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Sample records for hybridization probes targeting

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

    Science.gov (United States)

    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

  2. Fluoroscence in situ hybridization of chicken intestinal samples with bacterial rRNA targeted oligonucleotide probes

    DEFF Research Database (Denmark)

    Olsen, Katja Nyholm; Francesch, M.; Christensen, Henrik

    2006-01-01

    were hybridized for 24-72h, centrifuged, washed with pre-heated hybridization buffer, centrifuged and resuspended in Millipore quality water before filtration onto a 0.22 µm black polycarbonate filter. The probes used in this study were, LGC354A, LGC354B, LGC354C, Strc493, Bacto1080, Sal3, Chis150, EUB...

  3. Development and use of fluorescent 16S rRNA-targeted probes for the specific detection of Methylophaga species by in situ hybridization in marine sediments.

    Science.gov (United States)

    Janvier, Monique; Regnault, Béatrice; Grimont, Patrick

    2003-09-01

    Methylotrophic bacteria are widespread in nature. They may play an important role in the cycling of carbon and in the metabolism of dimethylsulfide in a marine environment. Bacteria belonging to the genus Methylophaga are a unique group of aerobic, halophilic, non-methane-utilizing methylotrophs. Two 16S rRNA-targeted oligonucleotide probes were developed for the specific detection of Methylophaga species, marine methylobacteria, by fluorescence in situ hybridization. Probe MPH-730 was highly specific for all members of the genus Methylophaga while probe MPHm-994 targeted exclusively M. marina. The application of these probes were demonstrated by the detection of Methylophaga species in enrichment cultures from various marine sediments. All isolates recovered were visualized by using the genus specific probe MPH-730. The results were confirmed by 16S rDNA sequencing which demonstrated that all selected isolates belong to Methylophaga. Five isolates could be detected by the M. marina-specific probe MPHm-994 and were confirmed by rRNA gene restriction pattern (ribotyping). With the development of these specific probes, fluorescence in situ hybridization shows that the genus Methylophaga is widespread in marine samples.

  4. Development of a 16S rRNA-targeted fluorescence in situ hybridization probe for quantification of the ammonia-oxidizer Nitrosotalea devanaterra and its relatives.

    Science.gov (United States)

    Restrepo-Ortiz, C X; Merbt, S N; Barrero-Canossa, J; Fuchs, B M; Casamayor, E O

    2018-04-28

    The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have high occurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential role in nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probe SAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequences in databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabilization for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditions were optimized on enrichment cultures of the target species N. devanaterra, as well as the non-target ammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competitor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design and application of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient, and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH protocol developed in the present study will help to improve the current understanding of the ecology and physiology of N. devanaterra and its relatives in natural environments. Copyright © 2018 Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

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

    1997-04-01

    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.

  6. 16S rRNA-targeted probes for specific detection of Thermoanaerobacterium spp., Thermoanaerobacterium thermosaccharolyticum, and Caldicellulosiruptor spp. by fluorescent in situ hybridization in biohydrogen producing systems

    Energy Technology Data Exchange (ETDEWEB)

    O-Thong, Sompong [Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bg 115, DK-2800, Kgs Lyngby (Denmark); Department of Biology, Faculty of Science, Thaksin University, Patthalung 93110 (Thailand); Prasertsan, Poonsuk [Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat-Yai, Songkhla 90112 (Thailand); Karakashev, Dimitar; Angelidaki, Irini [Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bg 115, DK-2800, Kgs Lyngby (Denmark)

    2008-11-15

    16S rRNA gene targeted oligonucleotide probes for specific detection of genera Thermoanaerobacterium (Tbm1282), Caldicellulosiruptor (Ccs432), and specie Thermoanaerobacterium thermosaccharolyticum (Tbmthsacc184) were designed and used to monitor the spatial distribution of hydrogen producing bacteria in sludge and granules from anaerobic reactors. The designed probes were checked for their specificity and then validated using fluorescence in situ hybridization with target microorganisms and non-target microorganisms. Thermoanaerobacterium spp., T. thermosaccharolyticum and Caldicellulosiruptor spp. were detected with the probes designed with coverage of 75%, 100% and 93%, respectively. Thermophilic (60 C) hydrogen producing reactors, one fed with sucrose and another, fed with palm oil mill effluent comprised of following major groups of hydrogen producers: Thermoanaerobacterium spp. (49% and 36%), T. thermosaccharolyticum (16% and 10%), phylum Firmicutes (low G+C) gram positive bacteria (15% and 27%). Extreme-thermophilic (70 C) hydrogen producing reactors, one fed with xylose and another, fed with lignocellulosic hydrolysate comprised of following major groups of hydrogen producers: Caldicellulosiruptor spp. (40.5% and 20.5%), phylum Firmicutes (low G+C) gram positive bacteria (17% and 20%), Archaea (7% and 8.5%), and Thermoanaerobacterium spp. (0% and 5%). Results obtained, showed good applicability of the probes Tbm1282, Tbmthsacc184 and Ccs432 for specific detection and quantification of thermophilic and extreme-thermophilic hydrogen producers in complex environments. (author)

  7. Expanding probe repertoire and improving reproducibility in human genomic hybridization

    Science.gov (United States)

    Dorman, Stephanie N.; Shirley, Ben C.; Knoll, Joan H. M.; Rogan, Peter K.

    2013-01-01

    Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance. A divide and conquer algorithm was implemented to design sc probes. With this approach, sc probes can include divergent repetitive elements, which hybridize to unique genomic targets under higher stringency experimental conditions. Genome-wide custom probe sets were created for fluorescent in situ hybridization (FISH) and microarray genomic hybridization. The scFISH probes were developed for detection of copy number changes within small tumour suppressor genes and oncogenes. The microarrays demonstrated increased reproducibility by eliminating cross-hybridization to repetitive sequences adjacent to probe targets. The genome-wide microarrays exhibited lower median coefficients of variation (17.8%) for two HapMap family trios. The coefficients of variations of commercial probes within 300 nt of a repetitive element were 48.3% higher than the nearest custom probe. Furthermore, the custom microarray called a chromosome 15q11.2q13 deletion more consistently. This method for sc probe design increases probe coverage for FISH and lowers variability in genomic microarrays. PMID:23376933

  8. Enzyme-free electrochemical detection of microRNA-21 using immobilized hairpin probes and a target-triggered hybridization chain reaction amplification strategy

    International Nuclear Information System (INIS)

    Liu, Hongying; Bei, Xiaoqiong; Xia, Qiuting; Fu, Yan; Zhang, Shi; Liu, Maochuan; Fan, Kai; Zhang, Mingzhen; Yang, Yong

    2016-01-01

    We describe a sensitive enzyme-free bioassay for the determination of microRNA-21. It is based on a combination of target-triggered hybridization chain reaction, tagging with CdTe quantum dots (QDs), and anodic stripping voltammetry. Firstly, a thiolated capture hairpin probe SH-HP1 was immobilized on the surface of a gold electrode. HP1 unfolds in the presence of microRNA-21. If hairpin probe 2 (HP2) is present, a HP1-HP2 complex will be formed which possesses an exposed stem of HP2, and microRNA is released in parallel. The released microRNA-21 triggers a hybridization chain reaction and this leads to form an exposed DNA segment of HP2 and cycle use microRNA-21. With the aid of assistant DNA A1 and A2, the exposed DNA segment of HP2 progressed to a long double strand. The strand is rich in CdTe QDs with the help of QDs-A1. Then, the attached QDs were dissolved with HNO 3 to give dissolved Cd(II) ions. Finally, the corresponding electrochemical current response of Cd(II) is monitored by anodic stripping voltammetry and used to quantify the concentration of microRNA-21. More microRNA-21 participated in this reaction increases the number of CdTe QDs, which results in increased electrochemical current. Thus, an ultrasensitive detection of microRNA-21 is accomplished by anodic stripping voltammetry. This gene assay displays a detection limit as low as 33 aM. It can discriminate between complementary DNA sequence and single-base mismatched DNA, indicating its high specificity. (author)

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

    OpenAIRE

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

    1994-01-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed prelim...

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

    Science.gov (United States)

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

    1994-09-01

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

  11. Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

    Science.gov (United States)

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

    2009-01-01

    A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

  12. Continuously tunable nucleic acid hybridization probes.

    Science.gov (United States)

    Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

    2015-12-01

    In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

  13. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    . This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)+ RNA accumulation within......In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues...

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization...... with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.......85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash...

  15. Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

    Science.gov (United States)

    Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

    2017-03-01

    This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

  16. 16S rRNA-targeted probes for specific detection of Thermoanaerobacterium spp., Thermoanaerobacterium thermosaccharolyticum, and Caldicellulosiruptor spp. by fluorescent in situ hybridization in biohydrogen producing systems

    DEFF Research Database (Denmark)

    O-Thong, Sompong; Prasertsan, P.; Karakashev, Dimitar Borisov

    2008-01-01

    % and 36%), T. thermosaccharolyticum (16% and 10%), phylum Firmicutes (low G+C) gram positive bacteria (15% and 27%). Extreme-thermophilic (70 °C) hydrogen producing reactors, one fed with xylose and another, fed with lignocellulosic hydrolysate comprised of following major groups of hydrogen producers......: Caldicellulosiruptor spp. (40.5% and 20.5%), phylum Firmicutes (low G+C) gram positive bacteria (17% and 20%), Archaea (7% and 8.5%), and Thermoanaerobacterium spp. (0% and 5%). Results obtained, showed good applicability of the probes Tbm1282, Tbmthsacc184 and Ccs432 for specific detection and quantification...... of thermophilic and extreme-thermophilic hydrogen producers in complex environments....

  17. Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

    Directory of Open Access Journals (Sweden)

    Ernault Pauline

    2003-05-01

    Full Text Available Abstract Background Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. Methods Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. Results Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. Conclusion We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more

  18. High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

    Science.gov (United States)

    Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

    2013-07-08

    Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

  19. Recent Advances in Target Characterization and Identification by Photoaffinity Probes

    Directory of Open Access Journals (Sweden)

    Sang J. Chung

    2013-08-01

    Full Text Available Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL. Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to their ability to form covalent bonds with the corresponding targets. Activity-based probe technology mainly relies on the chemical reactivity of the target proteins, thereby limiting the majority of the biological targets to enzymes or proteins which display reactive residues at the probe-binding site. In general, the probes should bear a reactive moiety such as an epoxide, a Michael acceptor, or a reactive alkyl halide in their structures. On the other hand, photoaffinity probes (PAPs are composed of a target-specific ligand and a photoactivatable functional group. When bound to the corresponding target proteins and activated with wavelength-specific light, PAPs generate highly reactive chemical species that covalently cross-link proximal amino acid residues. This process is better known as PAL and is widely employed to identify cellular targets of biologically active molecules. This review highlights recent advances in target identification by PAL, with a focus on the structure and chemistry of the photoaffinity probes developed in the recent decade, coupled to the target proteins identified using these probes.

  20. Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

    OpenAIRE

    Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

    2016-01-01

    Photoswitchable fluorescent proteins with controllable light?dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive ...

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

    Science.gov (United States)

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

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

  2. Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes.

    Science.gov (United States)

    Bowers, Holly A; Marin, Roman; Birch, James M; Scholin, Christopher A

    2017-12-01

    New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Electrogenerated chemiluminescence detection for deoxyribonucleic acid hybridization based on gold nanoparticles carrying multiple probes

    International Nuclear Information System (INIS)

    Wang Hui; Zhang Chengxiao; Li Yan; Qi Honglan

    2006-01-01

    A novel sensitive electrogenerated chemiluminescence (ECL) method for the detection deoxyribonucleic acid (DNA) hybridization based on gold nanoparticles carrying multiple probes was developed. Ruthenium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy) 2 (dcbpy)NHS) was used as a ECL label and gold nanoparticle as a carrier. Probe single strand DNA (ss-DNA) was self-assembled at the 3'-terminal with a thiol group to the surface of gold nanoparticle and covalently labeled at the 5'-terminal of a phosphate group with Ru(bpy) 2 (dcbpy)NHS and the resulting conjugate (Ru(bpy) 2 (dcbpy)NHS)-ss-DNA-Au, was taken as a ECL probe. When target analyte ss-DNA was immobilized on a gold electrode by self-assembled monolayer technique and then hybridized with the ECL probe to form a double-stranded DNA (ds-DNA), a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of the complementary sequence (target ss-DNA) in the range from 1.0 x 10 -11 to 1.0 x 10 -8 mol L -1 , and the linear regression equation was S = 57301 + 4579.6 lg C (unit of C is mol L -1 ). A detection limit of 5.0 x 10 -12 mol L -1 for target ss-DNA was achieved. The ECL signal generated from many reporters of ECL probe prepared is greatly amplified, compared to the convention scheme which is based on one reporter per hybridization event

  4. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

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

  5. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Science.gov (United States)

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

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  6. mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization.

    Science.gov (United States)

    Yilmaz, L Safak; Parnerkar, Shreyas; Noguera, Daniel R

    2011-02-01

    Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

  7. Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods

    International Nuclear Information System (INIS)

    Hunsaker, W.R.; Badri, H.; Lombardo, M.; Collins, M.L.

    1989-01-01

    A fourth capture is added to the reversible target capture procedure. This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the (capture probe) which contains a 3'-poly(dA) sequence and the (labeled probe) which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC

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

    Directory of Open Access Journals (Sweden)

    Gil Tae Hwang

    2018-01-01

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

  9. Systematic spatial bias in DNA microarray hybridization is caused by probe spot position-dependent variability in lateral diffusion.

    Science.gov (United States)

    Steger, Doris; Berry, David; Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

    2011-01-01

    The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization.

  10. Detection and quantitative analysis of actin mRNA by in situ hybridization with an oligodeoxynucleotide probe

    International Nuclear Information System (INIS)

    Taneja, K.; Singer, R.

    1987-01-01

    In situ hybridization is a useful method for localizing specific nucleic acid sequences intracellularly and for studying regulation of gene expression. Recently synthetic oligonucleotides have been successfully used as probes in this technique. Since they can be made easily to specific nucleic acid regions, they may be the best approach for analysis of a gene family of highly conserved sequences. They have analyzed these probes for the development of an in situ hybridization method. Oligonucleotides were made to different regions of chick beta-actin mRNA and used for detection of these sequences in a culture of chicken fibroblasts and myoblasts. They found that synthetic DNAs have different efficiencies of hybridization, indicating that not all target sequences are equivalent. They have investigated in detail a particular probe to the actin mRNA coding region and have optimized hybridization parameters. When hybridization was quantitated it was found that an oligonucleotide end labelled with 35 S or 32 P was capable of detecting several thousand messages per cell with a signal-to-noise ratio of 10:1. In situ hybridization confirmed the specificity of the hybridization as well as the background level. Increase in the number of oligonucleotides used should increase the signal-to-noise ratio-proportionately. Under particular circumstances the specificity of oligonucleotides make them an important reagent for in situ hybridization

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

    Science.gov (United States)

    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.

  12. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

    Science.gov (United States)

    Munir, Ahsan; Waseem, Hassan; Williams, Maggie R.; Stedtfeld, Robert D.; Gulari, Erdogan; Tiedje, James M.; Hashsham, Syed A.

    2017-01-01

    Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131). PMID:28555058

  13. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

    Directory of Open Access Journals (Sweden)

    Ahsan Munir

    2017-05-01

    Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

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

    Science.gov (United States)

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

    2015-08-10

    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.

  15. Sandwich nucleic acid hybridization: a method with a universally usable labeled probe for various specific tests

    International Nuclear Information System (INIS)

    Wolf, H.; Leser, U.; Haus, M.; Gu, S.Y.; Pathmanathan, R.

    1986-01-01

    The use of recombinant m13 phages as hybridization probes offers a considerable advantage over the commonly used recombinant plasmids as the preparation of the DNA probe is very simple and it can easily be labeled directly, e.g. with isotopes with long half-life like 125 I and used for hybridization. However, as the application of nucleic acid hybridization for diagnostic and epidemiological purposes becomes almost unavoidable, the logistic problems of keeping numerous individually labeled hybridization probes increase considerably and may reach prohibitory levels in less well-equipped laboratories. In a new sandwich technique, the first step involves hybridization with an unlabeled recombinant m13 DNA carrying an insert of the desired specificity. In a second step a universally usable labeled probe directed against the m13 part of the recombinant phage DNA is applied. This reduces considerably the problem of preparing and keeping multiple labeled probes in stock. (Auth.)

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

    DEFF Research Database (Denmark)

    Jakobsen, Ulla; Vogel, Stefan

    2016-01-01

    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...... 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....

  17. Photoelectronic characterization of IgG antibody molecule-quantum dot hybrid as biosensing probe

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hye-Weon; Kim, Sung-Jo; Kim, In S [School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712 (Korea, Republic of); Lee, Jinwook; Kim, Sungyoun, E-mail: iskim@gist.ac.kr [Center for Seawater Desalination Plant, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2010-10-22

    Quantum dot (QD)-based biomolecule hybrids have recently attracted much attention in specifically identifying and labeling target proteins. In this study, QD encapsulated with immunoglobulin antibodies, as a labeling building block in biosensors, was investigated to clarify the most efficient configuration and photoluminescence behavior. Both the biological recognition capacity and photoluminescence emitting signal of the antibody-coupled nanocrystal were validated through a photoelectrical characterization procedure. Derivation of the optimum number of antibody molecules to be packed onto the QD surface yielded the highest binding capacity for the target antigen. During formation of the bioactive layer, the intrinsic photoluminescence response of the QDs significantly decreased due to photoinduced hole transfer according to their rearranged electronic structure. The thorough study of this assembly provides a validation approach for the careful titration of biosensor probes for optimal reaction kinetics. Furthermore, it contributes to the development of an effective tool for the application and interpretation of QD-based labeling techniques.

  18. Photoelectronic characterization of IgG antibody molecule-quantum dot hybrid as biosensing probe

    International Nuclear Information System (INIS)

    Yu, Hye-Weon; Kim, Sung-Jo; Kim, In S; Lee, Jinwook; Kim, Sungyoun

    2010-01-01

    Quantum dot (QD)-based biomolecule hybrids have recently attracted much attention in specifically identifying and labeling target proteins. In this study, QD encapsulated with immunoglobulin antibodies, as a labeling building block in biosensors, was investigated to clarify the most efficient configuration and photoluminescence behavior. Both the biological recognition capacity and photoluminescence emitting signal of the antibody-coupled nanocrystal were validated through a photoelectrical characterization procedure. Derivation of the optimum number of antibody molecules to be packed onto the QD surface yielded the highest binding capacity for the target antigen. During formation of the bioactive layer, the intrinsic photoluminescence response of the QDs significantly decreased due to photoinduced hole transfer according to their rearranged electronic structure. The thorough study of this assembly provides a validation approach for the careful titration of biosensor probes for optimal reaction kinetics. Furthermore, it contributes to the development of an effective tool for the application and interpretation of QD-based labeling techniques.

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

    Science.gov (United States)

    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. © The Author(s) 2016.

  20. Electronic Detection of DNA Hybridization by Coupling Organic Field-Effect Transistor-Based Sensors and Hairpin-Shaped Probes

    Directory of Open Access Journals (Sweden)

    Corrado Napoli

    2018-03-01

    Full Text Available In this paper, the electronic transduction of DNA hybridization is presented by coupling organic charge-modulated field-effect transistors (OCMFETs and hairpin-shaped probes. These probes have shown interesting properties in terms of sensitivity and selectivity in other kinds of assays, in the form of molecular beacons (MBs. Their integration with organic-transistor based sensors, never explored before, paves the way to a new class of low-cost, easy-to-use, and portable genetic sensors with enhanced performances. Thanks to the peculiar characteristics of the employed sensor, measurements can be performed at relatively high ionic strengths, thus optimizing the probes’ functionality without affecting the detection ability of the device. A complete electrical characterization of the sensor is reported, including calibration with different target concentrations in the measurement environment and selectivity evaluation. In particular, DNA hybridization detection for target concentration as low as 100 pM is demonstrated.

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

    Science.gov (United States)

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

    2015-02-07

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

  2. Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

    Science.gov (United States)

    Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

    2012-01-01

    Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a

  3. Competitive reporter monitored amplification (CMA--quantification of molecular targets by real time monitoring of competitive reporter hybridization.

    Directory of Open Access Journals (Sweden)

    Thomas Ullrich

    Full Text Available BACKGROUND: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. METHODOLOGY AND PRINCIPAL FINDINGS: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. CONCLUSIONS AND SIGNIFICANCE: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2, we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls

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

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Carvalho, Daniel R; Guimarães, Nuno

    2016-01-01

    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......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...

  5. The BLAIRR Irradiation Facility Hybrid Spallation Target Optimization

    Energy Technology Data Exchange (ETDEWEB)

    Simos N.; Hanson A.; Brown, D.; Elbakhshawn, M.

    2016-04-11

    BLAIRR STUDY STATUS OVERVIEW Beamline Complex Evaluation/Assessment and Adaptation to the Goals Facility Radiological Constraints ? Large scale analyses of conventional facility and integrated shield (concrete, soil)Target Optimization and Design: Beam-target interaction optimization Hadronic interaction and energy deposition limitations Single phase and Hybrid target concepts Irradiation Damage Thermo-mechanical considerations Spallation neutron fluence optimization for (a) fast neutron irradiation damage (b) moderator/reflector studies, (c) NTOF potential and optimization (d) mono-energetic neutron beam

  6. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    Science.gov (United States)

    Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

  7. Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Directory of Open Access Journals (Sweden)

    Stougaard Magnus

    2007-11-01

    Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

  8. Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

    Science.gov (United States)

    Hommatsu, Manami; Okahashi, Hisamitsu; Ohta, Keisuke; Tamai, Yusuke; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2013-01-01

    A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.

  9. Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

    DEFF Research Database (Denmark)

    Yao, Danfeng; Kim, Junyoung; Yu, Fang

    2005-01-01

    at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA....../DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo...

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

    Science.gov (United States)

    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.

