TC-AuNPs, are able to protect the AuNPs from the aggregation induced by salt. It is demonstrated that the colorimetric method enabled the analysis of the telomerase activity in 1000 HeLa cells with the naked eye, and down to 100 HeLa cells with the aid of UV-Vis spectroscopy. This strategy is not only convenient and sensitive, but also has a tunable dynamic range. The platform is also applicable for the initial screening of a telomerase inhibitor to discover new anticancer drugs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05185d

  19. Onsite naked eye determination of cysteine and homocysteine using quencher displacement-induced fluorescence recovery of the dual-emission hybrid probes with desired intensity ratio.

    PubMed

    Wang, Kan; Qian, Jing; Jiang, Ding; Yang, Zhengting; Du, Xiaojiao; Wang, Kun

    2015-03-15

    Simple, inexpensive, portable sensing strategies for those clinically relevant molecules have attained a significant positive impact on the health care system. Herein, we have prepared a dual-emission ratiometric fluorescence probe with desired intensity ratio and demonstrated its efficiency for onsite naked eye determination of cysteine (Cys) and homocysteine (Hcy). The hybrid probe has been designed by hybridizing two differently sized CdTe quantum dots (QDs), in which the red-emitting CdTe QDs (rQDs) entrapped in the silica sphere acting as the reference signal, and the green-emitting CdTe QDs (gQDs) covalently attached on the silica surface serving as the response signal. When 1,10-phenanthroline with strong coordination ability to Cd atoms in gQDs was introduced, the fluorescence of the gQDs was effectively quenched, while the fluorescence of the rQDs stayed constant. Upon exposure to different contents of Cys or Hcy, the fluorescence of gQDs can be recovered gradually due to the displacement of the quencher. Based on the background signal of rQDs, the variations of the sensing system display continuous fluorescence color changes from red to green, which can be easily observed by the naked eye. The assay requires ∼20min and has a detection limit of 2.5 and 1.7μM for Cys and Hcy, respectively. Furthermore, we demonstrate that this sensing scheme can be fully integrated in a filter paper-based assay, thus enabling a potential point-of-care application featuring easy operation, low power consumption, and low fabrication costs. PMID:25461142

  20. Degradation of Acid Orange 7 Dye in Two Hybrid Plasma Discharge Reactors

    NASA Astrophysics Data System (ADS)

    Shen, Yongjun; Lei, Lecheng; Zhang, Xingwang; Ding, Jiandong

    2014-11-01

    To get an optimized pulsed electrical plasma discharge reactor and to increase the energy utilization efficiency in the removal of pollutants, two hybrid plasma discharge reactors were designed and optimized. The reactors were compared via the discharge characteristics, energy transfer efficiency, the yields of the active species and the energy utilization in dye wastewater degradation. The results showed that under the same AC input power, the characteristics of the discharge waveform of the point-to-plate reactor were better. Under the same AC input power, the two reactors both had almost the same peak voltage of 22 kV. The peak current of the point-to-plate reactor was 146 A, while that of the wire-to-cylinder reactor was only 48.8 A. The peak powers of the point-to-plate reactor and the wire-to-cylinder reactor were 1.38 MW and 1.01 MW, respectively. The energy per pulse of the point-to-plate reactor was 0.2221 J, which was about 29.4% higher than that of the wire-to-cylinder reactor (0.1716 J). To remove 50% Acid Orange 7 (AO7), the energy utilizations of the point-to-plate reactor and the wire-to-cylinder reactor were 1.02 × 10-9 mol/L and 0.61 × 10-9 mol/L, respectively. In the point-to-plate reactor, the concentration of hydrogen peroxide in pure water was 3.6 mmol/L after 40 min of discharge, which was higher than that of the wire-to-cylinder reactor (2.5 mmol/L). The concentration of liquid phase ozone in the point-to-plate reactor (5.7 × 10-2 mmol/L) was about 26.7% higher than that in the wire-to-cylinder reactor (4.5 × 10-2 mmol/L). The analysis results of the variance showed that the type of reactor and reaction time had significant impacts on the yields of the hydrogen peroxide and ozone. The main degradation intermediates of AO7 identified by gas chromatography and mass spectrometry (GCMS) were acetic acid, maleic anhydride, p-benzoquinone, phenol, benzoic acid, phthalic anhydride, coumarin and 2-naphthol. Proposed degradation pathways were

  1. High-resolution analysis of 16q22.1 in breast carcinoma using DNA amplifiable probes (multiplex amplifiable probe hybridization technique) and immunohistochemistry.

    PubMed

    Rakha, Emad A; Armour, John A L; Pinder, Sarah E; Paish, Claire E; Ellis, Ian O

    2005-05-01

    Loss of the chromosomal material at 16q22.1 is one of the most frequent genetic aberrations found in both lobular and low-grade nonlobular invasive carcinoma of the breast, indicating the presence of a tumour suppressor gene (TSG) at this region in these tumours. However, the TSG (s) at the 16q22.1 in the more frequent nonlobular carcinomas is still unknown. Multiplex Amplifiable Probe Hybridisation (MAPH) is a simple, accurate and a high-resolution technique that provides an alternative approach to DNA copy-number measurement. The aim of our study was to examine the most likely candidate genes at 16q22.1 using MAPH assay combined with protein expression analysis by immunohistochemistry. We identified deletion at 16q22.1 that involves some or all of these genes. We also noticed that the smallest region of deletion at 16q22.1 could be delineated to a 3 Mb region centromeric to the P-cadherin gene. Apart from the correlation between E-cadherin protein expression and its gene copy number, no correlation was detected between the expression of E2F-4, CTCF, TRF2 or P-cadherin with their gene's copy number. In the malignant tissues, no significant loss or decrease of protein expression of any gene other than E-cadherin was seen in association with any specific tumour type. No expression of VE-cadherin or Ksp-cadherin was detected in the normal and/or malignant tissues of the breast in these cases. However, there was a correlation between increased nuclear expression of E2F-4 and tumours with higher histological grade (p = 0.04) and positive lymph node disease (p = 0.02), suggesting that it may have an oncogenic rather than a tumour suppressor role. The malignant breast tissues also showed abnormal cytoplasmic cellular localisation of CTCF, compared to its expression in the normal parenchymal cells. In conclusion, we have demonstrated that MAPH is a potential technique for assessment of genomic imbalances in malignant tissues. Although our results support E-cadherin as the

  2. 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

  3. The Interaction of the Solar Wind with Solar Probe Plus - 3D Hybrid Simulation. Report 1; The Study for the Distance 4.5Rs

    NASA Technical Reports Server (NTRS)

    Lipatov, Alexander S.; Sittler, Edward C.; Hartle, Richard E.; Cooper, John F.

    2010-01-01

    Our report devotes a 3D numerical hybrid model of the interaction of the solar wind with the Solar Probe spacecraft. The SPP model includes 3 main parts, namely, a non-conducting heat shield, a support system, and cylindrical section or spacecraft bus that contains the particle analysis devices and antenna. One observes an excitation of the low frequency Alfven and whistler type wave directed by the magnetic field with an amplitude of about (0.06-0.6) V/m. The compression waves and the jumps in an electric field with an amplitude of about (0.15-0.7) V/m were also observed. The wave amplitudes are comparable to or greater than previously estimated max wave amplitudes that SPP is expected to measure. The results of our hybrid simulation will be useful for understanding the plasma environment near the SPP spacecraft at the distance 4.5 Rs. Future simulation will take into account the charging of the spacecraft, the charge separation effects, an outgassing from heat shield, a photoionization and an electron impact ionization effects near the spacecraft.

  4. Short Wavelength Electromagnetic Perturbations Excited Near the Solar Probe Plus Spacecraft in the Inner Heliosphere: 2.5D Hybrid Modeling

    NASA Technical Reports Server (NTRS)

    Lipatov, Alexander S.; Sittler, Edward C.; Hartle, Richard E.; Cooper, John F.

    2011-01-01

    A 2.5D numerical plasma model of the interaction of the solar wind (SW) with the Solar Probe Plus spacecraft (SPPSC) is presented. These results should be interpreted as a basic plasma model derived from the SW-interaction with the spacecraft (SC), which could have consequences for both plasma wave and electron plasma measurements on board the SC in the inner heliosphere. Compression waves and electric field jumps with amplitudes of about 1.5 V/m and (12-18) V/m were also observed. A strong polarization electric field was also observed in the wing of the plasma wake. However, 2.5D hybrid modeling did not show excitation of whistler/Alfven waves in the upstream connected with the bidirectional current closure that was observed in short-time 3D modeling SPPSC and near a tether in the ionosphere. The observed strong electromagnetic perturbations may be a crucial point in the electromagnetic measurements planned for the future Solar Probe Plus (SPP) mission. The results of modeling electromagnetic field perturbations in the SW due to shot noise in absence of SPPSC are also discussed.