  11. Quenching methods for background reduction in luminescence-based probe-target binding assays

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Hong [Los Alamos, NM; Goodwin, Peter M [Los Alamos, NM; Keller, Richard A [Los Alamos, NM; Nolan, Rhiannon L [Santa Fe, NM

    2007-04-10

    Background luminescence is reduced from a solution containing unbound luminescent probes, each having a first molecule that attaches to a target molecule and having an attached luminescent moiety, and luminescent probe/target adducts. Quenching capture reagent molecules are formed that are capable of forming an adduct with the unbound luminescent probes and having an attached quencher material effective to quench luminescence of the luminescent moiety. The quencher material of the capture reagent molecules is added to a solution of the luminescent probe/target adducts and binds in a proximity to the luminescent moiety of the unbound luminescent probes to quench luminescence from the luminescent moiety when the luminescent moiety is exposed to exciting illumination. The quencher capture reagent does not bind to probe molecules that are bound to target molecules and the probe/target adduct emission is not quenched.

  12. Detection of hepatitis A virus by hybridization with single-stranded RNA probes

    International Nuclear Information System (INIS)

    Xi, J.; Estes, M.K.; Metcalf, T.G.

    1987-01-01

    An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

  13. Action Potential Dynamics in Fine Axons Probed with an Axonally Targeted Optical Voltage Sensor.

    Science.gov (United States)

    Ma, Yihe; Bayguinov, Peter O; Jackson, Meyer B

    2017-01-01

    The complex and malleable conduction properties of axons determine how action potentials propagate through extensive axonal arbors to reach synaptic terminals. The excitability of axonal membranes plays a major role in neural circuit function, but because most axons are too thin for conventional electrical recording, their properties remain largely unexplored. To overcome this obstacle, we used a genetically encoded hybrid voltage sensor (hVOS) harboring an axonal targeting motif. Expressing this probe in transgenic mice enabled us to monitor voltage changes optically in two populations of axons in hippocampal slices, the large axons of dentate granule cells (mossy fibers) in the stratum lucidum of the CA3 region and the much finer axons of hilar mossy cells in the inner molecular layer of the dentate gyrus. Action potentials propagated with distinct velocities in each type of axon. Repetitive firing broadened action potentials in both populations, but at an intermediate frequency the degree of broadening differed. Repetitive firing also attenuated action potential amplitudes in both mossy cell and granule cell axons. These results indicate that the features of use-dependent action potential broadening, and possible failure, observed previously in large nerve terminals also appear in much finer unmyelinated axons. Subtle differences in the frequency dependences could influence the propagation of activity through different pathways to excite different populations of neurons. The axonally targeted hVOS probe used here opens up the diverse repertoire of neuronal processes to detailed biophysical study.

  14. Moth sex chromatin probed by comparative genomic hybridization (CGH)

    Czech Academy of Sciences Publication Activity Database

    Sahara, K.; Marec, František; Eickhoff, U.; Traut, W.

    2003-01-01

    Roč. 46, - (2003), s. 339-342 ISSN 0831-2796 R&D Projects: GA AV ČR IAA6007307 Institutional research plan: CEZ:AV0Z5007907 Keywords : Lepidoptera * comparative genomic hybridization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.861, year: 2003

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

    Science.gov (United States)

    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

  16. Phenylethynylpyrene excimer forming hybridization probes for fluorescence SNP detection

    DEFF Research Database (Denmark)

    Prokhorenko, Igor A.; Astakhova, Irina V.; Momynaliev, Kuvat T.

    2009-01-01

    Excimer formation is a unique feature of some fluorescent dyes (e.g., pyrene) which can be used for probing the proximity of biomolecules. Pyrene excimer fluorescence has previously been used for homogeneous detection of single nucleotide polymorphism (SNP) on DNA. 1-Phenylethynylpyrene (1-1-PEPy...

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

    Science.gov (United States)

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

    2010-01-01

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

  18. Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

    International Nuclear Information System (INIS)

    Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

    1992-01-01

    Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

  19. Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

    1992-01-01

    Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

  20. Hybridization-based biosensor containing hairpin probes and use thereof

    Science.gov (United States)

    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.

  1. Edge turbulence measurement in Heliotron J using a combination of hybrid probe system and fast cameras

    International Nuclear Information System (INIS)

    Nishino, N.; Zang, L.; Takeuchi, M.; Mizuuchi, T.; Ohshima, S.; Kasajima, K.; Sha, M.; Mukai, K.; Lee, H.Y.; Nagasaki, K.; Okada, H.; Minami, T.; Kobayashi, S.; Yamamoto, S.; Konoshima, S.; Nakamura, Y.; Sano, F.

    2013-01-01

    The hybrid probe system (a combination of Langmuir probes and magnetic probes), fast camera and gas puffing system were installed at the same toroidal section to study edge plasma turbulence/fluctuation in Heliotron J, especially blob (intermittent filament). Fast camera views the location of the probe head, so that the probe system yields the time evolution of the turbulence/fluctuation while the camera images the spatial profile. Gas puff at the same toroidal section was used to control the plasma density and simultaneous gas puff imaging technique. Using this combined system the filamentary structure associated with magnetic fluctuation was found in Heliotron J at the first time. The other kind of fluctuation was also observed at another experiment. This combination measurement enables us to distinguish MHD activity and electro-static activity

  2. Detection of TTV-DNA in PBMC using digoxigenin labelled probe by in situ hybridization

    International Nuclear Information System (INIS)

    Liu Yang; Qi Qige

    2002-01-01

    To determine TTV-DNA in PBMC in patients with viral hepatitis, a study of in situ hybridization using digoxigenin labelled probe by PCR method to the TTV ORF1 region was performed on PBMC. Results showed that the detection rate of TTV-DNA using double-stranded probe in TTV-DNA positive group in sera was 58.06 (18/31), and the detection rate of TTV-DNA using double-stranded probe in TTV-DNA negative group in sera was 27.59 (8/29). For TTV-DNA positive group detected by double- stranded probe, then we use negative- stranded probe to detect their replication. The detection rate was 22.2%(4/18). Conclusions: TTV can infect PBMC and replicate in PBMC

  3. A Molecularly Targeted Theranostic Probe for Ovarian Cancer

    Science.gov (United States)

    Chen, Wenxue; Bardhan, Rizia; Bartels, Marc; Perez-Torres, Carlos; Pautler, Robia G.; Halas, Naomi J.; Joshi, Amit

    2014-01-01

    Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in ovarian cancer because of its participation in signaling pathway regulating cellular proliferation, differentiation, motility, and survival. Currently, effective diagnostic and therapeutic schemes are lacking for treating ovarian cancer and consequently ovarian cancer has a high mortality rate. While HER2 receptor expression does not usually affect the survival rates of ovarian cancer to the same extent as in breast cancer, it can be employed as a docking site for directed nanotherapies in cases with de novo or acquired chemotherapy resistance. In this study, we have exploited a novel gold nanoshell-based complex (nanocomplex) for targeting, dual modal imaging, and photothermal therapy of HER2 overexpressing and drug resistant ovarian cancer OVCAR3 cells in vitro. The nanocomplexes are engineered to simultaneously provide contrast as fluorescence optical imaging probe and a magnetic resonance imaging (MRI) agent. Both immunofluorescence staining and MRI successfully demonstrate that nanocomplex-anti-HER2 conjugates specifically bind to OVCAR3 cells as opposed to the control, MDA-MB-231 cells, which have low HER2 expression. In addition, nanocomplexes targeted to OVCAR3 cells, when irradiated with near infrared (NIR) laser result in selective destruction of cancer cells through photothermal ablation. We also demonstrate that NIR light therapy and the nanocomplexes by themselves are non-cytotoxic in vitro. To the best of our knowledge, this is the first demonstration of a successful integration of dual modal bioimaging with photothermal cancer therapy for treatment of ovarian cancer. Based on their efficacy in vitro, these nanocomplexes are highly promising for image guided photo-thermal therapy of ovarian cancer as well as other HER2 overexpressing cancers. PMID:20371708

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Schwiertz, A; Le Blay, G; Blaut, M

    2000-01-01

    Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

  6. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

    International Nuclear Information System (INIS)

    Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

    1988-01-01

    Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with 32 P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays

  7. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

    1988-11-01

    Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with /sup 32/P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

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

    Science.gov (United States)

    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.

  9. In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition

    DEFF Research Database (Denmark)

    Kofoed, Michael Vedel; Stief, Peter; Poulsen, Morten

    can be designed to target a broader range of denitrifying bacteria; however, they require two-pass CARD-FISH, which may result in (too) high background fluorescence. In a first application example, habitat-specific polynucleotide probes were used to quantify bacteria expressing narG and nos...... reduction of nitrate to dinitrogen gas, is essential for the removal of fixed nitrogen from natural and engineered ecosystems. However, community structure and activity dynamics of denitrifying bacteria in most systems are poorly understood, partially due to difficulties in identifying and quantifying...... and catalyzed fluorescent reporter deposition (CARD-FISH). The general feasibility of the approach was first tested with pure cultures of Pseudomonas stutzeri and various denitrifying and nitrate-reducing isolates. Detailed studies of probe specificity and hybridization conditions using Clone-FISH of nar...

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated 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

  11. Synergistic Combination of Unquenching and Plasmonic Fluorescence Enhancement in Fluorogenic Nucleic Acid Hybridization Probes.

    Science.gov (United States)

    Vietz, Carolin; Lalkens, Birka; Acuna, Guillermo P; Tinnefeld, Philip

    2017-10-11

    Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes.

  12. Final report: Mapping Interactions in Hybrid Systems with Active Scanning Probes

    Energy Technology Data Exchange (ETDEWEB)

    Berezovsky, Jesse [Case Western Reserve Univ., Cleveland, OH (United States)

    2017-09-29

    This project aimed to study and map interactions between components of hybrid nanodevices using a novel scanning probe approach. To enable this work, we initially constructed a flexible experimental apparatus allowing for simultaneous scanning probe and confocal optical microscopy measurements. This setup was first used for all-optical measurements of nanostructures, with the focus then shifting to hybrid devices in which single coherent electron spins are coupled to micron-scale ferromagnetic elements, which may prove useful for addressing single spins, enhanced sensing, or spin-wave-mediated coupling of spins for quantum information applications. A significant breakthrough was the realization that it is not necessary to fabricate a magnetic structure on a scanning probe – instead a ferromagnetic vortex core can act as an integrated, solid state, scanning probe. The core of the vortex produces a very strong, localized fringe field which can be used analogously to an MFM tip. Unlike a traditional MFM tip, however, the vortex core is scanned within an integrated device (eliminating drift), and can be moved on vastly faster timescales. This approach allows the detailed investigation of interactions between single spins and complex driven ferromagnetic dynamics.

  13. Hybrid photonic-plasmonic near-field probe for efficient light conversion into the nanoscale hot spot.

    Science.gov (United States)

    Koshelev, Alexander; Munechika, Keiko; Cabrini, Stefano

    2017-11-01

    In this Letter, we present a design and simulations of the novel hybrid photonic-plasmonic near-field probe. Near-field optics is a unique imaging tool that provides optical images with resolution down to tens of nanometers. One of the main limitations of this technology is its low light sensitivity. The presented hybrid probe solves this problem by combining a campanile plasmonic probe with the photonic layer, consisting of the diffractive optic element (DOE). The DOE is designed to match the plasmonic field at the broad side of the campanile probe with the fiber mode. This makes it possible to optimize the size of the campanile tip to convert light efficiently into the hot spot. The simulations show that the hybrid probe is ∼540 times more efficient compared with the conventional campanile on average in the 600-900 nm spectral range.

  14. Ultrasound probe and needle-guide calibration for robotic ultrasound scanning and needle targeting.

    Science.gov (United States)

    Kim, Chunwoo; Chang, Doyoung; Petrisor, Doru; Chirikjian, Gregory; Han, Misop; Stoianovici, Dan

    2013-06-01

    Image-to-robot registration is a typical step for robotic image-guided interventions. If the imaging device uses a portable imaging probe that is held by a robot, this registration is constant and has been commonly named probe calibration. The same applies to probes tracked by a position measurement device. We report a calibration method for 2-D ultrasound probes using robotic manipulation and a planar calibration rig. Moreover, a needle guide that is attached to the probe is also calibrated for ultrasound-guided needle targeting. The method is applied to a transrectal ultrasound (TRUS) probe for robot-assisted prostate biopsy. Validation experiments include TRUS-guided needle targeting accuracy tests. This paper outlines the entire process from the calibration to image-guided targeting. Freehand TRUS-guided prostate biopsy is the primary method of diagnosing prostate cancer, with over 1.2 million procedures performed annually in the U.S. alone. However, freehand biopsy is a highly challenging procedure with subjective quality control. As such, biopsy devices are emerging to assist the physician. Here, we present a method that uses robotic TRUS manipulation. A 2-D TRUS probe is supported by a 4-degree-of-freedom robot. The robot performs ultrasound scanning, enabling 3-D reconstructions. Based on the images, the robot orients a needle guide on target for biopsy. The biopsy is acquired manually through the guide. In vitro tests showed that the 3-D images were geometrically accurate, and an image-based needle targeting accuracy was 1.55 mm. These validate the probe calibration presented and the overall robotic system for needle targeting. Targeting accuracy is sufficient for targeting small, clinically significant prostatic cancer lesions, but actual in vivo targeting will include additional error components that will have to be determined.

  15. Imaging Voltage in Genetically Defined Neuronal Subpopulations with a Cre Recombinase-Targeted Hybrid Voltage Sensor.

    Science.gov (United States)

    Bayguinov, Peter O; Ma, Yihe; Gao, Yu; Zhao, Xinyu; Jackson, Meyer B

    2017-09-20

    Genetically encoded voltage indicators create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of genetically encoded voltage indicators requires a generalized strategy for targeting the probe to genetically defined populations of cells. To this end, we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase-dependent expression. Crossing this mouse with Cre drivers generated double transgenics expressing hVOS probe in GABAergic, parvalbumin, and calretinin interneurons, as well as hilar mossy cells, new adult-born neurons, and recently active neurons. In each case, imaging in brain slices from male or female animals revealed electrically evoked optical signals from multiple individual neurons in single trials. These imaging experiments revealed action potentials, dynamic aspects of dendritic integration, and trial-to-trial fluctuations in response latency. The rapid time response of hVOS imaging revealed action potentials with high temporal fidelity, and enabled accurate measurements of spike half-widths characteristic of each cell type. Simultaneous recording of rapid voltage changes in multiple neurons with a common genetic signature offers a powerful approach to the study of neural circuit function and the investigation of how neural networks encode, process, and store information. SIGNIFICANCE STATEMENT Genetically encoded voltage indicators hold great promise in the study of neural circuitry, but realizing their full potential depends on targeting the sensor to distinct cell types. Here we present a new mouse line that expresses a hybrid optical voltage sensor under the control of Cre recombinase. Crossing this line with Cre drivers generated double-transgenic mice, which express this sensor in targeted cell types. In

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Preliminary test of an imaging probe for nuclear medicine using hybrid pixel detectors

    International Nuclear Information System (INIS)

    Bertolucci, E.; Maiorino, M.; Mettivier, G.; Montesi, M.C.; Russo, P.

    2002-01-01

    We are investigating the feasibility of an intraoperative imaging probe for lymphoscintigraphy with Tc-99m tracer, for sentinel node radioguided surgery, using the Medipix series of hybrid detectors coupled to a collimator. These detectors are pixelated semiconductor detectors bump-bonded to the Medipix1 photon counting read-out chip (64x64 pixel, 170 μm pitch) or to the Medipix2 chip (256x256 pixel, 55 μm pitch), developed by the European Medipix collaboration. The pixel detector we plan to use in the final version of the probe is a semi-insulating GaAs detector or a 1-2 mm thick CdZnTe detector. For the preliminary tests presented here, we used 300-μm thick silicon detectors, hybridized via bump-bonding to the Medipix1 chip. We used a tungsten parallel-hole collimator (7 mm thick, matrix array of 64x64 100 μm circular holes with 170 μm pitch), and a 22, 60 and 122 keV point-like (1 mm diameter) radioactive sources, placed at various distances from the detector. These tests were conducted in order to investigate the general feasibility of this imaging probe and its resolving power. Measurements show the high resolution but low efficiency performance of the detector-collimator set, which is able to image the 122 keV source with <1 mm FWHM resolution

  18. Probing the transition state for nucleic acid hybridization using phi-value analysis.

    Science.gov (United States)

    Kim, Jandi; Shin, Jong-Shik

    2010-04-27

    Genetic regulation by noncoding RNA elements such as microRNA and small interfering RNA (siRNA) involves hybridization of a short single-stranded RNA with a complementary segment in a target mRNA. The physical basis of the hybridization process between the structured nucleic acids is not well understood primarily because of the lack of information about the transition-state structure. Here we use transition-state theory, inspired by phi-value analysis in protein folding studies, to provide quantitative analysis of the relationship between changes in the secondary structure stability and the activation free energy. Time course monitoring of the hybridization reaction was performed under pseudo-steady-state conditions using a single fluorophore. The phi-value analysis indicates that the native secondary structure remains intact in the transition state. The nativelike transition state was confirmed via examination of the salt dependence of the hybridization kinetics, indicating that the number of sodium ions associated with the transition state was not substantially affected by changes in the native secondary structure. These results propose that hybridization between structured nucleic acids undergoes a transition state leading to formation of a nucleation complex and then is followed by sequential displacement of preexisting base pairings involving successive small energy barriers. The proposed mechanism might provide new insight into physical processes during small RNA-mediated gene silencing, which is essential to selection of a target mRNA segment for siRNA design.

  19. Multiple target sound quality balance for hybrid electric powertrain noise

    Science.gov (United States)

    Mosquera-Sánchez, J. A.; Sarrazin, M.; Janssens, K.; de Oliveira, L. P. R.; Desmet, W.

    2018-01-01

    The integration of the electric motor to the powertrain in hybrid electric vehicles (HEVs) presents acoustic stimuli that elicit new perceptions. The large number of spectral components, as well as the wider bandwidth of this sort of noises, pose new challenges to current noise, vibration and harshness (NVH) approaches. This paper presents a framework for enhancing the sound quality (SQ) of the hybrid electric powertrain noise perceived inside the passenger compartment. Compared with current active sound quality control (ASQC) schemes, where the SQ improvement is just an effect of the control actions, the proposed technique features an optimization stage, which enables the NVH specialist to actively implement the amplitude balance of the tones that better fits into the auditory expectations. Since Loudness, Roughness, Sharpness and Tonality are the most relevant SQ metrics for interior HEV noise, they are used as performance metrics in the concurrent optimization analysis, which, eventually, drives the control design method. Thus, multichannel active sound profiling systems that feature cross-channel compensation schemes are guided by the multi-objective optimization stage, by means of optimal sets of amplitude gain factors that can be implemented at each single sensor location, while minimizing cross-channel effects that can either degrade the original SQ condition, or even hinder the implementation of independent SQ targets. The proposed framework is verified experimentally, with realistic stationary hybrid electric powertrain noise, showing SQ enhancement for multiple locations within a scaled vehicle mock-up. The results show total success rates in excess of 90%, which indicate that the proposed method is promising, not only for the improvement of the SQ of HEV noise, but also for a variety of periodic disturbances with similar features.

  20. Detection of DNA fingerprints of cultivated rice by hybridization with a human minisatellite DNA probe

    International Nuclear Information System (INIS)

    Dallas, J.F.

    1988-01-01

    A human minisatellite DNA probe detects several restriction fragment length polymorphisms in cultivars of Asian and African rice. Certain fragments appear to be inherited in a Mendelian fashion and may represent unlinked loci. The hybridization patterns appear to be cultivar-specific and largely unchanged after the regeneration of plants from tissue culture. The results suggest that these regions of the rice genome may be used to generate cultivar-specific DNA fingerprints. The demonstration of similarity between a human minisatellite sequence and polymorphic regions in the rice genome suggests that such regions also occur in the genomes of many other plant species

  1. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi

    International Nuclear Information System (INIS)

    Andrade, Antero Silva Ribeiro de; Moreno, Elizabeth Castro; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela; Fernandes, Octavio

    2002-01-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with 32 P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with 32 P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  2. Preclinical Study on GRPR-Targeted (68)Ga-Probes for PET Imaging of Prostate Cancer

    DEFF Research Database (Denmark)

    Sun, Yao; Ma, Xiaowei; Zhang, Zhe

    2016-01-01

    Gastrin-releasing peptide receptor (GRPR) targeted positron emission tomography (PET) is a highly promising approach for imaging of prostate cancer (PCa) in small animal models and patients. Developing a GRPR-targeted PET probe with excellent in vivo performance such as high tumor uptake, high...

  3. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    Science.gov (United States)

    Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

  4. Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

    International Nuclear Information System (INIS)

    Ragoussis, J.; Jones, T.A.; Sheer, D.; Shrimpton, A.E.; Goodfellow, P.N.; Trowsdale, J.; Ziegler, A.

    1991-01-01

    A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

    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. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  7. Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11

    DEFF Research Database (Denmark)

    Kim, Jun Young; Sahu, Srikanta; Yau, Yin Hoe

    2016-01-01

    Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly...... facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated...

  8. Optical response of a quantum dot-metal nanoparticle hybrid interacting with a weak probe field.

    Science.gov (United States)

    Kosionis, Spyridon G; Terzis, Andreas F; Sadeghi, Seyed M; Paspalakis, Emmanuel

    2013-01-30

    We study optical effects in a hybrid system composed of a semiconductor quantum dot and a spherical metal nanoparticle that interacts with a weak probe electromagnetic field. We use modified nonlinear density matrix equations for the description of the optical properties of the system and obtain a closed-form expression for the linear susceptibilities of the quantum dot, the metal nanoparticle, and the total system. We then investigate the dependence of the susceptibility on the interparticle distance as well as on the material parameters of the hybrid system. We find that the susceptibility of the quantum dot exhibits optical transparency for specific frequencies. In addition, we show that there is a range of frequencies of the applied field for which the susceptibility of the semiconductor quantum dot leads to gain. This suggests that in such a hybrid system quantum coherence can reverse the course of energy transfer, allowing flow of energy from the metallic nanoparticle to the quantum dot. We also explore the susceptibility of the metal nanoparticle and show that it is strongly influenced by the presence of the quantum dot.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  10. Sperm-Hybrid Micromotor for Targeted Drug Delivery.

    Science.gov (United States)

    Xu, Haifeng; Medina-Sánchez, Mariana; Magdanz, Veronika; Schwarz, Lukas; Hebenstreit, Franziska; Schmidt, Oliver G

    2018-01-23

    A sperm-driven micromotor is presented as a targeted drug delivery system, which is appealing to potentially treat diseases in the female reproductive tract. This system is demonstrated to be an efficient drug delivery vehicle by first loading a motile sperm cell with an anticancer drug (doxorubicin hydrochloride), guiding it magnetically, to an in vitro cultured tumor spheroid, and finally freeing the sperm cell to deliver the drug locally. The sperm release mechanism is designed to liberate the sperm when the biohybrid micromotor hits the tumor walls, allowing it to swim into the tumor and deliver the drug through the sperm-cancer cell membrane fusion. In our experiments, the sperm cells exhibited a high drug encapsulation capability and drug carrying stability, conveniently minimizing  toxic side effects and unwanted drug accumulation in healthy tissues. Overall, sperm cells are excellent candidates to operate in physiological environments, as they neither express pathogenic proteins nor proliferate to form undesirable colonies, unlike other cells or microorganisms. This sperm-hybrid micromotor is a biocompatible platform with potential application in gynecological healthcare, treating or detecting cancer or other diseases in the female reproductive system.

  11. Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints.

    Science.gov (United States)

    Heuer, H; Hartung, K; Wieland, G; Kramer, I; Smalla, K

    1999-03-01

    Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5' exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.

  12. Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

    Science.gov (United States)

    Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

    2017-01-01

    Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

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

    International Nuclear Information System (INIS)

    Maria Lina R; Budiman Bela; Andi Yasmon

    2009-01-01

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

  14. Prediction of the optimum hybridization conditions of dot-blot-SNP analysis using estimated melting temperature of oligonucleotide probes.

    Science.gov (United States)

    Shiokai, Sachiko; Kitashiba, Hiroyasu; Nishio, Takeshi

    2010-08-01

    Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared T (m) values estimated from nucleotide sequences using the DINAMelt web server, measured T (m) values, and hybridization conditions yielding allele-specific signals. The estimated T (m) values were comparable to the measured T (m) values with small differences of less than 3 degrees C for most of the probes. There were differences of approximately 14 degrees C between the specific signal detection conditions and estimated T (m) values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0x SSC corresponded to a difference of approximately 5 degrees C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.

  15. Low temperature excitonic spectroscopy and dynamics as a probe of quality in hybrid perovskite thin films.

    Science.gov (United States)

    Sarang, Som; Ishihara, Hidetaka; Chen, Yen-Chang; Lin, Oliver; Gopinathan, Ajay; Tung, Vincent C; Ghosh, Sayantani

    2016-10-19

    We have developed a framework for using temperature dependent static and dynamic photoluminescence (PL) of hybrid organic-inorganic perovskites (PVSKs) to characterize lattice defects in thin films, based on the presence of nanodomains at low temperature. Our high-stability PVSK films are fabricated using a novel continuous liquid interface propagation technique, and in the tetragonal phase (T > 120 K), they exhibit bi-exponential recombination from free charge carriers with an average PL lifetime of ∼200 ns. Below 120 K, the emergence of the orthorhombic phase is accompanied by a reduction in lifetimes by an order of magnitude, which we establish to be the result of a crossover from free carrier to exciton-dominated radiative recombination. Analysis of the PL as a function of excitation power at different temperatures provides direct evidence that the exciton binding energy is different in the two phases, and using these results, we present a theoretical approach to estimate this variable binding energy. Our findings explain this anomalous low temperature behavior for the first time, attributing it to an inherent fundamental property of the hybrid PVSKs that can be used as an effective probe of thin film quality.

  16. Biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres for controlled drug release.

    Science.gov (United States)

    Du, Pengcheng; Zeng, Jin; Mu, Bin; Liu, Peng

    2013-05-06

    Well-defined biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres have been accomplished via the layer-by-layer (LbL) self-assembly technique. The hybrid shell was fabricated by the electrostatic interaction between the polyelectrolyte cation, chitosan (CS), and the hybrid anion, citrate modified ferroferric oxide nanoparticles (Fe3O4-CA), onto the uniform polystyrene sulfonate microsphere templates. Then the magnetic hybrid core/shell composite particles were modified with a linear, functional poly(ethylene glycol) (PEG) monoterminated with a biotargeting molecule (folic acid (FA)). Afterward the dual targeting hybrid hollow microspheres were obtained after etching the templates by dialysis. The dual targeting hybrid hollow microspheres exhibit exciting pH response and stability in high salt-concentration media. Their pH-dependent controlled release of the drug molecule (anticancer drug, doxorubicin (DOX)) was also investigated in different human body fluids. As expected, the cell viability of the HepG2 cells which decreased more rapidly was treated by the FA modified hybrid hollow microspheres rather than the unmodified one in the in vitro study. The dual-targeting hybrid hollow microspheres demonstrate selective killing of the tumor cells. The precise magnetic and molecular targeting properties and pH-dependent controlled release offers promise for cancer treatment.

  17. Multifunctional gadolinium-based dendritic macromolecules as liver targeting imaging probes.

    Science.gov (United States)

    Luo, Kui; Liu, Gang; He, Bin; Wu, Yao; Gong, Qingyong; Song, Bin; Ai, Hua; Gu, Zhongwei

    2011-04-01

    The quest for highly efficient and safe contrast agents has become the key factor for successful application of magnetic resonance imaging (MRI). The gadolinium (Gd) based dendritic macromolecules, with precise and tunable nanoscopic sizes, are excellent candidates as multivalent MRI probes. In this paper, a novel series of Gd-based multifunctional peptide dendritic probes (generation 2, 3, and 4) possessing highly controlled structures and single molecular weight were designed and prepared as liver MRI probes. These macromolecular Gd-ligand agents exhibited up to 3-fold increase in T(1) relaxivity comparing to Gd-DTPA complexes. No obvious in vitro cytotoxicity was observed from the measured concentrations. These dendritic probes were further functionalized with multiple galactosyl moieties and led to much higher cell uptake in vitro as demonstrated in T(1)-weighted scans. During in vivo animal studies, the probes provided better signal intensity (SI) enhancement in mouse liver, especially at 60 min post-injection, with the most efficient enhancement from the galactosyl moiety decorated third generation dendrimer. The imaging results were verified with analysis of Gd content in liver tissues. The design strategy of multifunctional Gd-ligand peptide dendritic macromolecules in this study may be used for developing other sensitive MRI probes with targeting capability. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno

    2015-01-01

    acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization...... step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion......In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo...

  19. The performance of a fiber optic displacement sensor for different types of probes and targets

    International Nuclear Information System (INIS)

    Yasin, M; Harun, S W; Abdul-Rashid, H A; Kusminarto; Karyono; Ahmad, H

    2008-01-01

    A simple fiber optic displacement sensor is presented using a multimode plastic bundled fiber and the intensity modulation technique. The performance of the sensor is compared for different types of probes and targets. The probe with the largest receiving core diameter demonstrates the highest linearity range, and increasing the number of receiving cores increases the sensitivity of the sensor. With a stainless steel target and the concentric bundled fiber with 16 receiving fibers as a probe, the sensitivity of the sensor is found to be 0.0220 mV/μm over 150 to 550 μm range and – 0.0061 mV/μm over 1100 to 2000 μm range. The target with a higher reflectivity shows a higher sensitivity. The linearity range for the front slope is almost similar for all targets tested. However, for the back slope, lower reflectivity objects have a relatively higher linearity range with the highest range of 1600 μm being obtained using plastic and aluminum targets. The simplicity of the design, high degree of sensitivity, dynamic range, non-contact measurement and low cost of the fabrication make it suitable for applications in industries for position control and micro displacement measurement in the hazardous regions

  20. UPAR targeted molecular imaging of cancers with small molecule-based probes.

    Science.gov (United States)

    Ding, Feng; Chen, Seng; Zhang, Wanshu; Tu, Yufeng; Sun, Yao

    2017-10-15

    Molecular imaging can allow the non-invasive characterization and measurement of biological and biochemical processes at the molecular and cellular levels in living subjects. The imaging of specific molecular targets that are associated with cancers could allow for the earlier diagnosis and better treatment of diseases. Small molecule-based probes play prominent roles in biomedical research and have high clinical translation ability. Here, with an emphasis on small molecule-based probes, we review some recent developments in biomarkers, imaging techniques and multimodal imaging in molecular imaging and highlight the successful applications for molecular imaging of cancers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Gene probes: principles and protocols

    National Research Council Canada - National Science Library

    Aquino de Muro, Marilena; Rapley, Ralph

    2002-01-01

    ... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

  2. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    Science.gov (United States)

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe

    Directory of Open Access Journals (Sweden)

    Ying Chen

    2017-08-01

    Full Text Available Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress. Keywords: Lipid-derived electrophile, 4-hydroxy-2-nonenal, Chemoproteomics, Aminooxy probe, Activity-based protein profiling

  4. Improved detection of genetic markers of antimicrobial resistance by hybridization probe-based melting curve analysis using primers to mask proximal mutations: examples include the influenza H275Y substitution.

    Science.gov (United States)

    Whiley, David M; Jacob, Kevin; Nakos, Jennifer; Bletchly, Cheryl; Nimmo, Graeme R; Nissen, Michael D; Sloots, Theo P

    2012-06-01

    Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, the performance of these methods can be undermined by sequence variation in the regions flanking the codon of interest. This is a problem encountered more broadly in microbial diagnostics. In this study, we developed a modification of hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations in the sequence targets of hybridization probes, so as to limit the potential for sequence variation to interfere with typing. The approach was applied to the H275Y alteration of the influenza A (H1N1) 2009 strain, as well as a Neisseria gonorrhoeae mutation associated with antimicrobial resistance. Assay performances were assessed using influenza A and N. gonorrhoeae strains characterized by DNA sequencing. The modified hybridization probe-based approach proved successful in limiting the effects of proximal mutations, with the results of melting curve analyses being 100% consistent with the results of DNA sequencing for all influenza A and N. gonorrhoeae strains tested. Notably, these included influenza A and N. gonorrhoeae strains exhibiting additional mutations in hybridization probe targets. Of particular interest was that the H275Y assay correctly typed influenza A strains harbouring a T822C nucleotide substitution, previously shown to interfere with H275Y typing methods. Overall our modified hybridization probe-based approach provides a simple means of circumventing problems caused by sequence variation, and offers improved detection of the influenza A H275Y alteration and potentially other resistance mechanisms.

  5. Expression of proto-oncogenes in non-Hodgkin's lymphomas by in situ hybridization with biotinylated DNA probes

    International Nuclear Information System (INIS)

    Hamatani, Kiyohiro; Yoshida, Kuniko; Abe, Masumi; Shimaoka, Katsutaro; Shiku, Hiroshi; Akiyama, Mitoshi; Kondo, Hisayoshi.

    1989-11-01

    Expression of six proto-oncogenes (fos, myc, myb, Ki-ras, Ha-ras, and N-ras) in 43 cases of non-Hodgkin's lymphoma was analyzed by means of in situ hybridization. Biotinylated DNA probes of the six oncogenes and those of the immunoglobulin H-chain (IgH) gene and the T cell receptor β-chain (TCRβ) gene were used. The results of in situ hybridization performed under blind conditions by IgH and TCRβ gene probes were compatible with those of typing by cell surface markers. The nuclear protein-related proto-oncogenes, fos myc, and myb, were expressed in about 70 % - 80 % of all cases regardless of phenotypes, histology or histologic grade. On the contrary, genes of the ras family were expressed in fewer cases except for the Ki-ras gene which was more frequently expressed by cases of the T cell immunophenotype with a high malignancy grade. The results of dot hybridization with RNA extracted from some cases were compatible with those of in situ hybridization, further demonstrating the specificity of in situ hybridization. (author)

  6. Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).

    Science.gov (United States)

    Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

    2006-09-01

    Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

  7. Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

    Science.gov (United States)

    Zeppa, Pio; Sosa Fernandez, Laura Virginia; Cozzolino, Immacolata; Ronga, Valentina; Genesio, Rita; Salatiello, Maria; Picardi, Marco; Malapelle, Umberto; Troncone, Giancarlo; Vigliar, Elena

    2012-12-25

    The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples. Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR. The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society. Copyright © 2012 American Cancer Society.

  8. Targeting of porous hybrid silica nanoparticles to cancer cells

    NARCIS (Netherlands)

    Rosenholm, J.M.; Meinander, A.; Peuhu, E.; Niemi, R.; Eriksson, J.E.; Sahlgren, C.; Lindén, M.

    2009-01-01

    Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of polyethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in

  9. Linking probe thermodynamics to microarray quantification

    International Nuclear Information System (INIS)

    Li, Shuzhao; Pozhitkov, Alexander; Brouwer, Marius

    2010-01-01

    Understanding the difference in probe properties holds the key to absolute quantification of DNA microarrays. So far, Langmuir-like models have failed to link sequence-specific properties to hybridization signals in the presence of a complex hybridization background. Data from washing experiments indicate that the post-hybridization washing has no major effect on the specifically bound targets, which give the final signals. Thus, the amount of specific targets bound to probes is likely determined before washing, by the competition against nonspecific binding. Our competitive hybridization model is a viable alternative to Langmuir-like models. (comment)

  10. Hyaluronan functionalizing QDs as turn-on fluorescent probe for targeted recognition CD44 receptor

    Science.gov (United States)

    Zhou, Shang; Huo, Danqun; Hou, Changjun; Yang, Mei; Fa, Huanbao

    2017-09-01

    The recognition of tumor markers in living cancer cells has attracted increasing interest. In the present study, the turn-on fluorescence probe was designed based on the fluorescence of thiolated chitosan-coated CdTe QDs (CdTe/TCS QDs) quenched by hyaluronan, which could provide the low background signal for sensitive cellular imaging. This system is expected to offer specific recognition of CD44 receptor over other substances owing to the specific affinity of hyaluronan and CD44 receptor ( 8-9 kcal/mol). The probe is stable in aqueous and has little toxicity to living cells; thus, it can be utilized for targeted cancer cell imaging. The living lung cancer cell imaging experiments further demonstrate its value in recognizing cell-surface CD44 receptor with turn-on mode. In addition, the probe can be used to recognize and differentiate the subtypes of lung cancer cells based on the difference of CD44 expression on the surface of lung cancer cells. And, the western blot test further confirmed that the expression level of the CD44 receptor in lung cancer cells is different. Therefore, this probe may be potentially applied in recognizing lung cancer cells with higher contrast and sensitivity and provide new tools for cancer prognosis and therapy. [Figure not available: see fulltext.

  11. Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe.

    Science.gov (United States)

    Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie

    2017-10-02

    Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

    Science.gov (United States)

    Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina; Figueiredo, Céu; Wengel, Jesper; Filipe Azevedo, Nuno

    2013-01-01

    The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2’-O-methyl RNAs (2’OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others. PMID:24278398

  13. Transforming a Targeted Porphyrin Theranostic Agent into a PET Imaging Probe for Cancer

    Directory of Open Access Journals (Sweden)

    Jiyun Shi, Tracy W.B. Liu, Juan Chen, David Green, David Jaffray, Brian C. Wilson, Fan Wang, Gang Zheng

    2011-01-01

    Full Text Available Porphyrin based photosensitizers are useful agents for photodynamic therapy (PDT and fluorescence imaging of cancer. Porphyrins are also excellent metal chelators forming highly stable metallo-complexes making them efficient delivery vehicles for radioisotopes. Here we investigated the possibility of incorporating 64Cu into a porphyrin-peptide-folate (PPF probe developed previously as folate receptor (FR targeted fluorescent/PDT agent, and evaluated the potential of turning the resulting 64Cu-PPF into a positron emission tomography (PET probe for cancer imaging. Noninvasive PET imaging followed by radioassay evaluated the tumor accumulation, pharmacokinetics and biodistribution of 64Cu-PPF. 64Cu-PPF uptake in FR-positive tumors was visible on small-animal PET images with high tumor-to-muscle ratio (8.88 ± 3.60 observed after 24 h. Competitive blocking studies confirmed the FR-mediated tracer uptake by the tumor. The ease of efficient 64Cu-radiolabeling of PPF while retaining its favorable biodistribution, pharmacokinetics and selective tumor uptake, provides a robust strategy to transform tumor-targeted porphyrin-based photosensitizers into PET imaging probes.

  14. Exogenous Molecular Probes for Targeted Imaging in Cancer: Focus on Multi-modal Imaging

    International Nuclear Information System (INIS)

    Joshi, Bishnu P.; Wang, Thomas D.

    2010-01-01

    Cancer is one of the major causes of mortality and morbidity in our healthcare system. Molecular imaging is an emerging methodology for the early detection of cancer, guidance of therapy, and monitoring of response. The development of new instruments and exogenous molecular probes that can be labeled for multi-modality imaging is critical to this process. Today, molecular imaging is at a crossroad, and new targeted imaging agents are expected to broadly expand our ability to detect and manage cancer. This integrated imaging strategy will permit clinicians to not only localize lesions within the body but also to manage their therapy by visualizing the expression and activity of specific molecules. This information is expected to have a major impact on drug development and understanding of basic cancer biology. At this time, a number of molecular probes have been developed by conjugating various labels to affinity ligands for targeting in different imaging modalities. This review will describe the current status of exogenous molecular probes for optical, scintigraphic, MRI and ultrasound imaging platforms. Furthermore, we will also shed light on how these techniques can be used synergistically in multi-modal platforms and how these techniques are being employed in current research

  15. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia; Melo, Maria N., E-mail: melo@icb.ufmg.br [Departamento de Parasitologia. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil)

    2011-07-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with {sup 32}P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  16. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    International Nuclear Information System (INIS)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R.

    2011-01-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with 32 P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  17. Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

    NARCIS (Netherlands)

    Klein, M.; Eslami Mosallam, B.; Gonzalez Arroyo, Dylan; Depken, S.M.

    2018-01-01

    Due to their specificity, efficiency, and ease of programming, CRISPR-associated nucleases are popular tools for genome editing. On the genomic scale, these nucleases still show considerable off-target activity though, posing a serious obstacle to the development of therapies. Off targeting is often

  18. Investigations of ultrafast charge dynamics in laser-irradiated targets by a self probing technique employing laser driven protons

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, H. [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom); Kar, S., E-mail: s.kar@qub.ac.uk [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom); Cantono, G. [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom); Department of Physics “E. Fermi”, Largo B. Pontecorvo 3, 56127 Pisa (Italy); Consiglio Nazionale delle Ricerche, Istituto Nazionale di Ottica, Research Unit Adriano Gozzini, via G. Moruzzi 1, Pisa 56124 (Italy); Nersisyan, G. [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom); Brauckmann, S. [Institut für Laser-und Plasmaphysik, Heinrich-Heine-Universität, Düsseldorf (Germany); Doria, D.; Gwynne, D. [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom); Macchi, A. [Department of Physics “E. Fermi”, Largo B. Pontecorvo 3, 56127 Pisa (Italy); Consiglio Nazionale delle Ricerche, Istituto Nazionale di Ottica, Research Unit Adriano Gozzini, via G. Moruzzi 1, Pisa 56124 (Italy); Naughton, K. [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom); Willi, O. [Institut für Laser-und Plasmaphysik, Heinrich-Heine-Universität, Düsseldorf (Germany); Lewis, C.L.S.; Borghesi, M. [School of Mathematics and Physics, Queen' s University Belfast, BT7 1NN (United Kingdom)

    2016-09-01

    The divergent and broadband proton beams produced by the target normal sheath acceleration mechanism provide the unique opportunity to probe, in a point-projection imaging scheme, the dynamics of the transient electric and magnetic fields produced during laser-plasma interactions. Commonly such experimental setup entails two intense laser beams, where the interaction produced by one beam is probed with the protons produced by the second. We present here experimental studies of the ultra-fast charge dynamics along a wire connected to laser irradiated target carried out by employing a ‘self’ proton probing arrangement – i.e. by connecting the wire to the target generating the probe protons. The experimental data shows that an electromagnetic pulse carrying a significant amount of charge is launched along the wire, which travels as a unified pulse of 10s of ps duration with a velocity close to speed of light. The experimental capabilities and the analysis procedure of this specific type of proton probing technique are discussed. - Highlights: • Prompt charging of laser irradiated target generates ultra-short EM pulses. • Its ultrafast propagation along a wire was studied by self-proton probing technique. • Self-proton probing technique is the proton probing with one laser pulse. • Pulse temporal profile and speed along the wire were measured with high resolution.