  5. Re-appraisal of the phylogeny and fluorescence in situ hybridization probes for the analysis of the Competibacteraceae in wastewater treatment systems.

    PubMed

    McIlroy, Simon J; Nittami, Tadashi; Kanai, Eri; Fukuda, Junji; Saunders, Aaron M; Nielsen, Per Halkjaer

    2015-04-01

    Members of the family Competibacteraceae are common in wastewater treatment plants (WWTPs) designed for enhanced biological phosphorus removal (EBPR) and are putatively deleterious to the process of P removal. Their ability to accumulate large amounts of polyhydroxyalkanoates is also suggested to be of potential commercial interest for bioplastic production. In this study we have updated the 16S rRNA-based phylogeny of the Competibacter and the Plasticicumulans lineages. The former is delineated by 13 clades including two described genera; 'Ca. Competibacter' and 'Ca. Contendobacter'. The oligonucleotide probes used for detection of the family by fluorescence in situ hybridization (FISH) were re-evaluated and designed for coverage of these clades. Surveys of full-scale WWTPs based on 16S rRNA gene amplicon sequencing and FISH analysis indicate that a number of member clades always coexist, with their relative abundances varying substantially between and temporally within plants. The hypothesis that these differences are based on niche partitioning is supported by marked phenotypic differences between clades. An in-depth understanding of the ecology of the family requires further studies of the metabolism of individual clades in situ. The proposed phylogeny and FISH probes will provide the foundation for such studies. PMID:25224028

  6. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions.

    PubMed

    Hua, Mengjuan; Wang, Chengquan; Qian, Jing; Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping; Wang, Kun

    2015-08-12

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg(2+). The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg(2+), the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg(2+) in a broad linear range of 10 nM-22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg(2+) contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg(2+) quantification related biological systems. PMID:26320973

  7. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments. PMID:24827529

  8. Detection of DNA hybridization and extension reactions by an extended-gate field-effect transistor: characterizations of immobilized DNA-probes and role of applying a superimposed high-frequency voltage onto a reference electrode.

    PubMed

    Kamahori, Masao; Ishige, Yu; Shimoda, Maki

    2008-02-28

    As we have already shown in a previous publication [Kamahori, M., Ihige, Y., Shimoda, M., 2007. Anal. Sci. 23, 75-79], an extended-gate field-effect transistor (FET) sensor with a gold electrode, on which both DNA probes and 6-hydroxyl-1-hexanethiol (6-HHT) molecules are immobilized, can detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to a reference electrode. However, kinetic parameters such as the dissociation constant (K(d)(s)) and the apparent DNA-probe concentration (C(probe)(s)) on a surface were not clarified. In addition, the role of applying the superimposed high-frequency voltage was not considered in detail. In this study, the values of K(d)(s) and C(probe)(s) were estimated using a method involving single-base extension reaction combined with bioluminescence detection. The value of K(d)(s) on the surface was 0.38 microM, which was about six times that in a liquid phase. The value of C(probe)(s), which expressed the upper detection limit for the solid phase reaction, was 0.079 microM at a DNA-probe density of 2.6 x 10(12)molecules/cm(2). We found that applying the superimposed high-frequency voltage accelerated the DNA molecules to reach the gold surface. Also, the distance between the DNA-probes immobilized on the gold surface was controlled to be over 6 nm by applying a method of competitive reaction with DNA probes and 6-HHT molecules. This space was sufficient to enable the immobilized DNA-probes to lie down on the 6-HHT monolayer in the space between them. Thus, the FET sensor could detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to the DNA-probes density-controlling gold surface. PMID:18054478

  9. Hybrid molecularly imprinted poly(methacrylic acid-TRIM)-silica chemically modified with (3-glycidyloxypropyl)trimethoxysilane for the extraction of folic acid in aqueous medium.

    PubMed

    de Oliveira, Fernanda Midori; Segatelli, Mariana Gava; Tarley, César Ricardo Teixeira

    2016-02-01

    In the present study a hybrid molecularly imprinted poly(methacrylic acid-trimethylolpropane trimethacrylate)-silica (MIP) was synthesized and modified with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) with posterior opening of epoxy ring to provide hydrophilic properties of material in the extraction of folic acid from aqueous medium. The chemical and structural aggregates of hybrid material were characterized by means of Fourier Transform Infrared (FT-IR), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Thermogravimetric analysis (TGA) and textural data. Selectivity data of MIP were compared to non-imprinted polymer (NIP) through competitive sorption studies in the presence of caffeine, paracetamol or 4-aminobenzamide yielding relative selectivity coefficients (k′) higher than one unit, thus confirming the selective character of MIP even in the presence of structurally smaller compounds than the folic acid. The lower hydrophobic sorption by bovine serum albumin (BSA) in the MIP as compared to unmodified MIP proves the hydrophilicity of polymer surface by using GPTMS with opening ring. Under acid medium(pH 1.5) the sorption of folic acid onto MIP from batch experiments was higher than the one achieved for NIP. Equilibrium sorption of folic acid was reached at 120 min for MIP, NIP and MIP without GPTMS and kinetic sorption data were well described by pseudo-second-order, Elovich and intraparticle diffusion models. Thus, these results indicate the existence of different binding energy sites in the polymers and a complex mechanism consisting of both surface sorption and intraparticle transport of folic acid within the pores of polymers. PMID:26652418

  10. Measurement of vapor pressures and heats of sublimation of dicarboxylic acids using atmospheric solids analysis probe mass spectrometry.

    PubMed

    Bruns, Emily A; Greaves, John; Finlayson-Pitts, Barbara J

    2012-06-21

    Vapor pressures of low volatility compounds are important parameters in several atmospheric processes, including the formation of new particles and the partitioning of compounds between the gas phase and particles. Understanding these processes is critical for elucidating the impacts of aerosols on climate, visibility, and human health. Dicarboxylic acids are an important class of compounds in the atmosphere for which reported vapor pressures often vary by more than an order of magnitude. In this study, atmospheric solids analysis probe mass spectrometry (ASAP-MS), a relatively new atmospheric pressure ionization technique, is applied for the first time to the measurement of vapor pressures and heats of sublimation of a series of dicarboxylic acids. Pyrene was also studied because its vapor pressures and heat of sublimation are relatively well-known. The heats of sublimation measured using ASAP-MS were in good agreement with published values. The vapor pressures, assuming an evaporation coefficient of unity, were typically within a factor of ∼3 lower than published values made at similar temperatures for most of the acids. The underestimation may be due to diffusional constraints resulting from evaporation at atmospheric pressure. However, this study establishes that ASAP-MS is a promising new technique for such measurements. PMID:22432524

  11. 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

  12. Insertion of fluorescent fatty acid probes into the outer membranes of the pathogenic spirochaetes Treponema pallidum and Borrelia burgdorferi.

    PubMed

    Cox, D L; Radolf, J D

    2001-05-01

    The authors examined the ability of octadecanoyl (C(18)), hexadecanoyl (C(16)) and dodecanoyl (C(12)) fatty acid (FA) conjugates of 5-aminofluorescein (OAF, HAF and DAF, respectively) to insert into the outer membranes (OMs) of Treponema pallidum, Borrelia burgdorferi and Escherichia coli. Biophysical studies have demonstrated that these compounds stably insert into phospholipid bilayers with the acyl chain within the hydrophobic interior of the apical leaflet and the hydrophilic fluorescein moiety near the phospholipid head groups. Consistent with the known poor intrinsic permeability of the E. coli OM to hydrophobic compounds and surfactants, E. coli was not labelled with any of the FA probes. OAF inserted more readily into OMs of B. burgdorferi than into those of T. pallidum, although both organisms were completely labelled at concentrations at or below 2 microg ml(-1). Intact spirochaetes were labelled with OAF but not with antibodies against known periplasmic antigens, thereby confirming that the probe interacted exclusively with the spirochaetal OMs. Separate experiments in which organisms were cooled to 4 degrees C (i.e. below the OM phase-transition temperatures) indicated that labelling with OAF was due to insertion of the probe into the OMs. B. burgdorferi, but not T. pallidum, was labelled by relatively high concentrations of HAF and DAF. Taken as a whole, these findings support the prediction that the lack of lipopolysaccharide renders T. pallidum and B. burgdorferi OMs markedly more permeable to lipophilic compounds than their Gram-negative bacterial counterparts. The data also raise the intriguing possibility that these two pathogenic spirochaetes obtain long-chain FAs, nutrients they are unable to synthesize, by direct permeation of their OMs. PMID:11320119

  13. Computer simulations of the electron spin resonance spectra of steroid and fatty acid nitroxide probes in bilayer systems

    NASA Astrophysics Data System (ADS)

    Eviatar, Hadass; van der Heide, Uulke A.; Levine, Yehudi K.

    1995-02-01

    Monte Carlo dynamics (MCD) techniques are used to simulate the orientational behavior and rotational motion of probe molecules in lipid bilayers. The trajectories of molecular orientations generated from the simulations are then used to calculate the order parameters and the orientational time correlation functions. The behavior of the time correlation functions is compared with the predictions of the rotational diffusion (RDM) and the compound motion (CM) models. The MCD trajectories are also used to produce electron-spin resonance (ESR) spectra, employing a recently developed time-domain algorithm. Two questions which have been the subject of debate in the literature are addressed. The first question concerns the discrepancy between the ability of motional models to describe ESR spectra and fluorescence depolarization measurements on rigid molecules in vesicles—while the RDM does an excellent job of fitting the former, the latter require the CM to describe them properly. It is argued that the key to resolving this lies in the fact that the ESR line shapes are sensitive to the tumbling motions of the long molecular axes as well as to rotational motions about them, while fluorescence anisotropy is blind for the latter. The rotation about the long molecular axis introduces a fast decay into the correlation functions in a way independent of the tumbling motion of the axis. The second question concerns the fidelity of reporting by fatty acid spin probes in lipid bilayers. It is shown that the motion of the bulky hydrophillic doxyl group does not, in fact, reflect the motion of the chains about it and consequently these spin probes cannot be considered good reporters for these applications.

  14. 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.

  15. Development of a lauric acid/albumin hybrid iron oxide nanoparticle system with improved biocompatibility

    PubMed Central

    Zaloga, Jan; Janko, Christina; Nowak, Johannes; Matuszak, Jasmin; Knaup, Sabine; Eberbeck, Dietmar; Tietze, Rainer; Unterweger, Harald; Friedrich, Ralf P; Duerr, Stephan; Heimke-Brinck, Ralph; Baum, Eva; Cicha, Iwona; Dörje, Frank; Odenbach, Stefan; Lyer, Stefan; Lee, Geoffrey; Alexiou, Christoph

    2014-01-01

    The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantum interference device. Using flow cytometry, we further investigated the effects of the different types of nanoparticle coating on morphology, viability, and DNA integrity of Jurkat cells. We showed that by addition of bovine serum albumin, the toxicity of nanoparticles is greatly reduced. We also investigated the effect of the particles on the growth of primary human endothelial cells to further demonstrate the biocompatibility of the particles. As proof of principle, we showed that the hybrid-coated particles are able to carry payloads of up to 800 μg/mL of the cytostatic drug mitoxantrone while still staying colloidally stable. The drug-loaded system exhibited excellent therapeutic potential in vitro, exceeding that of free mitoxantrone. In conclusion, we have synthesized a biocompatible ferrofluid that shows great potential for clinical

  16. Development of a lauric acid/albumin hybrid iron oxide nanoparticle system with improved biocompatibility.

    PubMed

    Zaloga, Jan; Janko, Christina; Nowak, Johannes; Matuszak, Jasmin; Knaup, Sabine; Eberbeck, Dietmar; Tietze, Rainer; Unterweger, Harald; Friedrich, Ralf P; Duerr, Stephan; Heimke-Brinck, Ralph; Baum, Eva; Cicha, Iwona; Dörje, Frank; Odenbach, Stefan; Lyer, Stefan; Lee, Geoffrey; Alexiou, Christoph

    2014-01-01

    The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantum interference device. Using flow cytometry, we further investigated the effects of the different types of nanoparticle coating on morphology, viability, and DNA integrity of Jurkat cells. We showed that by addition of bovine serum albumin, the toxicity of nanoparticles is greatly reduced. We also investigated the effect of the particles on the growth of primary human endothelial cells to further demonstrate the biocompatibility of the particles. As proof of principle, we showed that the hybrid-coated particles are able to carry payloads of up to 800 μg/mL of the cytostatic drug mitoxantrone while still staying colloidally stable. The drug-loaded system exhibited excellent therapeutic potential in vitro, exceeding that of free mitoxantrone. In conclusion, we have synthesized a biocompatible ferrofluid that shows great potential for clinical

  17. 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

  18. 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.

  19. Proficient Detection of Multi-Drug-Resistant Mycobacterium tuberculosis by Padlock Probes and Lateral Flow Nucleic Acid Biosensors.

    PubMed

    Pavankumar, Asalapuram R; Engström, Anna; Liu, Jie; Herthnek, David; Nilsson, Mats

    2016-04-19

    Tuberculosis is a major communicable disease. Its causative agent, Mycobacterium tuberculosis, becomes resistant to antibiotics by acquisition of point mutations in the chromosome. Multi-drug-resistant tuberculosis (MDR-TB) is an increasing public health threat, and prompt detection of such strains is of critical importance. As rolling circle amplification of padlock probes can be used to robustly distinguish single-nucleotide variants, we combined this technique with a sensitive lateral flow nucleic acid biosensor to develop a rapid molecular diagnostic test for MDR-TB. A proof-of-concept test was established for detection of the most common mutations [rpoB 531 (TCG/TTG) and katG 315 (AGC/ACC)] causing MDR-TB and verification of loss of the respective wild type. The molecular diagnostic test produces visual signals corresponding to the respective genotypes on lateral flow strips in approximately 75 min. By detecting only two mutations, the test can detect about 60% of all MDR-TB cases. The padlock probe-lateral flow (PLP-LF) test is the first of its kind and can ideally be performed at resource-limited clinical laboratories. Rapid information about the drug-susceptibility pattern can assist clinicians to choose suitable treatment regimens and take appropriate infection control actions rather than prescribing empirical treatment, thereby helping to control the spread of MDR-TB in the community. PMID:26985774

  20. QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

    PubMed Central

    Karabiber, Fethullah; McGinnis, Jennifer L.; Favorov, Oleg V.; Weeks, Kevin M.

    2013-01-01

    Chemical probing of RNA and DNA structure is a widely used and highly informative approach for examining nucleic acid structure and for evaluating interactions with protein and small-molecule ligands. Use of capillary electrophoresis to analyze chemical probing experiments yields hundreds of nucleotides of information per experiment and can be performed on automated instruments. Extraction of the information from capillary electrophoresis electropherograms is a computationally intensive multistep analytical process, and no current software provides rapid, automated, and accurate data analysis. To overcome this bottleneck, we developed a platform-independent, user-friendly software package, QuShape, that yields quantitatively accurate nucleotide reactivity information with minimal user supervision. QuShape incorporates newly developed algorithms for signal decay correction, alignment of time-varying signals within and across capillaries and relative to the RNA nucleotide sequence, and signal scaling across channels or experiments. An analysis-by-reference option enables multiple, related experiments to be fully analyzed in minutes. We illustrate the usefulness and robustness of QuShape by analysis of RNA SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) experiments. PMID:23188808

  1. 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

  2. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    SciTech Connect

    Fehm, Thomas Felix; Razansky, Daniel; Deán-Ben, Xosé Luís

    2014-10-27

    Ultrasonography and optoacoustic imaging share powerful advantages related to the natural aptitude for real-time image rendering with high resolution, the hand-held operation, and lack of ionizing radiation. The two methods also possess very different yet highly complementary advantages of the mechanical and optical contrast in living tissues. Nonetheless, efficient integration of these modalities remains challenging owing to the fundamental differences in the underlying physical contrast, optimal signal acquisition, and image reconstruction approaches. We report on a method for hybrid acquisition and reconstruction of three-dimensional pulse-echo ultrasound and optoacoustic images in real time based on passive ultrasound generation with an optical absorber, thus avoiding the hardware complexity of active ultrasound generation. In this way, complete hybrid datasets are generated with a single laser interrogation pulse, resulting in simultaneous rendering of ultrasound and optoacoustic images at an unprecedented rate of 10 volumetric frames per second. Performance is subsequently showcased in phantom experiments and in-vivo measurements from a healthy human volunteer, confirming general clinical applicability of the method.

  3. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    NASA Astrophysics Data System (ADS)

    Fehm, Thomas Felix; Deán-Ben, Xosé Luís; Razansky, Daniel

    2014-10-01

    Ultrasonography and optoacoustic imaging share powerful advantages related to the natural aptitude for real-time image rendering with high resolution, the hand-held operation, and lack of ionizing radiation. The two methods also possess very different yet highly complementary advantages of the mechanical and optical contrast in living tissues. Nonetheless, efficient integration of these modalities remains challenging owing to the fundamental differences in the underlying physical contrast, optimal signal acquisition, and image reconstruction approaches. We report on a method for hybrid acquisition and reconstruction of three-dimensional pulse-echo ultrasound and optoacoustic images in real time based on passive ultrasound generation with an optical absorber, thus avoiding the hardware complexity of active ultrasound generation. In this way, complete hybrid datasets are generated with a single laser interrogation pulse, resulting in simultaneous rendering of ultrasound and optoacoustic images at an unprecedented rate of 10 volumetric frames per second. Performance is subsequently showcased in phantom experiments and in-vivo measurements from a healthy human volunteer, confirming general clinical applicability of the method.

  4. Multifunctional compact hybrid Au nanoshells: a new generation of nanoplasmonic probes for biosensing, imaging, and controlled release.