  19. Human biodistribution and radiation dosimetry of novel PET probes targeting the deoxyribonucleoside salvage pathway

    Energy Technology Data Exchange (ETDEWEB)

    Schwarzenberg, Johannes [David Geffen School of Medicine, University of California, Department of Molecular and Medical Pharmacology, Ahmanson Biological Imaging Division, Los Angeles, CA (United States); Medical University of Vienna, Department of Pediatrics, Vienna (Austria); Radu, Caius G.; Tran, Andrew Q.; Phelps, Michael E.; Satyamurthy, Nagichettiar [David Geffen School of Medicine, University of California, Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, Los Angeles, CA (United States); Benz, Matthias; Fueger, Barbara; Czernin, Johannes; Schiepers, Christiaan [David Geffen School of Medicine, University of California, Department of Molecular and Medical Pharmacology, Ahmanson Biological Imaging Division, Los Angeles, CA (United States); Witte, Owen N. [David Geffen School of Medicine, University of California, Howard Hughes Medical Institute and Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, CA (United States)

    2011-04-15

    Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway involved in the production and maintenance of a balanced pool of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis. dCK phosphorylates and therefore activates nucleoside analogs such as cytarabine, gemcitabine, decitabine, cladribine, and clofarabine that are used routinely in cancer therapy. Imaging probes that target dCK might allow stratifying patients into likely responders and nonresponders with dCK-dependent prodrugs. Here we present the biodistribution and radiation dosimetry of three fluorinated dCK substrates, {sup 18}F-FAC, L-{sup 18}F-FAC, and L-{sup 18}F-FMAC, developed for positron emission tomography (PET) imaging of dCK activity in vivo. PET studies were performed in nine healthy human volunteers, three for each probe. After a transmission scan, the radiopharmaceutical was injected intravenously and three sequential emission scans acquired from the base of the skull to mid-thigh. Regions of interest encompassing visible organs were drawn on the first PET scan and copied to the subsequent scans. Activity in target organs was determined and absorbed dose estimated with OLINDA/EXM. The standardized uptake value was calculated for various organs at different times. Renal excretion was common to all three probes. Bone marrow had higher uptake for L-{sup 18}F-FAC and L-{sup 18}F-FMAC than {sup 18}F-FAC. Prominent liver uptake was seen in L-{sup 18}F-FMAC and L-{sup 18}F-FAC, whereas splenic activity was highest for {sup 18}F-FAC. Muscle uptake was also highest for {sup 18}F-FAC. The critical organ was the bladder wall for all three probes. The effective dose was 0.00524, 0.00755, and 0.00910 mSv/MBq for {sup 18}F-FAC, L-{sup 18}F-FAC, and L-{sup 18}F-FMAC, respectively. The biodistribution of {sup 18}F-FAC, L-{sup 18}F-FAC, and L-{sup 18}F-FMAC in humans reveals similarities and differences. Differences may be explained by different probe

  20. A Novel Loss Recovery and Tracking Scheme for Maneuvering Target in Hybrid WSNs.

    Science.gov (United States)

    Qian, Hanwang; Fu, Pengcheng; Li, Baoqing; Liu, Jianpo; Yuan, Xiaobing

    2018-01-25

    Tracking a mobile target, which aims to timely monitor the invasion of specific target, is one of the most prominent applications in wireless sensor networks (WSNs). Traditional tracking methods in WSNs only based on static sensor nodes (SNs) have several critical problems. For example, to void the loss of mobile target, many SNs must be active to track the target in all possible directions, resulting in excessive energy consumption. Additionally, when entering coverage holes in the monitoring area, the mobile target may be missing and then its state is unknown during this period. To tackle these problems, in this paper, a few mobile sensor nodes (MNs) are introduced to cooperate with SNs to form a hybrid WSN due to their stronger abilities and less constrained energy. Then, we propose a valid target tracking scheme for hybrid WSNs to dynamically schedule the MNs and SNs. Moreover, a novel loss recovery mechanism is proposed to find the lost target and recover the tracking with fewer SNs awakened. Furthermore, to improve the robustness and accuracy of the recovery mechanism, an adaptive unscented Kalman filter (AUKF) algorithm is raised to dynamically adjust the process noise covariance. Simulation results demonstrate that our tracking scheme for maneuvering target in hybrid WSNs can not only track the target effectively even if the target is lost but also maintain an excellent accuracy and robustness with fewer activated nodes.

  1. Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris.

    Science.gov (United States)

    Dedysh, S N; Derakshani, M; Liesack, W

    2001-10-01

    Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.

  2. Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria.

    Science.gov (United States)

    Gulledge, J; Ahmad, A; Steudler, P A; Pomerantz, W J; Cavanaugh, C M

    2001-10-01

    Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

  3. Progresses in optimization strategy for radiolabeled molecular probes targeting integrin αvβ3

    International Nuclear Information System (INIS)

    Chen Haojun; Wu Hua

    2012-01-01

    Tumor angiogenesis is critical in the growth, invasion and metastasis of malignant tumors. The integrins, which express on many types of tumor cells and activated vascular endothelial cells, play an important role in regulation of the tumor angiogenesis. RGD peptide, which contains Arg-Gly-Asp sequence, binds specifically to integrin α v β 3 . Therefore, the radiolabeled RGD peptides may have broad application prospects in radionuclide imaging and therapy. Major research interests include the selection of radionuclides, modification and improvement of RGD structures. In this article, we give a review on research progresses in optimization strategy for radiolabeled molecular probes targeting integrin α v β 3 . (authors)

  4. Design of mitochondria-targeted colorimetric and ratiometric fluorescent probes for rapid detection of SO2 derivatives in living cells

    Science.gov (United States)

    Yang, Yutao; Zhou, Tingting; Bai, Bozan; Yin, Caixia; Xu, Wenzhi; Li, Wei

    2018-05-01

    Two mitochondria-targeted colorimetric and ratiometric fluorescent probes for SO2 derivatives were constructed based on the SO2 derivatives-triggered Michael addition reaction. The probes exhibit high specificity toward HSO3-/SO32- by interrupting their conjugation system resulting in a large ratiometric blue shift of 46-121 nm in their emission spectrum. The two well-resolved emission bands can ensure accurate detection of HSO3-. The detection limits were calculated to be 1.09 and 1.35 μM. Importantly, probe 1 and probe 2 were successfully used to fluorescence ratiometric imaging of endogenous HSO3- in BT-474 cells.

  5. Characterisation of target plasma required for REB-plasma interaction studies using cylindrical Langmuir probes

    International Nuclear Information System (INIS)

    Roychowdhury, P.; Paithankar, A.S.; Iyyengar, S.K.; Rohatgi, V.K.

    1987-01-01

    The target plasma required for relativistic electron beam (REB)-plasma interaction studies has been generated by coaxial plasma gun. The measurement of electron density and temperature has been carried out using cylindrical Langmuir probes. Probes both oriented parallel and transverse to the flow have been used. The spatial as well as temporal variation of electron density and temperature have been studied. The typical electron density and temperature measured by probe were in the range of 9.0-3.5 x 10 13 cm -3 and 5-7 eV respectively. The typical e-folding decay time of density was 6.2 μs, while no appreciable change in electron temperature was observed until 10 μs after the peak density. The density decays by about 50% at distance of 30 cm from the gun. The plasma flow velocity has been measured by the time of flight technique and was found to be 2.5 x 10 6 cm s -1 . The plasma radius measured by dosimeter film, at distance of 30 cm from the gun was 3 cm. (author)

  6. An off-on fluorescence probe targeting mitochondria based on oxidation-reduction response for tumor cell and tissue imaging

    Science.gov (United States)

    Yao, Hanchun; Cao, Li; Zhao, Weiwei; Zhang, Suge; Zeng, Man; Du, Bin

    2017-10-01

    In this study, a tumor-targeting poly( d, l-lactic-co-glycolic acid) (PLGA) loaded "off-on" fluorescent probe nanoparticle (PFN) delivery system was developed to evaluate the region of tumor by off-on fluorescence. The biodegradability of the nanosize PFN delivery system readily released the probe under tumor acidic conditions. The probe with good biocompatibility was used to monitor the intracellular glutathione (GSH) of cancer cells and selectively localize to mitochondria for tumor imaging. The incorporated tumor-targeting probe was based on the molecular photoinduced electron transfer (PET) mechanism preventing fluorescence ("off" state) and could be easily released under tumor acidic conditions. However, the released tumor-targeting fluorescence probe molecule was selective towards GSH with high selectivity and an ultra-sensitivity for the mitochondria of cancer cells and tissues significantly increasing the probe molecule fluorescence signal ("on" state). The tumor-targeting fluorescence probe showed sensitivity to GSH avoiding interference from cysteine and homocysteine. The PFNs could enable fluorescence-guided cancer imaging during cancer therapy. This work may expand the biological applications of PFNs as a diagnostic reagent, which will be beneficial for fundamental research in tumor imaging. [Figure not available: see fulltext.

  7. Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

    Directory of Open Access Journals (Sweden)

    Takeshi Kubota

    Full Text Available 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.

  8. Development of tumor-targeted near infrared probes for fluorescence guided surgery.

    Science.gov (United States)

    Kelderhouse, Lindsay E; Chelvam, Venkatesh; Wayua, Charity; Mahalingam, Sakkarapalayam; Poh, Scott; Kularatne, Sumith A; Low, Philip S

    2013-06-19

    Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

  9. Measurement of the target current by inductive probe during laser interaction on terawatt laser system PALS

    Czech Academy of Sciences Publication Activity Database

    Cikhardt, Jakub; Krása, Josef; De Marco, Massimo; Pfeifer, Miroslav; Velyhan, Andriy; Krouský, Eduard; Cikhardtová, B.; Klír, Daniel; Řezáč, Karel; Ullschmied, Jiří; Skála, Jiří; Kubeš, P.; Kravárik, J.

    2014-01-01

    Roč. 85, č. 10 (2014), s. 103507-103507 ISSN 0034-6748 R&D Projects: GA MŠk LM2010014; GA MŠk(CZ) LG13029; GA ČR GAP205/12/0454; GA MŠk EE2.3.20.0279 Grant - others:LaserZdroj (OP VK 3)(XE) CZ.1.07/2.3.00/20.0279 Institutional support: RVO:61389021 ; RVO:68378271 Keywords : laser PALS * laser-target interaction * target current * inductive probe Subject RIV: BL - Plasma and Gas Discharge Physics; BL - Plasma and Gas Discharge Physics (FZU-D) Impact factor: 1.614, year: 2014 http://dx.doi.org/10.1063/1.4898016

  10. Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

    Directory of Open Access Journals (Sweden)

    Elisa Greotti

    2016-09-01

    Full Text Available Calcium ion (Ca2+ is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+] within its lumen ([Ca2+]ER can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2. The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.

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

    Directory of Open Access Journals (Sweden)

    Sílvia Fontenete

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

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

    Science.gov (United States)

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

    2015-01-01

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

  13. Molecular Imaging of Hepatocellular Carcinoma Xenografts with Epidermal Growth Factor Receptor Targeted Affibody Probes

    Directory of Open Access Journals (Sweden)

    Ping Zhao

    2013-01-01

    Full Text Available Hepatocellular carcinoma (HCC is a highly aggressive and lethal cancer. It is typically asymptomatic at the early stage, with only 10%–20% of HCC patients being diagnosed early enough for appropriate surgical treatment. The delayed diagnosis of HCC is associated with limited treatment options and much lower survival rates. Therefore, the early and accurate detection of HCC is crucial to improve its currently dismal prognosis. The epidermal growth factor receptor (EGFR has been reported to be involved in HCC tumorigenesis and to represent an attractive target for HCC imaging and therapy. In this study, an affibody molecule, Ac-Cys-ZEGFR:1907, targeting the extracellular domain of EGFR, was used for the first time to assess its potential to detect HCC xenografts. By evaluating radio- or fluorescent-labeled Ac-Cys-ZEGFR:1907 as a probe for positron emission tomography (PET or optical imaging of HCC, subcutaneous EGFR-positive HCC xenografts were found to be successfully imaged by the PET probe. Thus, affibody-based PET imaging of EGFR provides a promising approach for detecting HCC in vivo.

  14. Hybrid value foraging: How the value of targets shapes human foraging behavior.

    Science.gov (United States)

    Wolfe, Jeremy M; Cain, Matthew S; Alaoui-Soce, Abla

    2018-04-01

    In hybrid foraging, observers search visual displays for multiple instances of multiple target types. In previous hybrid foraging experiments, although there were multiple types of target, all instances of all targets had the same value. Under such conditions, behavior was well described by the marginal value theorem (MVT). Foragers left the current "patch" for the next patch when the instantaneous rate of collection dropped below their average rate of collection. An observer's specific target selections were shaped by previous target selections. Observers were biased toward picking another instance of the same target. In the present work, observers forage for instances of four target types whose value and prevalence can vary. If value is kept constant and prevalence manipulated, participants consistently show a preference for the most common targets. Patch-leaving behavior follows MVT. When value is manipulated, observers favor more valuable targets, though individual foraging strategies become more diverse, with some observers favoring the most valuable target types very strongly, sometimes moving to the next patch without collecting any of the less valuable targets.

  15. Targeting Interleukin-4 Receptor Alpha by Hybrid Peptide for Novel Biliary Tract Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Kahori Seto

    2014-01-01

    Full Text Available It is known that the interleukin-4 receptor α (IL-4Rα is highly expressed on the surface of various human solid tumors. We previously designed novel IL-4Rα-lytic hybrid peptide composed of binding peptide to IL-4Rα and cell-lytic peptide and reported that the designed IL-4Rα-lytic hybrid peptide exhibited cytotoxic and antitumor activity both in vitro and in vivo against the human pancreatic cancer cells expressing IL-4Rα. Here, we evaluated the antitumor activity of the IL-4Rα-lytic hybrid peptide as a novel molecular targeted therapy for human biliary tract cancer (BTC. The IL-4Rα-lytic hybrid peptide showed cytotoxic activity in six BTC cell lines with a concentration that killed 50% of all cells (IC50 as low as 5 μM. We also showed that IL-4Rα-lytic hybrid peptide in combination with gemcitabine exhibited synergistic cytotoxic activity in vitro. In addition, intravenous administration of IL-4Rα-lytic hybrid peptide significantly inhibited tumor growth in a xenograft model of human BTC in vivo. Taken together, these results indicated that the IL-4Rα-lytic hybrid peptide is a potent agent that might provide a novel therapy for patients with BTC.

  16. An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples.

    Science.gov (United States)

    Barr Fritcher, Emily G; Voss, Jesse S; Brankley, Shannon M; Campion, Michael B; Jenkins, Sarah M; Keeney, Matthew E; Henry, Michael R; Kerr, Sarah M; Chaiteerakij, Roongruedee; Pestova, Ekaterina V; Clayton, Amy C; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C; Kipp, Benjamin R

    2015-12-01

    Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of

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

    DEFF Research Database (Denmark)

    Hansen, K.W.; Ahring, Birgitte Kiær; Raskin, L.

    1999-01-01

    Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYE, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S, wolfei LYE was closely related...... 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...

  18. Identification of pathogenic Nocardia species by reverse line blot hybridization targeting the 16S rRNA and 16S-23S rRNA gene spacer regions.

    Science.gov (United States)

    Xiao, Meng; Kong, Fanrong; Sorrell, Tania C; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Sharon C A

    2010-02-01

    Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

  19. Targets and probes for non-invasive imaging of β-cells

    Energy Technology Data Exchange (ETDEWEB)

    Jodal, Andreas; Behe, Martin [Paul Scherrer Institut, Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Villigen (Switzerland); Schibli, Roger [Paul Scherrer Institut, Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Villigen (Switzerland); ETH Zurich, Department of Chemistry and Applied Biosciences, Zurich (Switzerland)

    2017-04-15

    β-cells, located in the islets of the pancreas, are responsible for production and secretion of insulin and play a crucial role in blood sugar regulation. Pathologic β-cells often cause serious medical conditions affecting blood glucose level, which severely impact life quality and are life-threatening if untreated. With 347 million patients, diabetes is one of the most prevalent diseases, and will continue to be one of the largest socioeconomic challenges in the future. The diagnosis still relies mainly on indirect methods like blood sugar measurements. A non-invasive diagnostic imaging modality would allow direct evaluation of β-cell mass and would be a huge step towards personalized medicine. Hyperinsulinism is another serious condition caused by β-cells that excessively secrete insulin, like for instance β-cell hyperplasia and insulinomas. Treatment options with drugs are normally not curative, whereas curative procedures usually consist of the resection of affected regions for which, however, an exact localization of the foci is necessary. In this review, we describe potential tracers under development for targeting β-cells with focus on radiotracers for PET and SPECT imaging, which allow the non-invasive visualization of β-cells. We discuss either the advantages or limitations for the various tracers and modalities. This article concludes with an outlook on future developments and discuss the potential of new imaging probes including dual probes that utilize functionalities for both a radioactive and optical moiety as well as for theranostic applications. (orig.)

  20. Targets and probes for non-invasive imaging of β-cells

    International Nuclear Information System (INIS)

    Jodal, Andreas; Behe, Martin; Schibli, Roger

    2017-01-01

    β-cells, located in the islets of the pancreas, are responsible for production and secretion of insulin and play a crucial role in blood sugar regulation. Pathologic β-cells often cause serious medical conditions affecting blood glucose level, which severely impact life quality and are life-threatening if untreated. With 347 million patients, diabetes is one of the most prevalent diseases, and will continue to be one of the largest socioeconomic challenges in the future. The diagnosis still relies mainly on indirect methods like blood sugar measurements. A non-invasive diagnostic imaging modality would allow direct evaluation of β-cell mass and would be a huge step towards personalized medicine. Hyperinsulinism is another serious condition caused by β-cells that excessively secrete insulin, like for instance β-cell hyperplasia and insulinomas. Treatment options with drugs are normally not curative, whereas curative procedures usually consist of the resection of affected regions for which, however, an exact localization of the foci is necessary. In this review, we describe potential tracers under development for targeting β-cells with focus on radiotracers for PET and SPECT imaging, which allow the non-invasive visualization of β-cells. We discuss either the advantages or limitations for the various tracers and modalities. This article concludes with an outlook on future developments and discuss the potential of new imaging probes including dual probes that utilize functionalities for both a radioactive and optical moiety as well as for theranostic applications. (orig.)

  1. Radiolabeled Probes Targeting Hypoxia-Inducible Factor-1-Active Tumor Microenvironments

    Directory of Open Access Journals (Sweden)

    Masashi Ueda

    2014-01-01

    Full Text Available Because tumor cells grow rapidly and randomly, hypoxic regions arise from the lack of oxygen supply in solid tumors. Hypoxic regions in tumors are known to be resistant to chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1 expressed in hypoxic regions regulates the expression of genes related to tumor growth, angiogenesis, metastasis, and therapy resistance. Thus, imaging of HIF-1-active regions in tumors is of great interest. HIF-1 activity is regulated by the expression and degradation of its α subunit (HIF-1α, which is degraded in the proteasome under normoxic conditions, but escapes degradation under hypoxic conditions, allowing it to activate transcription of HIF-1-target genes. Therefore, to image HIF-1-active regions, HIF-1-dependent reporter systems and injectable probes that are degraded in a manner similar to HIF-1α have been recently developed and used in preclinical studies. However, no probe currently used in clinical practice directly assesses HIF-1 activity. Whether the accumulation of 18F-FDG or 18F-FMISO can be utilized as an index of HIF-1 activity has been investigated in clinical studies. In this review, the current status of HIF-1 imaging in preclinical and clinical studies is discussed.

  2. Sampling Enrichment toward Target Structures Using Hybrid Molecular Dynamics-Monte Carlo Simulations.

    Directory of Open Access Journals (Sweden)

    Kecheng Yang

    Full Text Available Sampling enrichment toward a target state, an analogue of the improvement of sampling efficiency (SE, is critical in both the refinement of protein structures and the generation of near-native structure ensembles for the exploration of structure-function relationships. We developed a hybrid molecular dynamics (MD-Monte Carlo (MC approach to enrich the sampling toward the target structures. In this approach, the higher SE is achieved by perturbing the conventional MD simulations with a MC structure-acceptance judgment, which is based on the coincidence degree of small angle x-ray scattering (SAXS intensity profiles between the simulation structures and the target structure. We found that the hybrid simulations could significantly improve SE by making the top-ranked models much closer to the target structures both in the secondary and tertiary structures. Specifically, for the 20 mono-residue peptides, when the initial structures had the root-mean-squared deviation (RMSD from the target structure smaller than 7 Å, the hybrid MD-MC simulations afforded, on average, 0.83 Å and 1.73 Å in RMSD closer to the target than the parallel MD simulations at 310K and 370K, respectively. Meanwhile, the average SE values are also increased by 13.2% and 15.7%. The enrichment of sampling becomes more significant when the target states are gradually detectable in the MD-MC simulations in comparison with the parallel MD simulations, and provide >200% improvement in SE. We also performed a test of the hybrid MD-MC approach in the real protein system, the results showed that the SE for 3 out of 5 real proteins are improved. Overall, this work presents an efficient way of utilizing solution SAXS to improve protein structure prediction and refinement, as well as the generation of near native structures for function annotation.

  3. Mn-doped near-infrared quantum dots as multimodal targeted probes for pancreatic cancer imaging

    Science.gov (United States)

    Yong, Ken-Tye

    2009-01-01

    This work presents a novel approach to producing manganese (Mn)-doped quantum dots (Mnd-QDs) emitting in the near-infrared (NIR). Surface functionalization of Mnd-QDs with lysine makes them stably disperse in aqueous media and able to conjugate with targeting molecules. The nanoparticles were structurally and compositionally characterized and maintained a high photoluminescence quantum yield and displayed paramagnetism in water. The receptor-mediated delivery of bioconjugated Mnd-QDs into pancreatic cancer cells was demonstrated using the confocal microscopy technique. Cytotoxicity of Mnd-QDs on live cells has been evaluated. The NIR-emitting characteristic of the QDs has been exploited to acquire whole animal body imaging with high contrast signals. In addition, histological and blood analysis of mice have revealed that no long-term toxic effects arise from MnD-QDs. These studies suggest multimodal Mnd-QDs have the potentials as probes for early pancreatic cancer imaging and detection.

  4. Determination of spallation residues in thin target: toward an hybrid reactor lead target simulation

    International Nuclear Information System (INIS)

    Audouin, L.; Tassan-Got, L.; Bernas, M.; Rejmund, F.; Stephan, C.; Taieb, J.; Boudard, A.; Fernandez, B.; Legrain, R.; Leray, S.; Volant, C.; Wlazlo, W.; Benlliure, J.; Casajeros, E.; Pereira, J.; Czajkowski, S.

    2001-01-01

    The production of spallation primary residual nuclei in thin target has been studied by measurement of isotopic yields distributions for several systems. Issues relevant for the design of accelerator-driven systems are presented. Monte-Carlo code abilities to reproduce data are studied in details; it is shown that calculations do not reproduce data in a satisfactory way. Future work orientations leading to an improvement of thin targets calculations and ultimately to a thick target simulation are discussed. (author)

  5. Determination of spallation residues in thin target: toward an hybrid reactor lead target simulation

    Energy Technology Data Exchange (ETDEWEB)

    Audouin, L.; Tassan-Got, L.; Bernas, M.; Rejmund, F.; Stephan, C.; Taieb, J. [Paris-11 Univ., 91- Orsay (France). Inst. de Physique Nucleaire; Enqvist, T.; Armbruster, P.; Ricciardi, M.V.; Schmidt, K.H. [GSI, Planckstrasse 1, Darmstadt (Germany); Boudard, A.; Fernandez, B.; Legrain, R.; Leray, S.; Volant, C.; Wlazlo, W. [CEA Saclay, Dept. d' Astrophysique, de Physique des Particules, de Physique Nucleaire et de l' Instrumentation Associee, 91 - Gif sur Yvette (France); Benlliure, J.; Casajeros, E.; Pereira, J. [University of Santiago de Compostella (Spain); Czajkowski, S. [Centre d' Etudes Nucleaires de Bordeaux Gradignan, CENBG, CNRS-IN2P3, 33 - Gradignan (France)

    2001-07-01

    The production of spallation primary residual nuclei in thin target has been studied by measurement of isotopic yields distributions for several systems. Issues relevant for the design of accelerator-driven systems are presented. Monte-Carlo code abilities to reproduce data are studied in details; it is shown that calculations do not reproduce data in a satisfactory way. Future work orientations leading to an improvement of thin targets calculations and ultimately to a thick target simulation are discussed. (author)

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

    OpenAIRE

    Groben, R.; Medlin, Linda

    2005-01-01

    Fluorescently-labelled molecular probes were used to identify and characterise phytoplankton species using in situ hybridisation coupled with fluorescence microscopy and flow cytometry. The application of this technique is sometimes problematic, because of the many different species with which this method is to be used. Problems that may occur are: probe penetration versus maintanance of cell stability, strong autofluorescence and/or cell lost during the sample processing. Here we present a m...

  7. Selectivity on-target of bromodomain chemical probes by structure-guided medicinal chemistry and chemical biology.

    Science.gov (United States)

    Galdeano, Carles; Ciulli, Alessio

    2016-09-01

    Targeting epigenetic proteins is a rapidly growing area for medicinal chemistry and drug discovery. Recent years have seen an explosion of interest in developing small molecules binding to bromodomains, the readers of acetyl-lysine modifications. A plethora of co-crystal structures has motivated focused fragment-based design and optimization programs within both industry and academia. These efforts have yielded several compounds entering the clinic, and many more are increasingly being used as chemical probes to interrogate bromodomain biology. High selectivity of chemical probes is necessary to ensure biological activity is due to an on-target effect. Here, we review the state-of-the-art of bromodomain-targeting compounds, focusing on the structural basis for their on-target selectivity or lack thereof. We also highlight chemical biology approaches to enhance on-target selectivity.

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

    Science.gov (United States)

    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.

  9. Detection of mutations related to drug resistance in M. tuberculosis by dot blot hybridization and spoligotyping using specific radiolabelled probes

    International Nuclear Information System (INIS)

    El-Maghraby, T.K.; Abdelazeim, O.

    2002-01-01

    The present work has been conducted to determine the mutations related to drug resistance in M. tuberculosis in 63 Egyptian isolates using dot blot hybridization and spoligotyping. The PCR was done for amplification rpoB and katG genes in isolates. Dot blot hybridization were done to PCR products by using specific radiolabelled probes. Moreover, spoligotyping was done to know about the different strains found in Egypt. The results revealed that 58% from isolates had drug resistance to one or more of antituberculosis drugs. The results of spoligotyping have revealed that some Egyptian isolates are identical with the international code while the rest has not been identified yet. DNA sequencing was done to identify the mutation that not clear in dot blot hybridization. Early diagnosis of geno typing resistance to antituberculosis drugs is important as well as allow appropriate early patients management with few days of TB diagnosis. Using such strategy for early diagnosis of TB drug resistance allow and fast and potent patient's management

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

    Science.gov (United States)

    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.

  11. Targeted 2D/3D registration using ray normalization and a hybrid optimizer

    International Nuclear Information System (INIS)

    Dey, Joyoni; Napel, Sandy

    2006-01-01

    X-ray images are often used to guide minimally invasive procedures in interventional radiology. The use of a preoperatively obtained 3D volume can enhance the visualization needed for guiding catheters and other surgical devices. However, for intraoperative usefulness, the 3D dataset needs to be registered to the 2D x-ray images of the patient. We investigated the effect of targeting subvolumes of interest in the 3D datasets and registering the projections with C-arm x-ray images. We developed an intensity-based 2D/3D rigid-body registration using a Monte Carlo-based hybrid algorithm as the optimizer, using a single view for registration. Pattern intensity (PI) and mutual information (MI) were two metrics tested. We used normalization of the rays to address the problems due to truncation in 3D necessary for targeting. We tested the algorithm on a C-arm x-ray image of a pig's head and a 3D dataset reconstructed from multiple views of the C-arm. PI and MI were comparable in performance. For two subvolumes starting with a set of initial poses from +/-15 mm in x, from +/-3 mm (random), in y and z and +/-4 deg in the three angles, the robustness was 94% for PI and 91% for MI, with accuracy of 2.4 mm (PI) and 2.6 mm (MI), using the hybrid algorithm. The hybrid optimizer, when compared with a standard Powell's direction set method, increased the robustness from 59% (Powell) to 94% (hybrid). Another set of 50 random initial conditions from [+/-20] mm in x,y,z and [+/-10] deg in the three angles, yielded robustness of 84% (hybrid) versus 38% (Powell) using PI as metric, with accuracies 2.1 mm (hybrid) versus 2.0 mm (Powell)

  12. Synthesis and characterization of an MRI Gd-based probe designed to target the translocator protein

    International Nuclear Information System (INIS)

    Cerutti, Erika; Aime, Silvio; Damont, Annelaure; Dolle, Frederic; Baroni, Simona

    2013-01-01

    DPA-713 is the lead compound of a recently reported pyrazolo[1,5-a]pyrimidine acetamide series, targeting the translocator protein (TSPO 18 kDa), and as such, this structure, as well as closely related derivatives, have been already successfully used as positron emission tomography radioligands. On the basis of the pharmacological core of this ligands series, a new magnetic resonance imaging probe, coded DPA-C6-(Gd)DOTAMA was designed and successfully synthesized in six steps and 13% overall yield from DPA-713. The Gd-DOTA monoamide cage (DOTA = 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) represents the magnetic resonance imaging reporter, which is spaced from the phenyl-pyrazolo[1,5-a]pyrimidine acetamide moiety (DPA-713 motif) by a six carbon-atom chain. DPA-C6-(Gd)DOTAMA relaxometric characterization showed the typical behavior of a small-sized molecule (relaxivity value: 6.02 mM -1 s -1 at 20 MHz). The good hydrophilicity of the metal chelate makes DPAC6-(Gd)DOTAMA soluble in water, affecting thus its biodistribution with respect to the parent lipophilic DPA-713 molecule. For this reason, it was deemed of interest to load the probe to a large carrier in order to increase its residence lifetime in blood. Whereas DPA-C6-(Gd)DOTAMA binds to serum albumin with a low affinity constant, it can be entrapped into liposomes (both in the membrane and in the inner aqueous cavity). The stability of the supramolecular adduct formed by the Gd-complex and liposomes was assessed by a competition test with albumin. (authors)

  13. Computationally Probing the Performance of Hybrid, Heterogeneous, and Homogeneous Iridium-Based Catalysts for Water Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    García-Melchor, Max [SUNCAT Center for Interface Science and Catalysis, Department of Chemical Engineering, Stanford University, Stanford CA (United States); Vilella, Laia [Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST),Tarragona (Spain); Departament de Quimica, Universitat Autonoma de Barcelona, Barcelona (Spain); López, Núria [Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST), Tarragona (Spain); Vojvodic, Aleksandra [SUNCAT Center for Interface Science and Catalysis, SLAC National Accelerator Laboratory, Menlo Park CA (United States)

    2016-04-29

    An attractive strategy to improve the performance of water oxidation catalysts would be to anchor a homogeneous molecular catalyst on a heterogeneous solid surface to create a hybrid catalyst. The idea of this combined system is to take advantage of the individual properties of each of the two catalyst components. We use Density Functional Theory to determine the stability and activity of a model hybrid water oxidation catalyst consisting of a dimeric Ir complex attached on the IrO2(110) surface through two oxygen atoms. We find that homogeneous catalysts can be bound to its matrix oxide without losing significant activity. Hence, designing hybrid systems that benefit from both the high tunability of activity of homogeneous catalysts and the stability of heterogeneous systems seems feasible.

  14. Fabrication of genetically engineered polypeptide@quantum dots hybrid nanogels for targeted imaging

    Science.gov (United States)

    Yang, Jie; Yao, Ming-Hao; Zhao, Dong-Hui; Zhang, Xiao-Shuai; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2017-08-01

    Nanogels have been widely used as multifunctional drug delivery carriers because of high water content, biocompatibility, and high loading capability. We designed and biosynthesized two triblock artificial polypeptides PC10A and PC10ARGD as vehicles for encapsulating hydrophobic materials. These polypeptides can form nanogels by self-assembly when the concentration is below 2% ( w/ v). The physical properties of nanogels, including size, surface potential, and targeting domain, are able to be tuned. Hydrophobic materials from molecular size to nano-size can be loaded into the polypeptide nanogels to form hybrid nanogels. Hydrophobic quantum dots CdSe@ZnS below 10 nM were loaded into the polypeptide nanogels by ultrasonic treatment. Encapsulation endows hydrophobic QDs with good tunability of size, water solubility, stability, targeting, and biocompatibility. PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels showed excellent biocompatibility, which the cellular viabilities of HeLa and MCF-7 cells treated with 1% PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels contained 20 nM QDs were above 90 and 80%, respectively. PC10ARGD@QDs hybrid nanogels with an arginine-glycine-aspartic acid motif present efficient receptor-mediated endocytosis in α v β 3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy. These results demonstrate that such polypeptide nanogels as nanocarriers are expected to have great potential applications in biomedicine.

  15. 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

    International Nuclear Information System (INIS)

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

    1990-01-01

    We have synthesized 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

  16. 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

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    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.

  17. THE HYBRID APPROACH OF INFLATION TARGETING: WHAT OPPORTUNITIES FOR AN EMERGING ECONOMY LIKE TUNISIA?

    Directory of Open Access Journals (Sweden)

    Hella Guerchi Mehri

    2016-07-01

    Full Text Available After economic crises happening in many emerging countries, flexible exchange rates became a required theoretical condition helping to target inflation. Many countries stopped using exchange rate as an anchor for monetary policy and started using inflation targeting framework. In emerging countries, monetary authorities work to stabilize the exchange rate because of their “fear of floating”. They are against high volatility of interest rate allowing speculative attacks and causing free fluctuations of their national currency. To avoid uncontrolled market movements, they have to choose between active and public exchange rate management and tight inflation targeting. In the same vein, Central bank of Tunisia follows financial measures linked closely to inflation without focusing especially on monetary aggregates in order to study a possible transition to targeting inflation strategy. It uses a simple Taylor rule where interest rates adjustment are guided by the anticipated inflation deviation from its original target and also by the gap between observed and potential GDP.As an emerging economy with a high degree of financial vulnerability, and facing different shocks, Tunisia should adopt a hybrid rule of inflation targeting in an open economy. This hybrid rule explicitly takes into account the evolution of the exchange rate in the reaction function of the central bank.

  18. Probing photoinduced electron-transfer in graphene-dye hybrid materials for DSSC

    NARCIS (Netherlands)

    Guarracino, Paola; Gatti, Teresa; Canever, Nicolo; Abdu-Aguye, Mustapha; Loi, Maria Antonietta; Menna, Enzo; Franco, Lorenzo

    2017-01-01

    We investigated the photophysical properties of a newly synthesized hybrid material composed of a triphenylamine dye covalently bound to reduced graphene oxide, potentially relevant as a stable photosensitizer in dye-sensitized solar cells. The photophysical characterization has been carried out by

  19. Erythrocyte-derived nano-probes functionalized with antibodies for targeted near infrared fluorescence imaging of cancer cells

    OpenAIRE

    Anvari, Bahman; Mac, Jenny T.; Nunez, Vicente; Burns, Joshua M.; Guerrero, Yadir A.

    2016-01-01

    Constructs derived from mammalian cells are emerging as a new generation of nano-scale platforms for clinical imaging applications. Herein, we report successful engineering of hybrid nano-structures composed of erythrocyte-derived membranes doped with FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG), and surface-functionalized with antibodies to achieve molecular targeting. We demonstrate that these constructs can be used for targeted imaging of cancer cells in vitro. The...

  20. A RNA-DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    John C. Leach

    2016-03-01

    Full Text Available The side effects of radio- and chemo-therapy pose long-term challenges on a cancer patient’s health. It is, therefore, highly desirable to develop more effective therapies that can specifically target carcinoma cells without damaging normal and healthy cells. Tremendous efforts have been made in the past to develop targeted drug delivery systems for solid cancer treatment. In this study, a new aptamer, A10-3-J1, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA, was designed. A super paramagnetic iron oxide nanoparticle-aptamer-doxorubicin (SPIO-Apt-Dox was fabricated and employed as a targeted drug delivery platform for cancer therapy. This DNA RNA hybridized aptamer antitumor agent was able to enhance the cytotoxicity of targeted cells while minimizing collateral damage to non-targeted cells. This SPIO-Apt-Dox nanoparticle has specificity to PSMA+ prostate cancer cells. Aptamer inhibited nonspecific uptake of membrane-permeable doxorubic to the non-target cells, leading to reduced untargeted cytotoxicity and endocytic uptake while enhancing targeted cytotoxicity and endocytic uptake. The experimental results indicate that the drug delivery platform can yield statistically significant effectiveness being more cytotoxic to the targeted cells as opposed to the non-targeted cells.

  1. Multiply osmium-labeled reporter probes for electrochemical DNA hybridization assays: detection of trinucleotide repeats

    Czech Academy of Sciences Publication Activity Database

    Fojta, Miroslav; Havran, Luděk; Kizek, René; Paleček, Emil

    2004-01-01

    Roč. 20, č. 5 (2004), s. 985-994 ISSN 0956-5663 R&D Projects: GA MPO 1H-PK/42; GA AV ČR IAA4004108; GA AV ČR IBS5004355; GA AV ČR KJB4004302; GA AV ČR KSK4055109 Institutional research plan: CEZ:AV0Z5004920 Keywords : electrochemical sensors * DNA hybridization * DNA labeling Subject RIV: BO - Biophysics Impact factor: 3.251, year: 2004

  2. Fluctuation measurements by Langmuir probes during LHCD on ASDEX tokamak. [LHCD (Lower Hybrid Current Drive)

    Energy Technology Data Exchange (ETDEWEB)

    Stoeckel, J [Ceskoslovenska Akademie Ved, Prague (Czech Republic). Ustav Fyziky Plazmatu; Soeldner, F; Giannone, L.; Leuterer, F [Max-Planck-Institut fuer Plasmaphysik, Garching (Germany)

    1991-01-01

    The level of edge electrostatic fluctuations decreases and the global particle/energy confinement improves during lower hybrid current drive (LHCD) regimes on ASDEX, when the total power remains below the initial OH power level. For higher powers, the fluctuations increase noticeably, whereas the global confinement is returning to its OH value. The observed increase of fluctuations is poloidally asymmetric and is caused by local power deposition in front of the grill antenna. (author) 5 refs., 4 figs.

  3. Targeted delivery of 10-hydroxycamptothecin to human breast cancers by cyclic RGD-modified lipid-polymer hybrid nanoparticles.

    Science.gov (United States)

    Yang, Zhe; Luo, Xingen; Zhang, Xiaofang; Liu, Jie; Jiang, Qing

    2013-04-01

    Lipid-polymer hybrid nanoparticles (NPs) combining the positive attributes of both liposomes and polymeric NPs are increasingly being considered as promising candidates to carry therapeutic agents safely and efficiently into targeted sites. Herein, a modified emulsification technique was developed and optimized for the targeting lipid-polymer hybrid NPs fabrication; the surface properties and stability of the hybrid NPs were systematically investigated, which confirmed that the hybrid NPs consisted of a poly (lactide-co-glycolide) core with ∼90% surface coverage of the lipid monolayer and a ∼4.4 nm hydrated polyethylene glycol (PEG) shell. Optimization results showed that the lipid:polymer mass ratio and the lipid-PEG:lipid molar ratio could affect the size, lipid association efficiency and stability of hybrid NPs. Furthermore, a model chemotherapy drug, 10-hydroxycamptothecin, was encapsulated into hybrid NPs with a higher drug loading compared to PLGA NPs. Surface modification of the lipid layer and the PEG conjugated targeting ligand did not affect their drug release kinetics. Finally, the cytotoxicity and cellular uptake studies indicated that the lipid coverage and the c(RGDyk) conjugation of the hybrid NPs gained a significantly enhanced ability of cell killing and endocytosis. Our results suggested that lipid-polymer hybrid NPs prepared by the modified emulsion technique have great potential to be utilized as an engineered drug delivery system with precise control ability of surface targeting modification.

  4. Targeted delivery of 10-hydroxycamptothecin to human breast cancers by cyclic RGD-modified lipid–polymer hybrid nanoparticles

    International Nuclear Information System (INIS)

    Yang, Zhe; Luo, Xingen; Zhang, Xiaofang; Liu, Jie; Jiang, Qing

    2013-01-01

    Lipid–polymer hybrid nanoparticles (NPs) combining the positive attributes of both liposomes and polymeric NPs are increasingly being considered as promising candidates to carry therapeutic agents safely and efficiently into targeted sites. Herein, a modified emulsification technique was developed and optimized for the targeting lipid–polymer hybrid NPs fabrication; the surface properties and stability of the hybrid NPs were systematically investigated, which confirmed that the hybrid NPs consisted of a poly (lactide-co-glycolide) core with ∼90% surface coverage of the lipid monolayer and a ∼4.4 nm hydrated polyethylene glycol (PEG) shell. Optimization results showed that the lipid:polymer mass ratio and the lipid-PEG:lipid molar ratio could affect the size, lipid association efficiency and stability of hybrid NPs. Furthermore, a model chemotherapy drug, 10-hydroxycamptothecin, was encapsulated into hybrid NPs with a higher drug loading compared to PLGA NPs. Surface modification of the lipid layer and the PEG conjugated targeting ligand did not affect their drug release kinetics. Finally, the cytotoxicity and cellular uptake studies indicated that the lipid coverage and the c(RGDyk) conjugation of the hybrid NPs gained a significantly enhanced ability of cell killing and endocytosis. Our results suggested that lipid–polymer hybrid NPs prepared by the modified emulsion technique have great potential to be utilized as an engineered drug delivery system with precise control ability of surface targeting modification. (paper)

  5. The flush-mounted rail Langmuir probe array designed for the Alcator C-Mod vertical target plate divertor

    Science.gov (United States)

    Kuang, A. Q.; Brunner, D.; LaBombard, B.; Leccacorvi, R.; Vieira, R.

    2018-04-01

    An array of flush-mounted and toroidally elongated Langmuir probes (henceforth called rail probes) have been specifically designed for the Alcator C-Mod's vertical target plate divertor and operated over multiple campaigns. The "flush" geometry enables the tungsten electrodes to survive high heat flux conditions in which traditional "proud" tungsten electrodes suffer damage from melting. The toroidally elongated rail-like geometry reduces the influence of sheath expansion, which is an important effect to consider in the design and interpretation of flush-mounted Langmuir probes. The new rail probes successfully operated during C-Mod's FY2015 and FY2016 experimental campaigns with no evidence of damage, despite being regularly subjected to heat flux densities parallel to the magnetic field exceeding ˜1 GW m-2 for short periods of time. A comparison between rail and proud probe data indicates that sheath expansion effects were successfully mitigated by the rail design, extending the use of these Langmuir probes to incident magnetic field line angles as low as 0.5°.

  6. Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids.

    NARCIS (Netherlands)

    Meletiadis, J.; Melchers, W.J.G.; Meis, J.F.G.M.; Hurk, P.J.J.C. van den; Jannes, G.; Verweij, P.E.

    2003-01-01

    An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical

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

    International Nuclear Information System (INIS)

    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 35 S-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 35 S-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

  8. Integrin-Targeted Hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography for Imaging Tumor Progression and Early Response in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xiaopeng Ma

    2017-01-01

    Full Text Available Integrins play an important role in tumor progression, invasion and metastasis. Therefore we aimed to evaluate a preclinical imaging approach applying ανβ3 integrin targeted hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT for monitoring tumor progression as well as early therapy response in a syngeneic murine Non-Small Cell Lung Cancer (NSCLC model. Lewis Lung Carcinomas were grown orthotopically in C57BL/6 J mice and imaged in-vivo using a ανβ3 targeted near-infrared fluorescence (NIRF probe. ανβ3-targeted FMT-XCT was able to track tumor progression. Cilengitide was able to substantially block the binding of the NIRF probe and suppress the imaging signal. Additionally mice were treated with an established chemotherapy regimen of Cisplatin and Bevacizumab or with a novel MEK inhibitor (Refametinib for 2 weeks. While μCT revealed only a moderate slowdown of tumor growth, ανβ3 dependent signal decreased significantly compared to non-treated mice already at one week post treatment. ανβ3 targeted imaging might therefore become a promising tool for assessment of early therapy response in the future.

  9. Identification of waves by RF magnetic probes during lower hybrid wave injection experiments on the TST-2 spherical tokamak

    International Nuclear Information System (INIS)

    Shinya, Takahiro; Ejiri, Akira; Takase, Yuichi

    2014-01-01

    RF magnetic probes can be used to measure not only the wavevector, but also the polarization of waves in plasmas. A 5-channel RF magnetic probe (5ch-RFMP) was installed in the TST-2 spherical tokamak and the waves were studied in detail during lower hybrid wave injection experiments. From the polarization measurements, the poloidal RF magnetic field is found to be dominant. In addition to polarization, components of k perpendicular to the major radial direction were obtained from phase differences among the five channels. The radial wavenumber was obtained by scanning the radial position of the 5ch-RFMP on a shot by shot basis. The measured wavevector and polarization in the plasma edge region were consistent with those calculated from the wave equation for the slow wave branch. While the waves with small and large k ∥ were excited by the antenna, only the small k ∥ component was measured by the 5ch-RFMP; this suggests that the waves with larger k ∥ were absorbed by the plasma. (author)

  10. Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

    OpenAIRE

    Schwiertz, Andreas; Le Blay, Gwenaelle; Blaut, Michael

    2000-01-01

    Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none...

  11. Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

    Directory of Open Access Journals (Sweden)

    Trebesius Karlheinz

    2010-03-01

    Full Text Available Abstract Background Francisella (F. tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR have been developed but are restricted to reference laboratories. Results The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH assays were established using species- and subspecies-specific probes. Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. Conclusion We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples.

  12. Multi probes measurements at the PALS Facility Research Centre during high intense laser pulse interactions with various target materials

    Directory of Open Access Journals (Sweden)

    De Marco Massimo

    2018-01-01

    Full Text Available During the interaction of high intense laser pulse with solid target, a large amount of hot electrons is produced and a giant Electromagnetic Pulse (EMP is generated due to the current flowing into the system target–target holder, as well as due to the escaping charged particles in vacuum. EMP production for different target materials is investigated inside and outside the target chamber, using monopole antenna, super wide-band microstrip antenna and Moebius antenna. The EMP consists in a fast transient magnetic field lasting hundreds of nanosecond with frequencies ranging from MHz to tens of GHz. Measurements of magnetic field and return target current in the range of kA were carried out by an inductive target probe (Cikhardt J. et al. Rev. Sci. Instrum. 85 (2014 103507.