    PubMed

    Jin, Yongdong

    2014-01-21

    Gold nanoshells (AuNSs) with tunable localized surface plasmon resonance (LSPR) peaks in the near-infrared (NIR) region possess unique optical properties-particularly that soft tissues are "transparent" at these wavelengths-making them of great interest in cancer diagnosis and treatment. Since 1998 when Halas and co-workers invented the first generation of AuNS, with a silica core and Au shell, researchers have studied and designed AuNSs for theranostic-individualized, combination diagnosis and therapy-nanomedicine. As demand has increased for more powerful and practical theranostic applications, so has demand for the next generation of AuNSs-compact yet complex multifunctional AuNSs with finely integrated plasmonic and nonplasmonic inorganic components. For in vivo biomedical applications, such a hybrid AuNS offers the desirable optical properties of NIR LSPR. Size, however, has proved a more challenging parameter to control in hybrid AuNSs. The ideal size of therapeutic NPs is 10-100 nm. Larger particles have limited diffusion in the extracellular space, while particles less than 5 nm are rapidly cleared from the circulation through extravasation or renal clearance. Conventional methods of preparing AuNS have failed to obtain small-sized hybrid AuNSs with NIR LSPR responses. In this Account, we present a new class of multifunctional hybrid AuNSs with ultrathin AuNSs and varied, functional (nonplasmonic) core components ranging from "hard" semiconductor quantum dots (QDs), to superparamagnetic NPs, to "soft" liposomes made using poly-l-histidine as a template to direct Au deposition. The resultant hybrid AuNSs are uniform and compact (typically 15-60 nm) but also preserve the optical properties and shell-type NIR response necessary for biomedical use. We also demonstrate these particles' innovative plasmonic applications in biosensing, multimodal imaging and controlled release. More importantly, the magnetic-plasmonic Fe3O4/Au core-shell NP enables a new

  5. Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids.

    PubMed

    Magoulas, George E; Rigopoulos, Andreas; Piperigkou, Zoi; Gialeli, Chrysostomi; Karamanos, Nikos K; Takis, Panteleimon G; Troganis, Anastassios N; Chrissanthopoulos, Athanassios; Maroulis, George; Papaioannou, Dionissios

    2016-06-01

    Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl3-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256μM and periods of treatment of 24, 48 and 72h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC50 64-70μM) for the MDA-MB-231 cell line after 24-48h of treatment, but they were more selective and much more potent (IC50 4-16μM) for the MCF-7 cells after 48h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72h of treatment (IC50 1-2μM), probably as the result of slow hydrolysis of their methyl ester functions. PMID:27155809

  6. 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

  7. Protein adsorption on piezoelectric poly(L-lactic) acid thin films by scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Barroca, Nathalie; Vilarinho, Paula M.; Daniel-da-Silva, Ana Luisa; Wu, Aiying; Fernandes, Maria Helena; Gruverman, Alexei

    2011-03-01

    Up until now, no direct evidence of protein adsorption processes associated with polar activity of a piezoelectric has been reported. This work presents the experimental evidence of the protein adsorption process' dependence on the surface polarization of a piezoelectric by showing at the local scale that the process of protein adsorption is highly favored in the poled areas of a piezoelectric polymer such as poly(L-lactic) acid.

  8. 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

  9. A highly sensitive photoelectrochemical detection of perfluorooctanic acid with molecularly imprined polymer-functionalized nanoarchitectured hybrid of AgI-BiOI composite.

    PubMed

    Gong, Jingming; Fang, Tian; Peng, Dinghua; Li, Aimin; Zhang, Lizhi

    2015-11-15

    A rapid and ultrasensitive signal-off photoelectrochemical sensor has been developed under visible-light irradiation, for the detection of perfluorooctanoic acid (PFOA), especially low level PFOA present in environment, whereby a novel nanostructured probe made of molecularly imprinted polymer (MIP) modified AgI nanoparticles-BiOI nanoflake arrays (AgI-BiOINFs) is designed as the photoactive electrode (denoted as MIP@AgI-BiOINFs). Here, the unique nanoarchitectured hybrid of AgI-BiOINFs was first in situ synthesized via a facile successive ionic layer adsorption and reaction (SILAR) approach and then employed as a matrix to graft the recognition element of MIP. Such a newly designed PEC sensor exhibits high sensitivity and selectivity for the determination of PFOA. The PEC analysis is highly linear over the PFOA concentration ranging from 0.02 to 1000.0 ppb with a detection limit of 0.01 ppb (S/N=3). This value obtained by using the facile PEC sensor is comparable to the results obtained by using well-established liquid chromatography-tandem mass spectrometry (LC-MS/MS). Toward practical applications, this low-cost and sensitive assay was successfully applied to measure PFOA in real water samples. PMID:26092130

  10. Method for isolating chromosomal DNA in preparation for hybridization in suspension

    DOEpatents

    Lucas, Joe N.

    2000-01-01

    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. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

  11. Solid phase sequencing of double-stranded nucleic acids

    DOEpatents

    Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

    2002-01-01

    This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

  12. 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...

  13. The optimisation of grid designs for valve-regulated lead/acid batteries for hybrid electric vehicle applications

    NASA Astrophysics Data System (ADS)

    May, G. J.; Maleschitz, N.; Diermaier, H.; Haeupl, T.

    The design, construction and testing of valve-regulated lead/acid cells with grid designs optimised for high-rate partial state-of-charge cycling for hybrid electric vehicles are described. Computer modelling was used to develop the grid designs. This showed that designs with opposed tabs and terminals on the top and bottom of the cell were likely to have the best performance not only in terms of grid conductivity but also for uniformity of active material utilisation. Prototype cells were built and tested. Low rate performance was in line with the designs and the high-rate performance was substantially enhanced compared with conventional constructions. The cells were then tested to a shallow cycling regime and to a simplified hybrid electric vehicle cycle. The results showed excellent life under these conditions without the benefit of carbon or graphite additives to the negative active material that have also been shown to improve cycle life under these conditions.

  14. Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

    PubMed Central

    Hwang, Helen

    2014-01-01

    Single molecule studies of protein–nucleic acid interactions shed light on molecular mechanisms and kinetics involved in protein binding, translocation, and unwinding of DNA and RNA substrates. In this review, we provide an overview of a single molecule fluorescence method, termed “protein induced fluorescence enhancement” (PIFE). Unlike FRET where two dyes are required, PIFE employs a single dye attached to DNA or RNA to which an unlabeled protein is applied. We discuss both ensemble and single molecule studies in which PIFE was utilized. PMID:24056732

  15. Advanced nuclear magnetic resonance lanthanide probe analyses of short-range conformational interrelations controlling ribonucleic acid structures.

    PubMed

    Yokoyama, S; Inagaki, F; Miyazawa, T

    1981-05-12

    An advanced method was developed for lanthanide-probe analyses of the conformations of flexible biomolecules such as nucleotides. The new method is to determine structure parameters (such as internal-rotation angles) and population parameters for local conformational equilibria of flexible sites, together with standard deviations of these parameters. As the prominent advantage of this method, the interrelations among local conformations of flexible sites may be quantitatively elucidated from the experimental data of lanthanide-induced shifts and relaxations and vicinal coupling constants. As a structural unit of ribonucleic acids, the molecular conformations and conformational equilibria of uridine 3'-monophosphate in aqueous solution were analyzed. The stable local conformers about the C3'-O3' bond are the G+ (phi' = 281 +/- 11 degrees) and G- (phi' = 211 +/- 8 degrees) forms. The internal rotation about the C3'-O3' bond and the ribose-ring puckering are interrelated; 97 +/- 5% of the C3'-endo ribose ring is associated with the G- form while 70 +/- 22% o the C2'-endo ribose ring is associated with the G+ form. An interdependency also exists between the internal rotation about the C4'-C5' bond and the ribose-ring puckering. These short-range conformational interrelations are probably important in controlling the dynamic aspects of ribonucleic acid structures. PMID:6166319

  16. 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

  17. Interface engineering of hybrid perovskite solar cells with poly(3-thiophene acetic acid) under ambient conditions.

    PubMed

    Shit, Arnab; Nandi, Arun K

    2016-04-21

    The properties of methyl ammonium lead iodide (MAPbI3) perovskite solar cells with poly(3-thiophene acetic acid) (P3TAA) as a hole transporting material (HTM) and a dense layer of ZnO nanoparticle film as an electron transporting material (ETM) are described using the conventional ZnO (n)/perovskite (i)/P3TAA (p) (n-i-p) architecture. The FT-IR spectra of a MAPbI3/P3TAA mixture indicate a shift of the N-H stretching and the abolition of the N-H bending peak indicating the interaction between the components. UV-Vis spectra of the mixture exhibit a large red shift of the π-π* transition peak of the conjugated chain arising from the interaction causing an increase of the conjugation length. The cross-sectional SEM image of the device shows the sequence of the individual layers of ZnO, MAPbI3, P3TAA and Ag, respectively. The current density (J)-voltage (V) curves obtained upon illumination with a light of 100 mW cm(-2) indicate the average PCE to be 7.38 ± 0.59% under ambient conditions. The IPCE values of these cells reach about 63% across a broad range of wavelength (300-800 nm). The HOMO and the LUMO of P3TAA are measured using cyclic voltammetry and the optical band gap and the relative energy level of the components explain the operation of photocurrent in the cell. For comparison purposes a device using poly(3-hexyl thiophene) (P3HT) as the HTM is fabricated under similar conditions and it exhibits a lower PCE (5.85 ± 0.51%) than that of the P3TAA based device. The longevity of the P3TAA based cell is also found to be better than that of the P3HT based cell for storing in air. The UV-Vis and impedance spectral results clearly explain the above results, signifying the influence of the interface on the performance of hybrid solar cells. PMID:27020145

  18. 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)...

  19. 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

  20. Detection of {open_quotes}cryptic{close_quotes}karyotypic rearrangements in closely related primate species by fluorescence in situ hybridization (FISH) using human subtelomeric DNA probes

    SciTech Connect

    Youngblom, J.J.; Trask, B.J.; Friedman, C.

    1994-09-01

    Specific human subtelomeric DNA probes were used to reveal cryptic chromosomal rearrangements that cannot be detected by conventional high resolution cytogenetic techniques, or by chromosomal in situ suppression hybridization using whole chromosome paint analysis. Two cosmids containing different subtelomeric DNA sequences were derived from human chromosome 19 and designated as 7501 and 16432. Cosmid 7501 was hybridized to chromosomes from humans, chimpanzee, gorilla and orangutan. In humans, 7501 consistently labeled chromosomes 3q, 15q, and 19p. Additional chromosomes were labeled in different individuals, indicating a polymorphic distribution of this sequence in the human genome. In contrast, 7501 consistently and strongly labeled only the q arm terminus of chromosome 3 in both chimp and gorilla. The identification of the chromosome was made by two-color FISH analysis using human chromosome 4-specific paint and homologous to human chromosome 4. None of the human subjects showed labeling of chromosome 4 with 7501. This finding suggests that in the course of human evolution, subsequent to the divergence of humans and African apes, a cryptic translocation occurred between the ancestral human chromosome 4 and one or more of the other human chromosomes that now contain this DNA segment. In orangutan, 7501 labeled a single acrocentric chromosome pair, a distinctly different chromosome than that labeled in chimp and gorilla. Comparison of chromosome sites labeled with cosmid 16432 showed the distribution of signals on chromosome 1q arm is the same for humans and chimp, but different in the gorilla. Humans and chimps show distinct labeling on sites 1q terminus and 1q41-42. In gorilla, there is instead a large cluster of intense signal near the terminus of 1q that clearly does not extend all the way to the terminus. A paracentric inversion or an unequal cross-over event may account for the observed difference between these species.

  1. Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

    PubMed

    Combaret, Valérie; Iacono, Isabelle; Bréjon, Stéphanie; Schleiermacher, Gudrun; Pierron, Gäelle; Couturier, Jérôme; Bergeron, Christophe; Blay, Jean-Yves

    2012-12-01

    In cases of neuroblastoma, recurring genetic alterations--losses of the 1p, 3p, 4p, and 11q and/or gains of 1q, 2p, and 17q chromosome arms--are currently used to define the therapeutic strategy in therapeutic protocols for low- and intermediate-risk patients. Different genome-wide analysis techniques, such as array comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA), have been suggested for detecting chromosome segmental abnormalities. In this study, we compared the results of the two technologies in the analyses of the DNA of tumor samples from 91 neuroblastoma patients. Similar results were obtained with the two techniques for 75 samples (82%). In five cases (5.5%), the MLPA results were not interpretable. Discrepancies between the aCGH and MLPA results were observed in 11 cases (12%). Among the discrepancies, a 18q21.2-qter gain and 16p11.2 and 11q14.1-q14.3 losses were detected only by aCGH. The MLPA results showed that the 7p, 7q, and 14q chromosome arms were affected in six cases, while in two cases, 2p and 17q gains were observed; these results were confirmed by neither aCGH nor fluorescence in situ hybridization (FISH) analysis. Because of the higher sensitivity and specificity of genome-wide information, reasonable cost, and shorter time of aCGH analysis, we recommend the aCGH procedure for the analysis of genomic alterations in neuroblastoma. PMID:23265803

  2. A hybrid of ant colony optimization and minimization of metabolic adjustment to improve the production of succinic acid in Escherichia coli.

    PubMed

    Chong, Shiue Kee; Mohamad, Mohd Saberi; Mohamed Salleh, Abdul Hakim; Choon, Yee Wen; Chong, Chuii Khim; Deris, Safaai

    2014-06-01

    This paper presents a study on gene knockout strategies to identify candidate genes to be knocked out for improving the production of succinic acid in Escherichia coli. Succinic acid is widely used as a precursor for many chemicals, for example production of antibiotics, therapeutic proteins and food. However, the chemical syntheses of succinic acid using the traditional methods usually result in the production that is far below their theoretical maximums. In silico gene knockout strategies are commonly implemented to delete the gene in E. coli to overcome this problem. In this paper, a hybrid of Ant Colony Optimization (ACO) and Minimization of Metabolic Adjustment (MoMA) is proposed to identify gene knockout strategies to improve the production of succinic acid in E. coli. As a result, the hybrid algorithm generated a list of knockout genes, succinic acid production rate and growth rate for E. coli after gene knockout. The results of the hybrid algorithm were compared with the previous methods, OptKnock and MOMAKnock. It was found that the hybrid algorithm performed better than OptKnock and MOMAKnock in terms of the production rate. The information from the results produced from the hybrid algorithm can be used in wet laboratory experiments to increase the production of succinic acid in E. coli. PMID:24763079

  3. Growth, body composition, immune response and resistance to Streptococcus iniae of hybrid tilapia, Oreochromis niloticus x O. aureaus, fed diets containing various levels of linoleic and linolenic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of various levels of dietary linoleic (LA) and linolenic acids (LN) on growth, body proximate and fatty acid composition, immune response and resistance to Streptococcus iniae of juvenile, sex-reversed all-male hybrid tilapia, Oreochromis niloticus x O. areaus, were evaluated. A basal pu...

  4. 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

  5. Molecular Mechanisms in Morpholino-DNA Surface Hybridization

    PubMed Central

    Gong, Ping; Wang, Kang; Liu, Yatao; Shepard, Kenneth

    2010-01-01

    Synthetic nucleic acid mimics provide opportunity for redesigning the specificity and affinity of hybridization with natural DNA or RNA. Such redesign is of great interest for diagnostic applications where it can enhance the desired signal against a background of competing interactions. This report compares hybridization of DNA analyte strands with morpholinos (MOs), which are uncharged nucleic acid mimics, to the corresponding DNA-DNA case in solution and on surfaces. In solution, MO-DNA hybridization is found to be independent of counterion concentration, in contrast to DNA-DNA hybridization. On surfaces, when immobilized MO or DNA “probe” strands hybridize with complementary DNA “targets” from solution, both the MO-DNA and DNA-DNA processes depend on ionic strength but exhibit qualitatively different behaviors. At lower ionic strengths, MO-DNA surface hybridization exhibits hallmarks of kinetic limitations when separation between hybridized probe sites becomes comparable to target dimensions, whereas extents of DNA-DNA surface hybridization are instead consistent with limits imposed by buildup of surface (Donnan) potential. The two processes also fundamentally differ at high ionic strength, under conditions when electrostatic effects are weak. Here, variations in probe coverage have a much diminished impact on MO-DNA than on DNA-DNA hybridization for similarly crowded surface conditions. These various observations agree with a structural model of MO monolayers in which MO-DNA duplexes segregate to the buffer interface while unhybridized probes localize near the solid support. A general perspective is presented on using uncharged DNA analogues, which also include compounds such as peptide nucleic acids (PNA), in surface hybridization applications. PMID:20572663

  6. Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.

    PubMed

    Lederer, Florence; Vignaud, Caroline; North, Paul; Bodevin, Sabrina

    2016-09-01

    A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes. PMID:27155230

  7. An ultrasensitive DNA biosensor based on covalent immobilization of probe DNA on fern leaf-like α-Fe2O3 and chitosan Hybrid film using terephthalaldehyde as arm-linker.