  13. Novel targeted siRNA-loaded hybrid nanoparticles: preparation, characterization and in vitro evaluation.

    Science.gov (United States)

    Dim, Nneka; Perepelyuk, Maryna; Gomes, Olukayode; Thangavel, Chellappagounder; Liu, Yi; Den, Robert; Lakshmikuttyamma, Ashakumary; Shoyele, Sunday A

    2015-09-26

    siRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated. It was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells. This hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1

  14. Quantum dot tailored to single wall carbon nanotubes: a multifunctional hybrid nanoconstruct for cellular imaging and targeted photothermal therapy.

    Science.gov (United States)

    Nair, Lakshmi V; Nagaoka, Yutaka; Maekawa, Toru; Sakthikumar, D; Jayasree, Ramapurath S

    2014-07-23

    Hybrid nanomaterial based on quantum dots and SWCNTs is used for cellular imaging and photothermal therapy. Furthermore, the ligand conjugated hybrid system (FaQd@CNT) enables selective targeting in cancer cells. The imaging capability of quantum dots and the therapeutic potential of SWCNT are available in a single system with cancer targeting property. Heat generated by the system is found to be high enough to destroy cancer cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Study on sensitivity of southern blotting hybridization using a 32P-labeled probe of PCR products in detecting human cytomegalovirus

    International Nuclear Information System (INIS)

    Bu Hengfu; Chen Juan; Shen Rongsen; Ma Liren; Xu Yongqiang

    1996-01-01

    Southern blotting hybridization (SBH) using a 32 P-labeled probe is one of the most practical methods for genetic diagnosis of pathogen. On the basis of establishing PCR and nested PCR for detecting human cytomegalovirus (HCMV), a 32 P-labeled probe was prepared with the amplified products of 613 bp PCR outer primers and hybridized with 300 bp inner primer amplified product, resulting in increase in detecting sensitivity from 17 ng (in 1.2% agarose electrophoresis) before SBH to 500 pg (autoradiographed), in other words, increasing the sensitivity of detecting HCMV by 10 2 dilutions after using SBH. The method of PCR and SBH using a 32 P-labeled probe could detect less than 1 gene copy of HCMV, therefore, it is a rapid and reliable diagnosis method for detecting HCMV latent infection

  16. Folate-containing reduction-sensitive lipid-polymer hybrid nanoparticles for targeted delivery of doxorubicin.

    Science.gov (United States)

    Wu, Bo; Yu, Ping; Cui, Can; Wu, Ming; Zhang, Yang; Liu, Lei; Wang, Cai-Xia; Zhuo, Ren-Xi; Huang, Shi-Wen

    2015-04-01

    The development and evaluation of folate-targeted and reduction-triggered biodegradable nanoparticles are introduced to the research on targeted delivery of doxorubicin (DOX). This type of folate-targeted lipid-polymer hybrid nanoparticles (FLPNPs) is comprised of a poly(D,L-lactide-co-glycolide) (PLGA) core, a soybean lecithin monolayer, a monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) reduction-sensitive shell, and a folic acid-targeted ligand. FLPNPs exhibited high size stability but fast disassembly in a simulated cancer cell reductive environment. The experiments on the release process in vitro revealed that as a reduction-sensitive drug delivery system, FLPNPs released DOX faster in the presence of 10 mM dithiothreitol (DTT). Results from flow cytometry, confocal image and in vitro cytotoxicity assays revealed that FLPNPs further enhanced cell uptake and generated higher cytotoxicity against human epidermoid carcinoma in the oral cavity than non-targeted redox-sensitive and targeted redox-insensitive controls. Furthermore, in vivo animal experiments demonstrated that systemic administration of DOX-loaded FLPNPs remarkably reduced tumor growth. Experiments on biodistribution of DOX-loaded FLPNPs showed that an increasing amount of DOX accumulated in the tumor. Therefore, FLPNPs formulations have proved to be a stable, controllable and targeted anticancer drug delivery system.

  17. Hybrid ligand-alkylating agents targeting telomeric G-quadruplex structures.

    Science.gov (United States)

    Doria, Filippo; Nadai, Matteo; Folini, Marco; Di Antonio, Marco; Germani, Luca; Percivalle, Claudia; Sissi, Claudia; Zaffaroni, Nadia; Alcaro, Stefano; Artese, Anna; Richter, Sara N; Freccero, Mauro

    2012-04-14

    The synthesis, physico-chemical properties and biological effects of a new class of naphthalene diimides (NDIs) capable of reversibly binding telomeric DNA and alkylate it through an electrophilic quinone methide moiety (QM), are reported. FRET and circular dichroism assays showed a marked stabilization and selectivity towards telomeric G4 DNA folded in a hybrid topology. NDI-QMs' alkylating properties revealed a good reactivity on single nucleosides and selectivity towards telomeric G4. A selected NDI was able to significantly impair the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression. These findings points to our hybrid ligand-alkylating NDIs as possible tools for the development of novel targeted anticancer therapies. This journal is © The Royal Society of Chemistry 2012

  18. Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy.

    Science.gov (United States)

    Hammerer, Fabien; Poyer, Florent; Fourmois, Laura; Chen, Su; Garcia, Guillaume; Teulade-Fichou, Marie-Paule; Maillard, Philippe; Mahuteau-Betzer, Florence

    2018-01-01

    The proof of concept for two-photon activated photodynamic therapy has already been achieved for cancer treatment but the efficiency of this approach still heavily relies on the availability of photosensitizers combining high two-photon absorption and biocompatibility. In this line we recently reported on a series of porphyrin-triphenylamine hybrids which exhibit high singlet oxygen production quantum yield as well as high two-photon absorption cross-sections but with a very poor cellular internalization. We present herein new photosensitizers of the same porphyrin-triphenylamine hybrid series but bearing cationic charges which led to strongly enhanced water solubility and thus cellular penetration. In addition the new compounds have been found localized in mitochondria that are preferential target organelles for photodynamic therapy. Altogether the strongly improved properties of the new series combined with their specific mitochondrial localization lead to a significantly enhanced two-photon activated photodynamic therapy efficiency. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. "Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes

    Czech Academy of Sciences Publication Activity Database

    Fojta, Miroslav; Kostečka, Pavel; Trefulka, Mojmír; Havran, Luděk; Paleček, Emil

    2007-01-01

    Roč. 79, č. 3 (2007), s. 1022-1029 ISSN 0003-2700 R&D Projects: GA AV ČR(CZ) IAA4004402; GA ČR(CZ) GA203/05/0043; GA ČR(CZ) GA203/04/1325; GA MPO(CZ) 1H-PK/42; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA labeling * osmium tetroxide complexes * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 5.287, year: 2007

  20. Development of high-performance alkali-hybrid polarized 3He targets for electron scattering

    Science.gov (United States)

    Singh, Jaideep T.; Dolph, P. A. M.; Tobias, W. A.; Averett, T. D.; Kelleher, A.; Mooney, K. E.; Nelyubin, V. V.; Wang, Yunxiao; Zheng, Yuan; Cates, G. D.

    2015-05-01

    Background: Polarized 3He targets have been used as effective polarized neutron targets for electron scattering experiments for over twenty years. Over the last ten years, the effective luminosity of polarized 3He targets based on spin-exchange optical pumping has increased by over an order of magnitude. This has come about because of improvements in commercially-available lasers and an improved understanding of the physics behind the polarization process. Purpose: We present the development of high-performance polarized 3He targets for use in electron scattering experiments. Improvements in the performance of polarized 3He targets, target properties, and operating parameters are documented. Methods: We utilize the technique of alkali-hybrid spin-exchange optical pumping to polarize the 3He targets. Spectrally narrowed diode lasers used for the optical pumping greatly improved the performance. A simulation of the alkali-hybrid spin-exchange optical pumping process was developed to provide guidance in the design of the targets. Data was collected during the characterization of 24 separate glass target cells, each of which was constructed while preparing for one of four experiments at Jefferson Laboratory in Newport News, Virginia. Results: From the data obtained we made determinations of the so-called X -factors that quantify a temperature-dependent and as-yet poorly understood spin-relaxation mechanism that limits the maximum achievable 3He polarization to well under 100%. The presence of the X -factor spin-relaxation mechanism was clearly evident in our data. Good agreement between the simulation and the actual target performance was obtained by including details such as off-resonant optical pumping. Included in our results is a measurement of the K -3He spin-exchange rate coefficient kseK=(7.46 ±0.62 ) ×10-20cm3/s over the temperature range 503 K to 563 K. Conclusions: In order to achieve high performance under the operating conditions described in this paper

  1. Hybridized electronic states in potassium-doped picene probed by soft x-ray spectroscopies

    Directory of Open Access Journals (Sweden)

    Hiroyuki Yamane

    2012-12-01

    Full Text Available The electronic structure of the unoccupied and occupied states of potassium (K-doped and undoped picene crystalline films has been investigated by using the element-selective and bulk-sensitive photon-detection methods of X-ray absorption and emission spectroscopies. We observed the formation of the doping-induced unoccupied and occupied electronic states in K-doped picene. By applying the inner-shell resonant-excitation experiments, we observed the evidence for the orbital hybridization between K and picene near the Fermi energy. Furthermore, the resonant X-ray emission experiment suggests the presence of the Raman-active vibronic interaction in K-doped picene. These experimental evidences play a crucial role in the superconductivity of K-doped picene.

  2. Improving Probe Immobilization for Label-Free Capacitive Detection of DNA Hybridization on Microfabricated Gold Electrodes

    Directory of Open Access Journals (Sweden)

    Sandro Carrara

    2008-02-01

    Full Text Available Alternative approaches to labeled optical detection for DNA arrays are actively investigated for low-cost point-of-care applications. In this domain, label-free capacitive detection is one of the most intensely studied techniques. It is based on the idea to detect the Helmholtz ion layer displacements when molecular recognition occurs at the electrodes/solution interface. The sensing layer is usually prepared by using thiols terminated DNA single-strength oligonucleotide probes on top of the sensor electrodes. However, published data shows evident time drift, which greatly complicates signal conditioning and processing and ultimately increases the uncertainty in DNA recognition sensing. The aim of this work is to show that newly developed ethylene-glycol functionalized alkanethiols greatly reduce time drift, thereby significantly improving capacitance based label-free detection of DNA.

  3. On-site detection of Phytophthora spp.—single-stranded target DNA as the limiting factor to improve on-chip hybridization

    International Nuclear Information System (INIS)

    Schwenkbier, Lydia; Pollok, Sibyll; Popp, Jürgen; Weber, Karina; König, Stephan; Wagner, Stefan; Werres, Sabine; Weber, Jörg; Hentschel, Martin

    2014-01-01

    We report on a lab-on-a-chip approach for on-site detection of Phytophthora species that allows visual signal readout. The results demonstrate the significance of single-stranded DNA (ssDNA) generation in terms of improving the intensity of the hybridization signal and to improve the reliability of the method. Conventional PCR with subsequent heat denaturation, sodium hydroxide-based denaturation, lambda exonuclease digestion and two asymmetric PCR methods were investigated for the species P. fragariae, P. kernoviae, and P. ramorum. The positioning of the capture probe within the amplified yeast GTP-binding protein (YPT1) target DNA was also of interest because it significantly influences the intensity of the signal. Statistical tests were used to validate the impact of the ssDNA generation methods and the capture-target probe position. The single-stranded target DNA generated by Linear-After-The-Exponential PCR (LATE-PCR) was found to produce signal intensities comparable to post-PCR exonuclease treatment. The LATE-PCR is the best method for the on-site detection of Phytophthora because the enzymatic digestion after PCR is more laborious and time-consuming. (author)

  4. Hybrid protein-inorganic nanoparticles: From tumor-targeted drug delivery to cancer imaging.

    Science.gov (United States)

    Elzoghby, Ahmed O; Hemasa, Ayman L; Freag, May S

    2016-12-10

    Recently, a great interest has been paid to the development of hybrid protein-inorganic nanoparticles (NPs) for drug delivery and cancer diagnostics in order to combine the merits of both inorganic and protein nanocarriers. This review primarily discusses the most outstanding advances in the applications of the hybrids of naturally-occurring proteins with iron oxide, gadolinium, gold, silica, calcium phosphate NPs, carbon nanotubes, and quantum dots in drug delivery and cancer imaging. Various strategies that have been utilized for the preparation of protein-functionalized inorganic NPs and the mechanisms involved in the drug loading process are discussed. How can the protein functionalization overcome the limitations of colloidal stability, poor dispersibility and toxicity associated with inorganic NPs is also investigated. Moreover, issues relating to the influence of protein hybridization on the cellular uptake, tumor targeting efficiency, systemic circulation, mucosal penetration and skin permeation of inorganic NPs are highlighted. A special emphasis is devoted to the novel approaches utilizing the protein-inorganic nanohybrids in combined cancer therapy, tumor imaging, and theranostic applications as well as stimuli-responsive drug release from the nanohybrids. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Microdeletion and microduplication analysis of chinese conotruncal defects patients with targeted array comparative genomic hybridization.

    Directory of Open Access Journals (Sweden)

    Xiaohui Gong

    Full Text Available OBJECTIVE: The current study aimed to develop a reliable targeted array comparative genomic hybridization (aCGH to detect microdeletions and microduplications in congenital conotruncal defects (CTDs, especially on 22q11.2 region, and for some other chromosomal aberrations, such as 5p15-5p, 7q11.23 and 4p16.3. METHODS: Twenty-seven patients with CTDs, including 12 pulmonary atresia (PA, 10 double-outlet right ventricle (DORV, 3 transposition of great arteries (TGA, 1 tetralogy of Fallot (TOF and one ventricular septal defect (VSD, were enrolled in this study and screened for pathogenic copy number variations (CNVs, using Agilent 8 x 15K targeted aCGH. Real-time quantitative polymerase chain reaction (qPCR was performed to test the molecular results of targeted aCGH. RESULTS: Four of 27 patients (14.8% had 22q11.2 CNVs, 1 microdeletion and 3 microduplications. qPCR test confirmed the microdeletion and microduplication detected by the targeted aCGH. CONCLUSION: Chromosomal abnormalities were a well-known cause of multiple congenital anomalies (MCA. This aCGH using arrays with high-density coverage in the targeted regions can detect genomic imbalances including 22q11.2 and other 10 kinds CNVs effectively and quickly. This approach has the potential to be applied to detect aneuploidy and common microdeletion/microduplication syndromes on a single microarray.

  6. Spatially Probed Plasmonic Photothermic Nanoheater Enhanced Hybrid Polymeric-Metallic PVDF-Ag Nanogenerator.

    Science.gov (United States)

    Liow, Chi Hao; Lu, Xin; Tan, Chuan Fu; Chan, Kwok Hoe; Zeng, Kaiyang; Li, Shuzhou; Ho, Ghim Wei

    2018-02-01

    Surface plasmon-based photonics offers exciting opportunities to enable fine control of the site, span, and extent of mechanical harvesting. However, the interaction between plasmonic photothermic and piezoresponse still remains underexplored. Here, spatially localized and controllable piezoresponse of a hybrid self-polarized polymeric-metallic system that correlates to plasmonic light-to-heat modulation of the local strain is demonstrated. The piezoresponse is associated to the localized plasmons that serve as efficient nanoheaters leading to self-regulated strain via thermal expansion of the electroactive polymer. Moreover, the finite-difference time-domain simulation and linear thermal model also deduce the local strain to the surface plasmon heat absorption. The distinct plasmonic photothermic-piezoelectric phenomenon mediates not only localized external stimulus light response but also enhances dynamic piezoelectric energy harvesting. The present work highlights a promising surface plasmon coordinated piezoelectric response which underpins energy localization and transfer for diversified design of unique photothermic-piezotronic technology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. In situ hybridization of bat chromosomes with human (TTAGGGn probe, after previous digestion with Alu I

    Directory of Open Access Journals (Sweden)

    Karina de Cassia Faria

    2002-01-01

    Full Text Available The purpose of this work was to verify the ability of the enzyme Alu I to cleave and/or remove satellite DNA sequences from heterochromatic regions in chromosomes of bats, by identifying the occurrence of modifications in the pattern of fluorescence in situ hybridization with telomeric DNA. The localization and fluorescence intensity of the telomeric DNA sites of the Alu-digested and undigested chromosomes of species Eumops glaucinus, Carollia perspicillata, and Platyrrhinus lineatus were analyzed. Telomeric sequences were detected at the termini of chromosomes of all three species, although, in C. perspicillata, the signals were very faint or absent in most chromosomes. This finding was interpreted as being due to a reduced number of copies of the telomeric repeat, resulting from extensive telomeric association and/or rearrangements undergone by the chromosomes of Carollia. Fluorescent signals were also observed in centromeric and pericentromeric regions in several two-arm chromosomes of E. glaucinus and C. perspicillata. In E. glaucinus and P. lineatus, some interstitial and terminal telomeric sites were observed to be in association with regions of constitutive heterochromatin and ribosomal DNA (NORs. After digestion, these telomeric sites showed a significant decrease in signal intensity, indicating that enzyme Alu I cleaves and/or removes part of the satellite DNA present in these regions. These results suggest that the telomeric sequence is a component of the heterochromatin, and that the C-band- positive regions of bat chromosomes have a different DNA composition.

  8. A Pyridazine-Based Fluorescent Probe Targeting Aβ Plaques in Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Yong Dae Park

    2018-01-01

    Full Text Available Accumulation of β-amyloid (Aβ plaques comprising Aβ40 and Aβ42 in the brain is the most significant factor in the pathogenesis of Alzheimer’s disease (AD. Thus, the detection of Aβ plaques has increasingly attracted interest in the context of AD diagnosis. In the present study, a fluorescent pyridazine-based dye that can detect and image Aβ plaques was designed and synthesized, and its optical properties in the presence of Aβ aggregates were evaluated. An approximately 34-fold increase in emission intensity was exhibited by the fluorescent probe after binding with Aβ aggregates, for which it showed high affinity (KD = 0.35 µM. Moreover, the reasonable hydrophobic properties of the probe (log P = 2.94 allow it to penetrate the blood brain barrier (BBB. In addition, the pyridazine-based probe was used in the histological costaining of transgenic mouse (APP/PS1 brain sections to validate the selective binding of the probe to Aβ plaques. The results suggest that the pyridazine-based compound has the potential to serve as a fluorescent probe for the diagnosis of AD.

  9. Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

    Science.gov (United States)

    Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

    2005-08-01

    In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

  10. SU-E-I-81: Targeting of HER2-Expressing Tumors with Dual PET-MR Imaging Probes

    Energy Technology Data Exchange (ETDEWEB)

    Xu, P; Peng, Y; Sun, M; Yang, X [Suzhou Institute of Biomedical Engineering and Technology Chinese Academy o, Suzhou, Jiangsu (China)

    2015-06-15

    Purpose: The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Trastuzumab, effective in about 15 % of women with breast cancer, downregulates signalling through the Akt/PI3K and MAPK pathways.These pathways modulate metabolism which can be monitored by positron emission tomography (PET) and magnetic resonance imaging (MRI). Methods: The relationship between response of HER2 overexpressing tumours and changes in imaging PET or SPECT and MRI will be examined by a integrated bimodal imaging probe.Small (7 kDa) high-affinity anti-HER2 Affibody molecules and KCCYSL targeting peptide may be suitable tracers for visualization of HER2-expressing tumors. Peptide-conjugated iron oxide nanoparticles (Fe3O4 NPs) as MRI imaging and CB-TE2A as PET imaging are integrated into a single synthetic molecule in the HER2 positive cancer. Results: One of targeted contrast bimodal imaging probe agents was synthesized and evaluated to target HER2-expressing tumors in a HER2 positive rat model. We will report the newest results regarding the development of bimodal imaging probes. Conclusion: The preliminary results of the bimodal imaging probe presents high correlation of MRI signal and PET imaging intensity in vivo. This unique feature can hardly be obtained by single model contrast agents. It is envisioned that this bimodal agents can hold great potential for accurate detection of HER2-expressing tumors which are critical for clinical management of the disease.

  11. Monitoring Methanotrophic Bacteria in Hybrid Anaerobic-Aerobic Reactors with PCR and a Catabolic Gene Probe

    Science.gov (United States)

    Miguez, Carlos B.; Shen, Chun F.; Bourque, Denis; Guiot, Serge R.; Groleau, Denis

    1999-01-01

    We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on the mmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants. PMID:9925557

  12. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. MRI-guided targeting delivery of doxorubicin with reduction-responsive lipid-polymer hybrid nanoparticles

    Directory of Open Access Journals (Sweden)

    Wu B

    2017-09-01

    Full Text Available Bo Wu,1,2 Shu-Ting Lu,1 Kai Deng,2 Hui Yu,2 Can Cui,2 Yang Zhang,2 Ming Wu,2 Ren-Xi Zhuo,2 Hai-Bo Xu,1 Shi-Wen Huang2 1Department of Radiology, Zhongnan Hospital of Wuhan University, 2Key Laboratory of Biomedical Polymers, Ministry of Education, Department of Chemistry, Wuhan University, Wuhan, People’s Republic of China Abstract: In recent years, there has been increasing interest in developing a multifunctional nanoscale platform for cancer monitoring and chemotherapy. However, there is still a big challenge for current clinic contrast agents to improve their poor tumor selectivity and response. Herein, we report a new kind of Gd complex and folate-coated redox-sensitive lipid-polymer hybrid nanoparticle (Gd-FLPNP for tumor-targeted magnetic resonance imaging and therapy. Gd-FLPNPs can simultaneously accomplish diagnostic imaging, and specific targeting and controlled release of doxorubicin (DOX. They exhibit good monodispersity, excellent size stability, and a well-defined core-shell structure. Paramagnetic nanoparticles based on gadolinium-diethylenetriaminepentaacetic acid-bis-cetylamine have paramagnetic properties with an approximately two-fold enhancement in the longitudinal relaxivity compared to clinical used Magnevist. For targeted and reduction-sensitive drug delivery, Gd-FLPNPs released DOX faster and enhanced cell uptake in vitro, and exhibited better antitumor effect both in vitro and in vivo. Keywords: redox-sensitive, tumor-targeted, gadolinium, contrast agents, PLGA

  14. Evaluation of a fluorescence-labelled oligonucleotide tide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears

    DEFF Research Database (Denmark)

    Nordentoft, Steen; Christensen, H.; Wegener, Henrik Caspar

    1997-01-01

    with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin...

  15. Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography.

    Science.gov (United States)

    Mokarram, P; Rismanchi, M; Alizadeh Naeeni, M; Mirab Samiee, S; Paryan, M; Alipour, A; Honardar, Z; Kavousipour, S; Naghibalhossaini, F; Mostafavi-Pour, Z; Monabati, A; Hosseni, S V; Shamsdin, S A

    2014-05-01

    Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.

  16. An efficient hybrid technique in RCS predictions of complex targets at high frequencies

    Science.gov (United States)

    Algar, María-Jesús; Lozano, Lorena; Moreno, Javier; González, Iván; Cátedra, Felipe

    2017-09-01

    Most computer codes in Radar Cross Section (RCS) prediction use Physical Optics (PO) and Physical theory of Diffraction (PTD) combined with Geometrical Optics (GO) and Geometrical Theory of Diffraction (GTD). The latter approaches are computationally cheaper and much more accurate for curved surfaces, but not applicable for the computation of the RCS of all surfaces of a complex object due to the presence of caustic problems in the analysis of concave surfaces or flat surfaces in the far field. The main contribution of this paper is the development of a hybrid method based on a new combination of two asymptotic techniques: GTD and PO, considering the advantages and avoiding the disadvantages of each of them. A very efficient and accurate method to analyze the RCS of complex structures at high frequencies is obtained with the new combination. The proposed new method has been validated comparing RCS results obtained for some simple cases using the proposed approach and RCS using the rigorous technique of Method of Moments (MoM). Some complex cases have been examined at high frequencies contrasting the results with PO. This study shows the accuracy and the efficiency of the hybrid method and its suitability for the computation of the RCS at really large and complex targets at high frequencies.

  17. MRI-guided targeting delivery of doxorubicin with reduction-responsive lipid-polymer hybrid nanoparticles.

    Science.gov (United States)

    Wu, Bo; Lu, Shu-Ting; Deng, Kai; Yu, Hui; Cui, Can; Zhang, Yang; Wu, Ming; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

    2017-01-01

    In recent years, there has been increasing interest in developing a multifunctional nanoscale platform for cancer monitoring and chemotherapy. However, there is still a big challenge for current clinic contrast agents to improve their poor tumor selectivity and response. Herein, we report a new kind of Gd complex and folate-coated redox-sensitive lipid-polymer hybrid nanoparticle (Gd-FLPNP) for tumor-targeted magnetic resonance imaging and therapy. Gd-FLPNPs can simultaneously accomplish diagnostic imaging, and specific targeting and controlled release of doxorubicin (DOX). They exhibit good monodispersity, excellent size stability, and a well-defined core-shell structure. Paramagnetic nanoparticles based on gadolinium-diethylenetriaminepentaacetic acid-bis-cetylamine have paramagnetic properties with an approximately two-fold enhancement in the longitudinal relaxivity compared to clinical used Magnevist. For targeted and reduction-sensitive drug delivery, Gd-FLPNPs released DOX faster and enhanced cell uptake in vitro, and exhibited better antitumor effect both in vitro and in vivo.

  18. Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

    Science.gov (United States)

    Wang, Xuhua; Yao, Sheng; Ahn, Hyo-Yang; Zhang, Yuanwei; Bondar, Mykhailo V.; Torres, Joseph A.; Belfield, Kevin D.

    2010-01-01

    Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated. PMID:21258480

  19. A nanobody targeting carcinoembryonic antigen as a promising molecular probe for non-small cell lung cancer.

    Science.gov (United States)

    Wang, Hao; Meng, Ai-Min; Li, Sheng-Hua; Zhou, Xiao-Liang

    2017-07-01

    Carcinoembryonic antigen (CEA) is a biomarker and therapy target for non‑small cell lung cancer (NSCLC), which is the most common type of lung cancer. Nanobodies with high target specificity are promising candidates to function as anti‑CEA probes. In the present study, the targeting effects of an anti‑CEA nanobody obtained from phage display were investigated using technetium‑99 m (99mTc) and fluorescence labeling. In vitro binding and immunofluorescent staining assays, as well as in vivo blood clearance and biodistribution assays were performed. High specificity and affinity of the nanobody for CEA‑positive H460 cells was observed in vitro. The pharmacokinetics assay of the 99mTc‑nanobody in Wistar rats demonstrated that the nanobody had appropriate T1/2α and T1/2β, which were 20.2 and 143.5 min, respectively. The biodistribution assay using H460 xenograft‑bearing nude mice demonstrated a high ratio of signal in tumor compared with background, which confirmed that the nanobody may be useful as a molecular probe for CEA‑positive cancer, particularly in NSCLC.

  20. Folate-modified lipid–polymer hybrid nanoparticles for targeted paclitaxel delivery

    Directory of Open Access Journals (Sweden)

    Zhang L

    2015-03-01

    Full Text Available Linhua Zhang,1 Dunwan Zhu,1 Xia Dong,1 Hongfan Sun,1 Cunxian Song,1 Chun Wang,2 Deling Kong1 1Tianjin Key Laboratory of Biomaterials, Institute of Biomedical Engineering, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin, People’s Republic of China; 2Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA Abstract: The purpose of this study was to develop a novel lipid–polymer hybrid drug carrier comprised of folate (FA modified lipid-shell and polymer-core nanoparticles (FLPNPs for sustained, controlled, and targeted delivery of paclitaxel (PTX. The core-shell NPs consist of 1 a poly(ε-caprolactone hydrophobic core based on self-assembly of poly(ε-caprolactone–poly(ethylene glycol–poly(ε-caprolactone (PCL-PEG-PCL amphiphilic copolymers, 2 a lipid monolayer formed with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol-2000] (DSPE-PEG2000, 3 a targeting ligand (FA on the surface, and were prepared using a thin-film hydration and ultrasonic dispersion method. Transmission electron microscopy and dynamic light scattering analysis confirmed the coating of the lipid monolayer on the hydrophobic polymer core. Physicochemical characterizations of PTX-loaded FLPNPs, such as particle size and size distribution, zeta potential, morphology, drug loading content, encapsulation efficiency, and in vitro drug release, were also evaluated. Fluorescent microscopy proved the internalization efficiency and targeting ability of the folate conjugated on the lipid monolayer for the EMT6 cancer cells which overexpress folate receptor. In vitro cytotoxicity assay demonstrated that the cytotoxic effect of PTX-loaded FLPNPs was lower than that of Taxol®, but higher than that of PTX-loaded LPNPs (without folate conjugation. In EMT6 breast tumor model, intratumoral administration of PTX-loaded FLPNPs showed similar antitumor efficacy but low toxicity compared to Taxol®. More

  1. Design, synthesis and validation of integrin α2β1-targeted probe for microPET imaging of prostate cancer

    International Nuclear Information System (INIS)

    Huang, Chiun-Wei; Li, Zibo; Cai, Hancheng; Chen, Kai; Shahinian, Tony; Conti, Peter S.

    2011-01-01

    The ability of PET to aid in the diagnosis and management of recurrent and/or disseminated metastatic prostate cancer may be enhanced by the development of novel prognostic imaging probes. Accumulating experimental evidence indicates that overexpression of integrin α 2 β 1 may correlate with progression in human prostate cancer. In this study, 64 Cu-labeled integrin α 2 β 1 -targeted PET probes were designed and evaluated for the imaging of prostate cancer. DGEA peptides conjugated with a bifunctional chelator (BFC) were developed to image integrin α 2 β 1 expression with PET in a subcutaneous PC-3 xenograft model. The microPET images were reconstructed by a two-dimensional ordered subsets expectation maximum algorithm. The average radioactivity accumulation within a tumor or an organ was quantified from the multiple region of interest volumes. The PET tracer demonstrated prominent tumor uptake in the PC-3 xenograft (integrin α 2 β 1 -positive). The receptor specificity was confirmed in a blocking experiment. Moreover, the low tracer uptake in a CWR-22 tumor model (negative control) further confirmed the receptor specificity. The sarcophagine-conjugated DGEA peptide allows noninvasive imaging of tumor-associated α 2 β 1 expression, which may be a useful PET probe for evaluating the metastatic potential of prostate cancer. (orig.)

  2. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

    International Nuclear Information System (INIS)

    Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

    2016-01-01

    Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

  3. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen (Germany); Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany)

    2016-07-01

    Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

  4. Dual-acting of Hybrid Compounds - A New Dawn in the Discovery of Multi-target Drugs: Lead Generation Approaches.

    Science.gov (United States)

    Abdolmaleki, Azizeh; Ghasemi, Jahan B

    2017-01-01

    Finding high quality beginning compounds is a critical job at the start of the lead generation stage for multi-target drug discovery (MTDD). Designing hybrid compounds as selective multitarget chemical entity is a challenge, opportunity, and new idea to better act against specific multiple targets. One hybrid molecule is formed by two (or more) pharmacophore group's participation. So, these new compounds often exhibit two or more activities going about as multi-target drugs (mtdrugs) and may have superior safety or efficacy. Application of integrating a range of information and sophisticated new in silico, bioinformatics, structural biology, pharmacogenomics methods may be useful to discover/design, and synthesis of the new hybrid molecules. In this regard, many rational and screening approaches have followed by medicinal chemists for the lead generation in MTDD. Here, we review some popular lead generation approaches that have been used for designing multiple ligands (DMLs). This paper focuses on dual- acting chemical entities that incorporate a part of two drugs or bioactive compounds to compose hybrid molecules. Also, it presents some of key concepts and limitations/strengths of lead generation methods by comparing combination framework method with screening approaches. Besides, a number of examples to represent applications of hybrid molecules in the drug discovery are included. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform.

    Science.gov (United States)

    Gao, Wenhua; Zhang, An; Chen, Yunsheng; Chen, Zixuan; Chen, Yaowen; Lu, Fushen; Chen, Zhanguang

    2013-11-15

    Biosensor based on DNA hybridization holds great potential to get higher sensitivity as the optimal DNA hybridization efficiency can be achieved by controlling the distribution and orientation of probe strands on the transducer surface. In this work, an innovative strategy is reported to tap the sensitivity potential of current electrochemiluminescence (ECL) biosensing system by dispersedly anchoring the DNA beacons on the gold nanoparticles (GNPs) array which was electrodeposited on the glassy carbon electrode surface, rather than simply sprawling the coil-like strands onto planar gold surface. The strategy was developed by designing a "signal-on" ECL biosensing switch fabricated on the GNPs nanopatterned electrode surface for enhanced ultra-sensitivity detection of Hg(2+). A 57-mer hairpin-DNA labeled with ferrocene as ECL quencher and a 13-mer DNA labeled with Ru(bpy)3(2+) as reporter were hybridized to construct the signal generator in off-state. A 31-mer thymine (T)-rich capture-DNA was introduced to form T-T mismatches with the loop sequence of the hairpin-DNA in the presence of Hg(2+) and induce the stem-loop open, meanwhile the ECL "signal-on" was triggered. The peak sensitivity with the lowest detection limit of 0.1 nM was achieved with the optimal GNPs number density while exorbitant GNPs deposition resulted in sensitivity deterioration for the biosensor. We expect the present strategy could lead the renovation of the existing probe-immobilized ECL genosensor design to get an even higher sensitivity in ultralow level of target detection such as the identification of genetic diseases and disorders in basic research and clinical application. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Waves of visibility: probing the depth of inter-ocular suppression with transient and sustained targets

    Directory of Open Access Journals (Sweden)

    Lisandro eKaunitz

    2014-07-01

    Full Text Available In order to study non-conscious visual processing, researchers render otherwise consciously perceived images into invisible stimuli. Through the years, several psychophysical techniques have been developed for this purpose. Yet the comparison of experimental results across techniques remains a difficult task as the depth of suppression depends on the interactions between the type of stimuli and the suppression methods employed. This poses a limit to the inferences that researchers make about the extent of non-conscious processes. We investigated the mechanisms underlying inter-ocular suppression during continuous flash suppression (CFS and dichoptic visual masking using a transient onset target stimulus and a variety of stimulus / mask temporal manipulations. We show that target duration, timing of target onset, and mask frequency are key aspects of inter-ocular suppression during CFS with transient targets. The differences between our results and sustained target CFS studies suggest that two distinct mechanisms are involved in the detection of transient and prolonged target stimuli during CFS. Our results provide insight into the dynamics of CFS together with evidence for similarities between transient target CFS and dichoptic visual masking.

  7. Atomic Force Microscopy Probing of Receptor–Nanoparticle Interactions for Riboflavin Receptor Targeted Gold–Dendrimer Nanocomposites

    Science.gov (United States)

    2015-01-01

    Riboflavin receptors are overexpressed in malignant cells from certain human breast and prostate cancers, and they constitute a group of potential surface markers important for cancer targeted delivery of therapeutic agents and imaging molecules. Here we report on the fabrication and atomic force microscopy (AFM) characterization of a core–shell nanocomposite consisting of a gold nanoparticle (AuNP) coated with riboflavin receptor-targeting poly(amido amine) dendrimer. We designed this nanocomposite for potential applications such as a cancer targeted imaging material based on its surface plasmon resonance properties conferred by AuNP. We employed AFM as a technique for probing the binding interaction between the nanocomposite and riboflavin binding protein (RfBP) in solution. AFM enabled precise measurement of the AuNP height distribution before (13.5 nm) and after chemisorption of riboflavin-conjugated dendrimer (AuNP–dendrimer; 20.5 nm). Binding of RfBP to the AuNP–dendrimer caused a height increase to 26.7 nm, which decreased to 22.8 nm when coincubated with riboflavin as a competitive ligand, supporting interaction of AuNP–dendrimer and its target protein. In summary, physical determination of size distribution by AFM imaging can serve as a quantitative approach to monitor and characterize the nanoscale interaction between a dendrimer-covered AuNP and target protein molecules in vitro. PMID:24571134

  8. Whole-slide imaging is a robust alternative to traditional fluorescent microscopy for fluorescence in situ hybridization imaging using break-apart DNA probes.

    Science.gov (United States)

    Laurent, Camille; Guérin, Maxime; Frenois, François-Xavier; Thuries, Valérie; Jalabert, Laurence; Brousset, Pierre; Valmary-Degano, Séverine

    2013-08-01

    Fluorescence in situ hybridization is an indispensable technique used in routine pathology and for theranostic purposes. Because fluorescence in situ hybridization techniques require sophisticated microscopic workstations and long procedures of image acquisition with sometimes subjective and poorly reproducible results, we decided to test a whole-slide imaging system as an alternative approach. In this study, we used the latest generation of Pannoramic 250 Flash digital microscopes (P250 Flash digital microscopes; 3DHISTECH, Budapest, Hungary) to digitize fluorescence in situ hybridization slides of diffuse large B cells lymphoma cases for detecting MYC rearrangement. The P250 Flash digital microscope was found to be precise with better definition of split signals in cells containing MYC rearrangement with fewer truncated signals as compared to traditional fluorescence microscopy. This digital technique is easier thanks to the preview function, which allows almost immediate identification of the tumor area, and the panning and zooming functionalities as well as a shorter acquisition time. Moreover, fluorescence in situ hybridization analyses using the digital technique appeared to be more reproducible between pathologists. Finally, the digital technique also allowed prolonged conservation of photos. In conclusion, whole-slide imaging technologies represent rapid, robust, and highly sensitive methods for interpreting fluorescence in situ hybridization slides with break-apart probes. In addition, these techniques offer an easier way to interpret the signals and allow definitive storage of the images for pathology expert networks or e-learning databases. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Radioiodine labeled CdSe/CdS quantum dots. Lectin targeted dual probes

    Energy Technology Data Exchange (ETDEWEB)

    Akca, Ozlet; Unak, Perihan; Medine, E. Ilker; Kilcar, Ayfer Yurt; Ichedef, Cigdem [Ege Univ., Izmir (Turkey). Dept. of Nuclear Applications; Sakarya, Serhan [Adnan Menderes Univ., Aydin (Turkey). Dept. of Nuclear Medicine; Bekis, Recep [Dokuz Eyluel Univ., Izmir (Turkey). Dept. of Nuclear Medicine; Timur, Suna [Ege Univ., Izmir (Turkey). Biochemistry Dept.

    2014-11-01

    CdSe/CdS quantum dots (QD) were synthesized and bioconjugated with Sambucus nigra agglutinin (SNA) lectin (Lec). Mannose triflate and cysteamine molecules (MTC) were also utilized to prepare MTC-QDs and MTC-QDs-Lec probes as well as Lec bound QDs. Afterwards; potential use of these nanoparticles as radiolabeled fluorescence nano-probes for the cell imaging studies has been investigated. Biological activities of {sup 125}I{sup -}, {sup 125}I-MTC-QDs, MTC-QDs- Lec-{sup 125}I, QDs-Lec-{sup 125}I and Lec-{sup 125}I were examined on various cancer cell lines such as Caco-2, MCF-7 and A-549 in terms of cell incorporation. QDs-Lec-{sup 125}I exhibited the highest cell incorporation on whole cell lines. In addition, the QDs-Lec-{sup 131}I, was used for in vivo imaging. The whole body distribution of the radiolabeled QDs on New Zealand rabbits and Balb C mice were examined by taking dynamic and static images. Radioactivity cleared from the kidneys and the bladder, while significant amount radioactivity was retained in the heart and liver within 24 h.

  10. A new hybrid target concept for multi-keV X-ray sources

    International Nuclear Information System (INIS)

    Primout, M.; Babonneau, D.; Jacquet, L.; Villette, B.; Girard, F.; Brebion, D.; Stemmler, P.; Fournier, K.B.; Marrs, R.; May, M.J.; Heeter, R.F.; Wallace, R.J.; Nishimura, H.; Fujioka, S.; Tanabe, M.; Nagai, H.

    2013-01-01

    A novel concept for using hybrid targets to create multi-keV X-ray sources was tested on the GEKKO XII facility of the Osaka University and on the OMEGA facility of the University of Rochester. The sources were made via laser irradiation of a titanium foil placed at the end of a plastic cylinder, filled with a very low-density (2 and 5 mg/cm 3 ) silicon-dioxide aerogel that was designed to control the longitudinal expansion of the titanium plasma. Preliminary calculations were used to determine optimal conditions for the aerogel density, cylinder diameter and length that maximize multi-keV X-ray emission. The X-ray emission power was measured on OMEGA using absolutely calibrated broad-band, diode-based CEA diagnostics, in addition to high resolution crystal spectrometers. On GEKKO XII, the heat wave propagation velocity in the aerogel was also measured with an X-ray framing camera. The advantage of using the thermal wave generated in the aerogel to heat a solid material to increase the conversion efficiency has not been fully demonstrated in these experiments. However, it was shown that a 5 mg/cm 3 aerogel placed in front of a titanium foil can improve the x-ray conversion efficiency with respect to the case of 2 mg/cm 3 for some target diameter and length. (authors)

  11. Graphical representation of ribosomal RNA probe accessibility data using ARB software package

    Directory of Open Access Journals (Sweden)

    Amann Rudolf

    2005-03-01

    Full Text Available Abstract Background Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH, besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. Results The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool. Conclusion Using this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page http://www.arb-home.de.

  12. Analytical definition of fission chain reaction parameters for cylindrical uranium bar and energy release evaluations for HIF hybrid targets

    International Nuclear Information System (INIS)

    Imshennik, V.S.

    2006-01-01

    Within the conditions of Heavy-Ion Fusion (HIF) arises a possibility to obtain the fission chain reaction for a cylindrical HIF target. The paper contains the solution interpolated with the diffusion approximation in order to receive the general approximation expressions for the bar critical radius as well as for over-critical state. The obtained critical parameters generalized for uranium envelope are used for rough evaluation of the energy release in HIF hybrid targets [ru

  13. Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting Burkholderia Infections.

    Science.gov (United States)

    Capelli, Riccardo; Matterazzo, Elena; Amabili, Marco; Peri, Claudio; Gori, Alessandro; Gagni, Paola; Chiari, Marcella; Lertmemongkolchai, Ganjana; Cretich, Marina; Bolognesi, Martino; Colombo, Giorgio; Gourlay, Louise J

    2017-10-13

    Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 Å crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.

  14. Linear and nonlinear photophysics and bioimaging of an integrin-targeting water-soluble fluorenyl probe.

    Science.gov (United States)

    Morales, Alma R; Luchita, Gheorghe; Yanez, Ciceron O; Bondar, Mykhailo V; Przhonska, Olga V; Belfield, Kevin D

    2010-06-07

    Linear photophysical characterization and two-photon absorption (2PA) properties of a new water-soluble fluorene derivative, 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-bis{3-[2-(polyethyleneglycol-550-monomethylether-1-yl)]-4-(benzo[d]thiazol-2-yl)styryl}-9H-fluoren-9-yl)propanoic acid (1), were investigated in several organic solvents and water at room temperature. A comprehensive analysis of the steady-state absorption, emission and excitation anisotropy spectra revealed electronic structures of 1, including mutual orientation of the transition dipoles, relatively weak solvatochromic effects, high fluorescence quantum yield (approximately 0.5-1.0), and strong aggregation in water. The 2PA spectra of 1 were obtained in the 600-900 nm spectral range by two-photon induced fluorescence (2PF) and open aperture Z-scan methods using femtosecond laser sources. No discrete 2PA bands were apparent and values of the corresponding 2PA cross sections monotonically increased in the short wavelength range up to 3000 GM in organic solvents and approximately 6000 GM in aqueous solution, reflecting relatively high two-photon absorptivity. The 2PA efficiency of in water increased 2-3 times relative to aprotic solvents and can be explained by cooperative electronic effects of molecular aggregates of 1 produced in aqueous media. The carboxylic acid fluorenyl probe 1 was conjugated with the cyclic peptide RGDfK. Two-photon fluorescence microscopy (2PFM) imaging of U87MG cells (and MCF-7 as control), incubated with fluorene-RGD peptide conjugate 2, demonstrated high alpha(v)beta(3) integrin selectivity, making this probe particularly attractive for integrin imaging.