    PubMed

    Xu, Biyan; Zheng, Delun; Qiu, Weiwei; Gao, Feng; Jiang, Shaoxiong; Wang, Qingxiang

    2015-10-15

    In this work, a novel electrochemical DNA biosensor has been developed based on the hybrid film of fern leaf-like α-Fe2O3 microparticles and chitosan (CS). The fern leaf-like α-Fe2O3 microparticles were synthesized via a facile template-free hydrothermal method, and their morphologies were characterized by X-ray diffraction, energy dispersive spectrometry, scanning electron microscope, and transmission electron microscope. Electrochemical characterization assays revealed that the hybrid film modified electrode had remarkable synergistic effects of the large accessible surface area and high electrical conductivity of semiconductive Fe2O3, and the good film stability of CS. Based on the rich amino groups on CS, the CS-Fe2O3 hybrid film was employed as a functional matrix for probe DNA immobilization using terephthalaldehyde (TPA) as a bifunctional arm-linker. The hybridization capacity of the developed biosensor was evaluated with electrochemical impedance spectroscopy (EIS) using [Fe(CN)6](3-/4-) as the indicating probe. A wide dynamic detection range from 1.0 × 10(-14) to 1.0 × 10(-10)M with ultralow detection limit of 5.6 × 10(-15)M was achieved for the target DNA. The hybridization selectivity experiments further revealed that the biosensor could discriminate fully complementary sequences from one-base mismatched, three-base mismatched, and non-complementary sequences. Moreover, the biosensor showed the advantage of good regeneration ability and reproducibility. PMID:25982725

  8. Towards monitoring dysplastic progression in the oral cavity using a hybrid fiber-bundle imaging and spectroscopy probe

    PubMed Central

    Greening, Gage J.; James, Haley M.; Dierks, Mary K.; Vongkittiargorn, Nontapoth; Osterholm, Samantha M.; Rajaram, Narasimhan; Muldoon, Timothy J.

    2016-01-01

    Intraepithelial dysplasia of the oral mucosa typically originates in the proliferative cell layer at the basement membrane and extends to the upper epithelial layers as the disease progresses. Detection of malignancies typically occurs upon visual inspection by non-specialists at a late-stage. In this manuscript, we validate a quantitative hybrid imaging and spectroscopy microendoscope to monitor dysplastic progression within the oral cavity microenvironment in a phantom and pre-clinical study. We use an empirical model to quantify optical properties and sampling depth from sub-diffuse reflectance spectra (450–750 nm) at two source-detector separations (374 and 730 μm). Average errors in recovering reduced scattering (5–26 cm−1) and absorption coefficients (0–10 cm−1) in hemoglobin-based phantoms were approximately 2% and 6%, respectively. Next, a 300 μm-thick phantom tumor model was used to validate the probe’s ability to monitor progression of a proliferating optical heterogeneity. Finally, the technique was demonstrated on 13 healthy volunteers and volume-averaged optical coefficients, scattering exponent, hemoglobin concentration, oxygen saturation, and sampling depth are presented alongside a high-resolution microendoscopy image of oral mucosa from one volunteer. This multimodal microendoscopy approach encompasses both structural and spectroscopic reporters of perfusion within the tissue microenvironment and can potentially be used to monitor tumor response to therapy. PMID:27220821

  9. Ordering in bio-inorganic hybrid nanomaterials probed by in situ scanning transmission X-ray microscopy

    DOE PAGESBeta

    Lee, Jonathan R. I.; Bagge-Hansen, Michael; Tunuguntla, Ramya; Kim, Kyunghoon; Bangar, Mangesh; Willey, Trevor M.; Tran, Ich C.; Kilcoyne, David A.; Noy, Aleksandr; van Buuren, Tony

    2015-04-15

    Here, phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. Inmore » this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ~30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.« less

  10. "Fastening" porphyrin in highly cross-linked polyphosphazene hybrid nanoparticles: powerful red fluorescent probe for detecting mercury ion.

    PubMed

    Hu, Ying; Meng, Lingjie; Lu, Qinghua

    2014-04-22

    It is a significant issue to overcome the concentration-quenching effect of the small fluorescent probes and maintain the high fluorescent efficiency at high concentration for sensitive and selective fluorescent mark or detection. We developed a new strategy to "isolate" and "fasten" porphyrin moieties in a highly cross-linked poly(tetraphenylporphyrin-co-cyclotriphosphazene) (TPP-PZS) by the polycondensation of hexachlorocyclotriphosphazene (HCCP) and 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (TPP-(OH)4) in a suitable solvent. The resulting TPP-PZS particles were characterized with transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR), (31)P nuclear magnetic resonance (NMR), and ultraviolet and visible (UV-vis) absorption spectra. Remarkably, TPP-PZS particles obtained in acetone emitted a bright red fluorescence both in powder state and in solution because the aggregation of porphyrin moieties in "H-type" (face-to-face) and "J-type" (edge-to-edge) was effectively blocked. The fluorescent TPP-PZS particles also showed superior resistance to photobleaching, and had a high sensitivity and selectivity for the detection of Hg(2+) ions. The TPP-PZS particles were therefore used as an ideal material for preparing test strips to quickly detect/monitor the Hg(2+) ions in a facile way. PMID:24678932

  11. Probing the source and timing of rejuvenation and hybridization in post-caldera rhyolite magmas at Yellowstone Caldera

    NASA Astrophysics Data System (ADS)

    Till, C.; Vazquez, J. A.; Boyce, J. W.; Stelton, M. E.

    2013-12-01

    We find petrographic, isotopic and geochemical evidence for rejuvenation and recycling of subvolcanic intrusions within low δ18O intracaldera rhyolite lavas erupted following the formation of Yellowstone Caldera. In order to resolve the timing and compositional end-members involved in rejuvenation and hybridization of Yellowstone's subvolcanic magma reservoir, we have analyzed the Pb isotopic composition of clinopyroxene and sanidine phenocrysts and performed U-Pb dating of zircons from the South Biscuit Basin (SBB) and Scaup Lake (SCL) rhyolite lava flows. Both SBB and SCL erupted ca. 260 ka based on indistinguishable 40Ar/39Ar ages [1,2] and represent a renewed episode of postcaldera volcanism after a hiatus of ~200 kyr. Zoned phenocrysts of quartz, clinopyroxene, orthopyroxene, plagioclase, and sanidine and accessory zircon and Fe-Ti oxides characterize both SBB and SCL. SCL and SBB clinopyroxene have identical compositions and core-to-rim zoning patterns in Fe, Mg, and trace elements and commonly exhibit exsolution lamellae in their cores, suggesting that subsolidus conditions were attained during the early evolution of these rhyolites. The exsolution-bearing cores are mantled by a zone of relatively high Mg/Fe and low HREE & Rb, which is in turn overgrown by a rim with slightly lower Mg/Fe, and higher HREE & Rb. This zoning pattern suggests rejuvenation of subsolidus rhyolite by the influx of at least two less evolved, hotter silicic magmas. Diffusion modeling of Fe-Mg concentration profiles in clinopyroxene suggests that these events occurred on the order of 103 yrs prior to eruption. To identify the source of the rejuvenated rhyolite and delimit intra-grain variability, we analyzed the Pb-isotopic compositions of the SBB and SCL clinopyroxene zones via LA-MC-ICPMS. The different zones within SBB and SCL clinopyroxene yield indistinguishable Pb-isotope compositions with 208Pb/206Pb=2.165-2.185 and 207Pb/206Pb=0.882-0.890, which matches the Pb

  12. Probing the interaction of caffeic acid with ZnO nanoparticles.

    PubMed

    Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

    2016-05-01

    The binding of ZnO nanoparticles (NPs) and caffeic acid (CFA) was investigated using fluorescence quenching, UV/vis absorption spectrscopy, Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) at different temperatures. The study results indicated fluorescence quenching between ZnO NPs and CFA rationalized in terms of a static quenching mechanism or the formation of non-fluorescent CFA-ZnO. From fluorescence quenching spectral analysis, the binding constant (Ka ), number of binding sites (n) and thermodynamic properties were determined. Values of the quenching (KSV ) and binding (Ka ) constants decrease with increasing temperature and the number of binding sites n = 2. The thermodynamic parameters determined using Van't Hoff equation indicated that binding occurs spontaneously involving the hydrogen bond, and van der Waal's forces played a major role in the reaction of ZnO NPs with CFA. The FTIR, TEM and DLS measurements also indicated differences in the structure, morphology and size of CFA, ZnO NPs and their corresponding CFA-ZnO. Copyright © 2015 John Wiley & Sons, Ltd. PMID:27037967

  13. 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

  14. 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

  15. Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.

    PubMed

    Anguizola, Jeanethe; Debolt, Erin; Suresh, D; Hage, David S

    2016-05-15

    The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems. PMID:26468085

  16. Strong, Thermally Superinsulating Biopolymer-Silica Aerogel Hybrids by Cogelation of Silicic Acid with Pectin.

    PubMed

    Zhao, Shanyu; Malfait, Wim J; Demilecamps, Arnaud; Zhang, Yucheng; Brunner, Samuel; Huber, Lukas; Tingaut, Philippe; Rigacci, Arnaud; Budtova, Tatiana; Koebel, Matthias M

    2015-11-23

    Silica aerogels are excellent thermal insulators, but their brittle nature has prevented widespread application. To overcome these mechanical limitations, silica-biopolymer hybrids are a promising alternative. A one-pot process to monolithic, superinsulating pectin-silica hybrid aerogels is presented. Their structural and physical properties can be tuned by adjusting the gelation pH and pectin concentration. Hybrid aerogels made at pH 1.5 exhibit minimal dust release and vastly improved mechanical properties while remaining excellent thermal insulators. The change in the mechanical properties is directly linked to the observed "neck-free" nanoscale network structure with thicker struts. Such a design is superior to "neck-limited", classical inorganic aerogels. This new class of materials opens up new perspectives for novel silica-biopolymer nanocomposite aerogels. PMID:26447457

  17. Proton conductive inorganic-organic hybrid membranes functionalized with phosphonic acid for polymer electrolyte fuel cell

    NASA Astrophysics Data System (ADS)

    Umeda, Junji; Suzuki, Masashi; Kato, Masaki; Moriya, Makoto; Sakamoto, Wataru; Yogo, Toshinobu

    Proton conductive sol-gel derived hybrid membranes were synthesized from aromatic derivatives of methoxysilanes and ethyl 2-[3-(dihydroxyphosphoryl)-2-oxapropyl]acrylate (EPA). Two aromatic derivatives of methoxysilanes with different number of methoxy groups were used as the starting materials. Hybrid membranes from difunctional (methyldimethoxysilylmethyl)styrene (MDMSMS(D))/EPA revealed a higher chemical stability and mechanical properties than those from monofunctional (dimethylmethoxysilylmethyl)styrene (DMMSMS(M))/EPA. The membrane-electrode assembly (MEA) using the hybrid membranes as electrolytes worked as a fuel cell at 100 °C under saturated humidity. The DMMSMS(M)/EPA membrane-based MEA showed a larger current density than that from MDMSMS(D)/EPA. On the other hand, the MDMSMS(D)/EPA membrane-based MEA exhibited higher open circuit voltages than the DMMSMS(M)/EPA-based MEA, and was stable during fuel cell operation at 80 °C at least for 48 h.

  18. A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune–Albright syndrome

    PubMed Central

    Karadag, Abdullah; Riminucci, Mara; Bianco, Paolo; Cherman, Natasha; Kuznetsov, Sergei A.; Nguyen, Nga; Collins, Michael T.; Robey, Pamela G.; Fisher, Larry W.

    2004-01-01

    Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gsα protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer. PMID:15096559

  19. Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

    PubMed Central

    Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

    2016-01-01

    Background: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Methods: Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results: All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype. PMID:26960370

  20. Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces.

    PubMed

    Sanpool, O; Intapan, P M; Thanchomnang, T; Sri-Aroon, P; Lulitanond, V; Sadaow, L; Maleewong, W

    2012-09-01

    Schistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts. PMID:22717071

  1. Fabrication and structure analysis of poly(lactide-co-glycolic acid)/silk fibroin hybrid scaffold for wound dressing applications.

    PubMed

    Shahverdi, Sheida; Hajimiri, Mirhamed; Esfandiari, Mohammad Amin; Larijani, Bagher; Atyabi, Fatemeh; Rajabiani, Afsaneh; Dehpour, Ahmad Reza; Gharehaghaji, Ali Akbar; Dinarvand, Rassoul

    2014-10-01

    Silk fibroin (SF) and poly(lactide-co-glycolic acid) (PLGA) have been proved to be invaluable polymers in the field wound healing. This study aims at optimizing the electrospinning process of those polymers to make a hybrid membrane as a chronic wounds dressing. After characterizing the scaffolds, PLGA/SF (2:1), and PLGA scaffolds were selected for further study according to their superior tensile mechanical properties. The attachment and proliferation of mouse fibroblasts (L929) on scaffolds were measured using colorimetric assay and scanning electron microscopy. Furthermore, to evaluate the wound healing effect of the scaffolds in comparison with gauze and Comfeel(®) dressings, an excision wound model was conducted on diabetic rats. On the postoperative days of 3, 6, 9, 12, and 15, residual wound area was calculated using macroscopic data. In vitro results showed that the attachment and proliferation of L929 were significantly increased on PLGA/SF (2:1) hybrid scaffold. Animal study and histopathological evaluation outcomes confirmed the in vitro results as well. On day 15, the residual wound area in PLGA/SF (2:1) hybrid membrane group was significantly smaller than PLGA and control groups. This promising scaffold has the potential to be used for the upcoming development of wound dressings with or without biological drugs. PMID:25051110

  2. Enantiomer separation of acidic chiral compounds on a quinine-silica/zirconia hybrid monolith by capillary electrochromatography.

    PubMed

    Tran, Le Ngoc; Park, Jung Hag

    2015-05-29

    A weak anion-exchanger chiral selector, quinine-incorporated silica/zirconia hybrid monolithic (QUI-S/ZHM) capillary column was prepared by sol-gel technology. The performance of the QUI-S/ZHM column was investigated for enantioresolution of a set of acidic chiral drugs and dinitrobenzoyl (DNB)-amino acids by capillary electrochromatography in aqueous organic mobile phases composed of acetonitrile (ACN) and triethylammonium acetate (TEAA) buffer. Effects of several parameters including the ACN content, concentration and pH of the mobile phase on the chiral separation were examined. Baseline resolutions of all the compounds were obtained in the mobile phase consisting of 70:30 ACN/TEAA (10mM, pH 6) under applied voltage of -10kV at 25°C within 20min. PMID:25892638

  3. Synthesis and properties of poly(methyl methacrylate-2-acrylamido-2-methylpropane sulfonic acid)/PbS hybrid composite

    SciTech Connect

    Preda, N.; Rusen, E.; Musuc, A.; Enculescu, M.; Matei, E.; Marculescu, B.; Fruth, V.; Enculescu, I.

    2010-08-15

    The synthesis of a new hybrid composite based on PbS nanoparticles and poly(methyl methacrylate-2-acrylamido-2-methylpropane sulfonic acid) [P(MMA-AMPSA)] copolymer is reported. The chemical synthesis consists in two steps: (i) a surfactant-free emulsion copolymerization between methyl methacrylate and 2-acrylamido-2-methylpropane sulfonic acid and (ii) the generation of PbS particles in the presence of the P(MMA-AMPSA) latex, from the reaction between lead nitrate and thiourea. The composite was studied by scanning electron microscopy (SEM), X-ray diffraction, FTIR spectroscopy, thermogravimetric analysis and differential scanning calorimetry. The microstructure observed using SEM proves that the PbS nanoparticles are well dispersed in the copolymer matrix. The X-ray diffraction measurements demonstrate that the PbS nanoparticles have a cubic rock salt structure. It was also found that the inorganic semiconductor nanoparticles improve the thermal stability of the copolymer matrix.

  4. In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

    PubMed Central

    Alexandersen, S; Bloom, M E; Wolfinbarger, J; Race, R E

    1987-01-01

    Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis. Images PMID:3037104

  5. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

    PubMed Central

    Suchan, Tomasz; Pitteloud, Camille; Gerasimova, Nadezhda S.; Kostikova, Anna; Schmid, Sarah; Arrigo, Nils; Pajkovic, Mila; Ronikier, Michał; Alvarez, Nadir

    2016-01-01

    In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes

  6. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.

    PubMed

    Suchan, Tomasz; Pitteloud, Camille; Gerasimova, Nadezhda S; Kostikova, Anna; Schmid, Sarah; Arrigo, Nils; Pajkovic, Mila; Ronikier, Michał; Alvarez, Nadir

    2016-01-01

    In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes

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

    PubMed

    Sato, Takaya; Sato, Yusuke; Nishizawa, Seiichi

    2016-08-01

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

  8. 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. PMID:25546245

  9. 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

  10. Application of steered molecular dynamics (SMD) to study DNA drug complexes and probing helical propensity of amino acids

    NASA Astrophysics Data System (ADS)

    Orzechowski, Marek; Cieplak, Piotr

    2005-05-01

    We present the preliminary results of two computer experiments involving the application of an external force to molecular systems. In the first experiment we simulated the process of pulling out a simple intercalator, the 9-aminoacridine molecule, from its complex with a short DNA oligonucleotide in aqueous solution. Removing a drug from the DNA is assumed to be an opposite process to the complex formation. The force and energy profiles suggest that formation of the DNA-9-aminoacridine complex is preferred when the acridine approaches the DNA from the minor groove rather than the major groove side. For a given mode of pulling the intercalation process is also shown to be nucleotide sequence dependent. In another computer experiment we performed a series of molecular dynamics simulations for stretching short, containing 15 amino acids, helical polypeptides in aqueous solution using an external force. The purpose of these simulations is to check whether this type of approach is sensitive enough to probe the sequence dependent helical propensity of short polypeptides.

  11. Kinetic mechanisms in morpholino-DNA surface hybridization.

    PubMed

    Liu, Yatao; Irving, Damion; Qiao, Wanqiong; Ge, Dongbiao; Levicky, Rastislav

    2011-08-01

    Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or "probe", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes. PMID:21699181

  12. High-sensitivity ascorbic acid sensor using graphene sheet/graphene nanoribbon hybrid material as an enhanced electrochemical sensing platform.

    PubMed

    Lavanya, J; Gomathi, N

    2015-11-01

    A novel electrode material of graphene sheet/graphene nanoribbon (GS/GNR) hybrid material was developed by incorporating graphene nanoribbons into graphene nanosheets through simultaneous chemical reduction of graphene oxide sheets and graphene oxide ribbons. The structure and properties of synthesized GS/GNR were characterized by transmission electron microscope, scanning electron microscope, X-ray diffraction, Brunauer Emmett Teller measurements and Fourier transform infrared spectroscopy. This work compares the electro catalytic performance of the GS/GNR, chemically reduced graphene oxide sheets (CRGOS) and GS/carbon nanotube (CNT) by modifying the glassy carbon electrode (GCE) using ascorbic acid (AA) as analyte. The electrochemical impedance spectroscopy revealed that the charge transfer resistance of GS/GNR modified electrode was less than that of CRGOS modified electrode and bare GCE. The cyclic voltammetric sensing of GS/GNR modified electrode towards AA was negatively shifted (0.08 V) compared to CRGOS, GS/CNT modified electrode and bare GCE (0.222, 0.150 and 0.666 V). This catalytic oxidation allows an amperometric detection of AA with a detection limit of 230 nM and sensitivity of 22 nA μM(-1) cm(-2). GS/GNR modified GCE exhibited a high selectivity for ascorbic acid in the presence of other interferents like dopamine, uric acid and citric acid. PMID:26452874

  13. Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

    PubMed Central

    Voordouw, Gerrit; Voordouw, Johanna K.; Karkhoff-Schweizer, Roxann R.; Fedorak, Phillip M.; Westlake, Donald W. S.

    1991-01-01

    A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a “standard”) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples. Images PMID:16348574

  14. Ampholine-functionalized hybrid organic-inorganic silica material as sorbent for solid-phase extraction of acidic and basic compounds.

    PubMed

    Wang, Tingting; Chen, Yihui; Ma, Junfeng; Chen, Mingliang; Nie, Chenggang; Hu, Minjie; Li, Ying; Jia, Zhijian; Fang, Jianghua; Gao, Haoqi

    2013-09-20

    A novel sorbent for solid-phase extraction (SPE) was synthesized by chemical immobilization of ampholine on hybrid organic-inorganic silica material. The ampholine-functionalized hybrid organic-inorganic silica sorbent is consisted of aliphatic amine groups, carboxyl groups and long carbon chains, allowing for extraction of both acidic and basic compounds. The retention properties of the developed sorbent were evaluated for 1-hydroxy-2-naphthoic acid (HNA), 1-naphthoic acid (NA), 3-hydroxybenzoic acid (HBA), benzoic acid (BA), sorbic acid (SA), vanillic aldehyde (VA), butyl 4-hydroxybenzoate (BHB), propyl 4-hydroxybenzoate (PHB), ethyl 4-hydroxybenzoate (EHB), and methyl 4-hydroxybenzoate (MHB). The results show that such a sorbent has three types of interaction, i.e., electrostatic interaction, hydrophobic interaction, and hydrogen bonding, exhibiting high extraction efficiency towards the compounds tested. The adsorption capacities of the analytes ranged from 0.61 to 6.54μgmg(-1). The reproducibility of the sorbent preparation was evaluated at three spiking concentration levels, with relative standard deviations (RSDs) of 1.0-10.5%. The recoveries of ten acidic and basic compounds spiked in beverage Coca-Cola(®) sample ranged from 82.5% to 98.2% with RSDs less than 5.8%. Under optimum conditions, the ampholine-functionalized hybrid organic-inorganic silica sorbent rendered higher extraction efficiency for acidic compounds than that of the commercially available ampholine-functionalized silica particles, and was comparable to that of the commercial Oasis WAX and Oasis WCX. PMID:23953713

  15. Novel Thiosemicarbazide Hybrids with Amino Acids and Peptides Against Hepatocellular Carcinoma: A Molecular Designing Approach Towards Multikinase Inhibitor.

    PubMed

    Chacko, Shinu; Samanta, Subir

    2015-01-01

    Hepatocellular Carcinoma is the most common primary malignant tumor of the liver. Development of multidrug resistance is the main obstacle to the success of anticancer drugs. In this study, designing and docking study of thiosemicarbazide hybrids with amino acids or peptides against hepatocellular carcinoma was performed since hybrids of biologically active compounds with amino acids or peptides may show target specificity and lower toxicity. All the structures were drawn in 2D platform and converted to the 3D platform using ChemDraw 10.0. Evaluations of ADME properties were done by using QikProp 3.0 to check for the possibility of oral delivery. In silico prediction of LD50 values were performed using Pro-Tox webserver. Interestingly, it was found that conjugation with amino acids decreases toxicity and increases the therapeutic index of thiosemicarbazide. Finally, all the compounds were docked to the crystal structure of the Vascular Endothelial Growth Factor Receptor-2 and Checkpoint kinase-1 utilizing Glide 5.0, Schrödinger 8.5, to understand the interaction of ligands with the receptor. A significant number of derivatives have been found active in both the receptors and also displayed multikinase inhibitory activity similar to Sorafenib, against hepatocellular carcinoma. Further, wet lab synthesis, in vitro ADMET and biological screening studies need to be performed to prove that designed compounds are effective against hepatocellular carcinoma as predicted by molecular modeling. However, as predicted by molecular modeling, the efficacy of designed compounds against hepatocellular carcinoma, needs to be confirmed by wet lab synthesis, in vitro ADMET and biological screening studies. PMID:26526710

  16. Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells.

    PubMed

    Wang, Xuzhe; Zhou, Li; Qiang, Fei; Wang, Feiyi; Wang, Rui; Zhao, Chunchang

    2016-03-10

    A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4-10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells. PMID:26893093

  17. Self-assembly of nucleic acids, silk and hybrid materials thereof

    NASA Astrophysics Data System (ADS)

    Humenik, Martin; Scheibel, Thomas

    2014-12-01

    Top-down approaches based on etching techniques have almost reached their limits in terms of dimension. Therefore, novel assembly strategies and types of nanomaterials are required to allow technological advances. Self-assembly processes independent of external energy sources and unlimited in dimensional scaling have become a very promising approach. Here, we highlight recent developments in self-assembled DNA-polymer, silk-polymer and silk-DNA hybrids as promising materials with biotic and abiotic moieties for constructing complex hierarchical materials in ‘bottom-up’ approaches. DNA block copolymers assemble into nanostructures typically exposing a DNA corona which allows functionalization, labeling and higher levels of organization due to its specific addressable recognition properties. In contrast, self-assembly of natural silk proteins as well as their recombinant variants yields mechanically stable β-sheet rich nanostructures. The combination of silk with abiotic polymers gains hybrid materials with new functionalities. Together, the precision of DNA hybridization and robustness of silk fibrillar structures combine in novel conjugates enable processing of higher-order structures with nanoscale architecture and programmable functions.

  18. Alkyl substituent effects on gas-phase acidities - The influence of hybridization.

    NASA Technical Reports Server (NTRS)

    Brauman, J. I.; Blair, L. K.

    1971-01-01

    Exploration of the effect on acidity of alkyl groups bonded to trigonal and digonal carbon. Some results on the relative acidities of toluene and p-xylene, and acetylene and substitute acetylenes, as determined by ion cyclotron resonance (icr) spectroscopy, are described. Some limitations of the CNDO/2 calculation method are discussed.

  19. A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics.

    PubMed Central

    Mazumder, A; Majlessi, M; Becker, M M

    1998-01-01

    We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets. The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Reaction of AE with sodium sulfite renders it non-chemiluminescent. When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent. In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent. Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence. Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets. The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques. PMID:9518495

  20. Investigation of the distribution of acidity strength in zeolites by temperature-programmed desorption of probe molecules. 2. Dealuminated Y-type zeolites

    SciTech Connect

    Karge, H.G.; Dondur, V. ); Weitkamp, J. )

    1991-01-10

    The acidity of dealuminated hydrogen forms of Y-type zeolites (Si/Al = 2.4-8.6) is determined by temperature-programmed desorption of ammonia or pyridine, which is monitored through a mass spectrometer. Four types of acidic sites are indicated by ammonia, viz., weak Broensted and/or Lewis centers and medium and strong Broensted and strong Lewis sites. In contrast, pyridine, after sample activation at 675 K, probed only two types of sites, i.e., medium and strong Broensted sites. This difference is ascribed to different accessibility of sites for the two probe molecules. From the desorption spectra (i) the fractional coverage of the various sites, (ii) the most frequent energies of activation, {anti E}{sub d}, for desorption, and (iii) the probability functions of the activation energies are derived by using a previously described method of evaluation.