  15. Target Monitoring and Plasma Diagnosis using 2ω probe beam for CANDY

    International Nuclear Information System (INIS)

    Ishii, Katsuhiro; Mori, Yoshitaka; Hanayama, Ryohei; Nakayama, Suisei; Okihara, Shinichiro; Fujita, Kazuhisa; Nishimura, Yasuhiko; Sekina, Takashi; Sato, Nakahiro; Kurita, Takashi; Kawashima, Toshiyuki; Kan, Hirofumi; Komeda, Osamu; Nakamura, Naoki; Kondo, Takuya; Fujine, Manabu; Azuma, Hirozumi; Hioki, Tatsumi; Kakeno, Mitsutaka; Motohiro, Tomoyoshi

    2016-01-01

    We have developed the shadowgraph and interferometer with second-harmonic of heating pulses laser to observe target and plasma in highly-repetitive fusion reaction experiments. In the deuterated polystyrene ((C 8 D 8 )n double foil experiment, we confirm implosion plasma and plasma collision. In target injection experiment at a 1 Hz rate, we measure the position of the flying deuterated polystyrene beads at the moment of laser pulse illumination and observe the plasma generation by counter-illumination by 0.63 J, 800 nm, and 104 fs laser pulses. (paper)

  16. An electrochemical impedance biosensor for Hg2+ detection based on DNA hydrogel by coupling with DNAzyme-assisted target recycling and hybridization chain reaction.

    Science.gov (United States)

    Cai, Wei; Xie, Shunbi; Zhang, Jin; Tang, Dianyong; Tang, Ying

    2017-12-15

    In this work, an electrochemical impedance biosensor for high sensitive detection of Hg 2+ was presented by coupling with Hg 2+ -induced activation of Mg 2+ -specific DNAzyme (Mg 2+ -DNAzyme) for target cycling and hybridization chain reaction (HCR) assembled DNA hydrogel for signal amplification. Firstly, we synthesized two different copolymer chains P1 and P2 by modifying hairpin DNA H3 and H4 with acrylamide polymer, respectively. Subsequently, Hg 2+ was served as trigger to activate the Mg 2+ -DNAzyme for selectively cleavage ribonucleobase-modified substrate in the presence of Mg 2+ . The partial substrate strand could dissociate from DNAzyme structure, and hybridize with capture probe H1 to expose its concealed sequence for further hybridization. With the help of the exposed sequence, the HCR between hairpin DNA H3 and H4 in P1 and P2 was initiated, and assembled a layer of DNA cross-linked hydrogel on the electrode surface. The formed non-conductive DNA hydrogel film could greatly hinder the interfacial electronic transfer which provided a possibility for us to construct a high sensitive impedance biosensor for Hg 2+ detection. Under the optimal conditions, the impedance biosensor showed an excellent sensitivity and selectivity toward Hg 2+ in a concentration range of 0.1pM - 10nM with a detection limit of 0.042pM Moreover, the real sample analysis reveal that the proposed biosensor is capable of discriminating Hg 2+ ions in reliable and quantitative manners, indicating this method has a promising potential for preliminary application in routine tests. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, N.; Wienberg, J.; Ermert, K. [Universitaet Muenchen (Germany)] [and others

    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.

  18. Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

    Science.gov (United States)

    Oerther, D B; de los Reyes, F L; de los Reyes, M F; Raskin, L

    2001-10-01

    Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

  19. Probing the exotic structure of {sup 8}B by its elastic scattering and breakup reaction on nuclear targets

    Energy Technology Data Exchange (ETDEWEB)

    Lukyanov, V.K.; Zemlyanaya, E.V.; Lukyanov, K.V. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Kadrev, D.N.; Antonov, A.N.; Gaidarov, M.K. [Bulgarian Academy of Sciences, Institute for Nuclear Research and Nuclear Energy, Sofia (Bulgaria); Spasova, K. [Bulgarian Academy of Sciences, Institute for Nuclear Research and Nuclear Energy, Sofia (Bulgaria); University ' ' Ep. K. Preslavski' ' , Shumen (Bulgaria)

    2017-02-15

    The structure of the exotic {sup 8}B nucleus is studied by means of elastic scattering, as well as its breakup on nuclear targets. We present microscopic calculations of the optical potentials (OPs) and cross sections of elastic scattering of {sup 8}B on {sup 12}C, {sup 58}Ni, and {sup 208}Pb targets at energies 20 < E < 170 MeV. The density distributions of {sup 8}B obtained within the variational Monte Carlo (VMC) model and the three-cluster model (3CM) are used to construct the potentials. The real part of the hybrid OP is calculated using the folding model with the direct and exchange terms included, while the imaginary part is obtained on the base of the high-energy approximation (HEA) and also taken to be equal to the microscopic real part of the OP. In this model the only free parameters are the depths of the real and imaginary parts of OP obtained by fitting the elastic scattering experimental data. A dependence of their values on the model density of {sup 8} B is found. In addition, the cluster model, in which {sup 8}B consists of a p-halo and the {sup 7} Be core, is applied to calculate the breakup cross sections of the {sup 8}B nucleus on {sup 9}Be, {sup 12}C, and {sup 197}Au targets, as well as momentum distributions of {sup 7}Be fragments, and a comparison with the existing experimental data is made. (orig.)

  20. Development of TaqMan probes targeting the four major celiac disease epitopes found in α-gliadin sequences of spelt (Triticum aestivum ssp. spelta) and bread wheat (Triticum aestivum ssp. aestivum).

    Science.gov (United States)

    Dubois, Benjamin; Bertin, Pierre; Muhovski, Yordan; Escarnot, Emmanuelle; Mingeot, Dominique

    2017-01-01

    Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat ( Triticum aestivum ssp. aestivum ) and spelt ( T. aestivum ssp. spelta ). Among them, the α-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed α-gliadins of spelt and bread wheat. Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed α-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed α-gliadins. The results obtained in this study provide useful tools to study the immunogenic potential of expressed α-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the

  1. Molecular Imaging of Cancer Using X-ray Computed Tomography with Protease Targeted Iodinated Activity-Based Probes.

    Science.gov (United States)

    Gaikwad, Hanmant K; Tsvirkun, Darya; Ben-Nun, Yael; Merquiol, Emmanuelle; Popovtzer, Rachela; Blum, Galia

    2018-03-14

    X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT.

  2. 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

    International Nuclear Information System (INIS)

    Hua, Mengjuan; Wang, Chengquan; Qian, Jing; Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping; Wang, Kun

    2015-01-01

    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. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg 2+ detection. • The Hg 2+ content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg 2+ detection. • The ratiometric probe is of good simplicity, low toxicity, and excellent stability

  3. Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model

    Science.gov (United States)

    Li, Shen; Bradley, Philip

    2013-01-01

    When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily non-specific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules while treating the majority of the solvent implicitly. Comparing the performance of this model to its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein sidechain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems. PMID:23444044

  4. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end.......PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  5. Physical consequences of the mitochondrial targeting of single-walled carbon nanotubes probed computationally

    Science.gov (United States)

    Chistyakov, V. A.; Zolotukhin, P. V.; Prazdnova, E. V.; Alperovich, I.; Soldatov, A. V.

    2015-06-01

    Experiments by F. Zhou and coworkers (2010) [16] showed that mitochondria are the main target of the cellular accumulation of single-walled carbon nanotubes (SWCNTs). Our in silico experiments, based on geometrical optimization of the system consisting of SWCNT+proton within Density Functional Theory, revealed that protons can bind to the outer side of SWCNT so generating a positive charge. Calculation results allow one to propose the following mechanism of SWCNTs mitochondrial targeting. SWCNTs enter the space between inner and outer membranes of mitochondria, where the excess of protons has been formed by diffusion. In this compartment SWCNTs are loaded with protons and acquire positive charges distributed over their surface. Protonation of hydrophobic SWCNTs can also be carried out within the mitochondrial membrane through interaction with the protonated ubiquinone. Such "charge loaded" particles can be transferred as "Sculachev ions" through the inner membrane of the mitochondria due to the potential difference generated by the inner membrane. Physiological consequences of the described mechanism are discussed.

  6. Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

    Science.gov (United States)

    Tymoczko, Jakub; Schuhmann, Wolfgang; Gebala, Magdalena

    2014-12-24

    Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.

  7. Probe into rational target volume of nasopharyngeal carcinoma having been treated with conventional radiotherapy

    International Nuclear Information System (INIS)

    Zheng Yingjie; Zhao Chong; Lu Lixia; Wu Shaoxiong; Cui Nianji; Chen Fujin

    2006-01-01

    Objective: To analyze the local control rate and the dosimetric patterns of local recurrence in nasopharyngeal carcinoma (NPC) patients having been treated with standardized conventional radiotherapy and to evaluate the delineation of rational target volume. Methods: From Jan. 2000 to Dec. 2000, 476 patients with untreated NPC were treated by standardized conventional radiotherapy alone at the Sun Yat-sen University Cancer Center. The radiation ports were designed on a X-ray simulator. The nasopharyngeal lesion demonstrated by CT scan and the subclinical spread regions adjacent to the nasopharynx were defined as the target volume. Kaplan- Meier method was used to calculate the cumulative local recurrence rate. For patients with local recurrence, the primary and recurrent local tumor volumes(V nx , V recur ) were delineated with three-dimensional treatment planning system(3DTPS), and the dataset of radiation ports and delivered prescription dose to the 3DTPS were transferred according to the first treatment. The dose of radiation received by V recur was calculated and analyzed with dose- volume histogram(DVH). Local recurrence was classified as: 1. 'in-port' with 95% or more of the recurrence volume ( recur V 95 ) was within the 95% isodose; 2. 'marginal' with 20% to 95% of recur V 95 within the 95% isodose; 3. o utside w ith only less than 20% of recur V 95 within the 95% isodose curve. Results: With the median follow- up of 42.5 months (range 8-54 months), 52 patients developed local recurrence. The 1-, 2-, 3 and 4-year cumulative local failure rate was 0.6%, 3.9%, 8.7% and 11.5%, respectively. Among the 42 local recurrent patients who could be analyzed by 3DTPS, 52% were in-port, 40% were marginal and 7% were outside. For most of the marginal recurrence and all the outside recurrence patients, the main reason of recurrence were related to the unreasonable design of the radiation port and inaccuracy in the interpretation image findings. Conclusions: The outcome of

  8. In vivo type 2 cannabinoid receptor-targeted tumor optical imaging using a near infrared fluorescent probe.

    Science.gov (United States)

    Zhang, Shaojuan; Shao, Pin; Bai, Mingfeng

    2013-11-20

    The type 2 cannabinoid receptor (CB2R) plays a vital role in carcinogenesis and progression and is emerging as a therapeutic target for cancers. However, the exact role of CB2R in cancer progression and therapy remains unclear. This has driven the increasing efforts to study CB2R and cancers using molecular imaging tools. In addition, many types of cancers overexpress CB2R, and the expression levels of CB2R appear to be associated with tumor aggressiveness. Such upregulation of the receptor in cancer cells provides opportunities for CB2R-targeted imaging with high contrast and for therapy with low side effects. In the present study, we report the first in vivo tumor-targeted optical imaging using a novel CB2R-targeted near-infrared probe. In vitro cell fluorescent imaging and a competitive binding assay indicated specific binding of NIR760-mbc94 to CB2R in CB2-mid delayed brain tumor (DBT) cells. NIR760-mbc94 also preferentially labeled CB2-mid DBT tumors in vivo, with a 3.7-fold tumor-to-normal contrast enhancement at 72 h postinjection, whereas the fluorescence signal from the tumors of the mice treated with NIR760 free dye was nearly at the background level at the same time point. SR144528, a CB2R competitor, significantly inhibited tumor uptake of NIR760-mbc94, indicating that NIR760-mbc94 binds to CB2R specifically. In summary, NIR760-mbc94 specifically binds to CB2R in vitro and in vivo and appears to be a promising molecular tool that may have great potential for use in diagnostic imaging of CB2R-positive cancers and therapeutic monitoring as well as in elucidating the role of CB2R in cancer progression and therapy.

  9. Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

    Directory of Open Access Journals (Sweden)

    Kazuya Shiogama

    2013-01-01

    Full Text Available Background. In situ hybridization (ISH with high sensitivity has been requested to demonstrate hepatitis C virus (HCV RNA in formalin-fixed, paraffin-embedded (FFPE sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82% of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73% of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions. HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

  10. Gene probes : principles and protocols [Methods in molecular biology, v. 179

    National Research Council Canada - National Science Library

    Rapley, Ralph; Aquino de Muro, Marilena

    2002-01-01

    ... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

  11. Detection of Misdistribution of Tyrosinase from Melanosomes to Lysosomes and Its Upregulation under Psoralen/Ultraviolet A with a Melanosome-Targeting Tyrosinase Fluorescent Probe.

    Science.gov (United States)

    Zhou, Jin; Shi, Wen; Li, Lihong; Gong, Qiuyu; Wu, Xiaofeng; Li, Xiaohua; Ma, Huimin

    2016-04-19

    Tyrosinase is regarded as an important biomarker of melanoma cancer, and its metabolism is closely related to some severe skin diseases such as vitiligo. Since tyrosinase is mainly located in the melanosomes of melanocytes, a probe that can specifically detect and image tysosinase in melanosomes would be in urgent demand to study the behavior of the enzyme in cells, but unfortunately, no melanosome-targeting tyrosinase fluorescent probe has been reported so far to the best of our knowledge. In this work, we have developed such a new probe, Mela-TYR, which bears morpholine as a melanosome-targeting group and 4-aminophenol as a tyrosinase reaction group. The probe exhibits not only a highly sensitive and selective off-on response to tyrosinase via oxidization cleavage, but also an accurate targeting ability toward the acidic organelles of melanosomes and lyososomes, which is validated by colocalization experiments with mCherry-tagged melanosomes as well as DND-99 (a commercial dye). The probe has been used to image the relative contents of tyrosinase in different cells. Notably, because of the tyrosinase deficiency in normal lysosomes, the probe only fluoresces in melanosomes in principle although it can accumulate in other acidic organelles like lysosomes. By virtue of this property, the misdistribution of tyrosinase from melanosomes to lysosomes in murine melanoma B16 cells under the stimulation of inulavosin is imaged in real time for the first time. Moreover, the upregulation of melanosomal tyrosinase in live B16 cells under the stimulation of psoralen/ultraviolet A is detected with our probe, and this upregulation is further verified by standard colorimetric assay. The probe provides a simple, visual method to study the metabolism of tyrosinase in cells and shows great potential in clinical diagnosis and treatments of tyrosinase-associated diseases.

  12. 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)

    Science.gov (United States)

    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

  13. The Interaction of the Solar Wind with Solar Probe Plus - 3D Hybrid Simulation. Report 1; The Study for the Distance 4.5Rs

    Science.gov (United States)

    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.

  14. A lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells based on a 1,8-naphthalimide derivative

    Science.gov (United States)

    Liang, Beibei; Wang, Baiyan; Ma, Qiujuan; Xie, Caixia; Li, Xian; Wang, Suiping

    2018-03-01

    Biological thiols, like cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in biological systems and in lysosomal processes. Highly selective probes for detecting biological thiols in lysomes of living cells are rare. In this work, a lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells was designed and synthesized based on a 1,8-naphthalimide derivative. The probe has a 4-(2-aminoethyl)morpholine unit as a lysosome-targetable group and an acrylate group as the thiol recognition unit as well as a fluorescence quencher. In the absence of biothiols, the probe displayed weak fluorescence due to the photoinduced electron transfer (PET) process. Upon the addition of biothiols, the probe exhibited an enhanced fluorescence emission centered at 550 nm due to cleavage of the acrylate moiety. The probe had high selectivity toward biothiols. Moreover, the probe features fast response time, excitation in the visible region and ability of working in a wide pH range. The linear response range covers a concentration range of Cys from 1.5 × 10- 7 to 1.0 × 10- 5 mol·L- 1 and the detection limit is 6.9 × 10- 8 mol·L- 1 for Cys. The probe has been successfully applied to the confocal imaging of biothiols in lysosomes of A549 cells with low cell toxicity. Furthermore, the method was successfully applied to the determination of thiols in a complex multicomponent mixture such as human serum, which suggests our proposed method has great potential for diagnostic purposes. All of such good properties prove it can be used to monitor biothiols in lysosomes of living cells and to be a good fluorescent probe for the selective detection of thiols.

  15. Probing the onset of laser-induced relativistic transparency in massive targets

    Science.gov (United States)

    Wang, Tao; Wagner, Craig; Toncian, Toma; Dyer, Gilliss; Arefiev, Alexey; Ditmire, Todd

    2017-10-01

    We have investigated a novel approach of using harmonics of the laser frequency generated in the plasma to detect the onset of relativistic transparency induced by an intense laser pulse. The onset of the transparency is directly associated with a forward motion of a relativistically adjusted critical surface. The corresponding velocity is relativistic, so the harmonics generated at this critical surface are noticeably shifted. Using particle-in-cell simulations, we have confirmed that the resulting shift greatly exceeds the shift produced during a hole-boring process when the relativistic transparency plays no role, which allows us to clearly identify the onset of the relativistic transparency. Experiments that we have carried out at the Texas Petawatt laser showcase this approach. The 3rd harmonic signal detected in experiments with massive targets irradiated at laser intensities around 1020 W/cm2 has a pronounced shift associated with the relativistic transparency. The shift represents a recession of the relativistically adjusted critical surface with a velocity close to 0.2 c. This approach opens a new possibility of detecting changes in the optical properties of matter induced by intense laser pulses even when no transmission of the laser pulse takes place. This research was supported part by NSF (Grant No. 1632777) and NNSA (Cont. No. DE-NA0002008). Simulations were performed using HPC resources at TACC at the University of Texas.

  16. A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes☆

    Science.gov (United States)

    Pun, Pamela Boon Li; Logan, Angela; Darley-Usmar, Victor; Chacko, Balu; Johnson, Michelle S.; Huang, Guang W.; Rogatti, Sebastian; Prime, Tracy A.; Methner, Carmen; Krieg, Thomas; Fearnley, Ian M.; Larsen, Lesley; Larsen, David S.; Menger, Katja E.; Collins, Yvonne; James, Andrew M.; Kumar, G.D. Kishore; Hartley, Richard C.; Smith, Robin A.J.; Murphy, Michael P.

    2014-01-01

    The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging. PMID:24316194

  17. A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes.

    Science.gov (United States)

    Pun, Pamela Boon Li; Logan, Angela; Darley-Usmar, Victor; Chacko, Balu; Johnson, Michelle S; Huang, Guang W; Rogatti, Sebastian; Prime, Tracy A; Methner, Carmen; Krieg, Thomas; Fearnley, Ian M; Larsen, Lesley; Larsen, David S; Menger, Katja E; Collins, Yvonne; James, Andrew M; Kumar, G D Kishore; Hartley, Richard C; Smith, Robin A J; Murphy, Michael P

    2014-02-01

    The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles

    DEFF Research Database (Denmark)

    Ragelle, Héloïse; Colombo, Stefano; Pourcelle, Vincent

    2015-01-01

    chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse...... that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards...

  19. Structural Probing of Off-Target G Protein-Coupled Receptor Activities within a Series of Adenosine/Adenine Congeners

    Science.gov (United States)

    Paoletta, Silvia; Tosh, Dilip K.; Salvemini, Daniela; Jacobson, Kenneth A.

    2014-01-01

    We studied patterns of off-target receptor interactions, mostly at G protein-coupled receptors (GPCRs) in the µM range, of nucleoside derivatives that are highly engineered for nM interaction with adenosine receptors (ARs). Because of the considerable interest of using AR ligands for treating diseases of the CNS, we used the Psychoactive Drug Screening Program (PDSP) for probing promiscuity of these adenosine/adenine congeners at 41 diverse receptors, channels and a transporter. The step-wise truncation of rigidified, trisubstituted (at N6, C2, and 5′ positions) nucleosides revealed unanticipated interactions mainly with biogenic amine receptors, such as adrenergic receptors and serotonergic receptors, with affinities as high as 61 nM. The unmasking of consistent sets of structure activity relationship (SAR) at novel sites suggested similarities between receptor families in molecular recognition. Extensive molecular modeling of the GPCRs affected suggested binding modes of the ligands that supported the patterns of SAR at individual receptors. In some cases, the ligand docking mode closely resembled AR binding and in other cases the ligand assumed different orientations. The recognition patterns for different GPCRs were clustered according to which substituent groups were tolerated and explained in light of the complementarity with the receptor binding site. Thus, some likely off-target interactions, a concern for secondary drug effects, can be predicted for analogues of this set of substructures, aiding the design of additional structural analogues that either eliminate or accentuate certain off-target activities. Moreover, similar analyses could be performed for unrelated structural families for other GPCRs. PMID:24859150

  20. Interfacial micropore defect formation in PEDOT:PSS-Si hybrid solar cells probed by TOF-SIMS 3D chemical imaging.

    Science.gov (United States)

    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.

  1. Allele specific hybridization using oligonucleotide probes of very high specific activity: Discrimination of the human β/sup A/ and β/sup S/-globin genes

    International Nuclear Information System (INIS)

    Studencki, A.B.; Wallace, R.B.

    1984-01-01

    The repair activity of E. coli DNA polymerase I (Klenow fragment) was used to prepare nonadecanucleotide hybridization probes which were complementary either to the normal human β-globin (β/sup A/) or to the sickle cell human β-globin (β/sup S/) gene. Template directed polymerization of highly radiolabeled α-/sup 32/P-deoxyribonucleoside triphosphates (3200, 5000 and/or 7800 Ci/mmol) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 - 2.0 x 10/sup 10/ dpm/μg. The extremely high specific activities of these probes made it possible to detect the β/sup A/ and β/sup S/ single copy gene sequences in as little as 1 μg of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states. This means that it was possible to detect 0.5 - 1.0 x 10/sup -18/ moles of a given single copy sequence

  2. Fast reciprocating probe system for local scrape-off layer measurements in front of the lower hybrid launcher on JT-60U

    International Nuclear Information System (INIS)

    Asakura, N.; Tsuji-Iio, S.; Ikeda, Y.; Neyatani, Y.; Seki, M.

    1995-01-01

    A fast reciprocating probe system with a long drive shaft was incorporated into a multi-junction lower hybrid (LH) wave launcher on JT-60U in order to investigate an improved coupling mechanism of the radio frequency wave to the core plasma. The system has been operated reliably over a horizontal scan of 25 cm in 1.5 s using a compact pneumatic cylinder drive and springs. A double probe measurement provided the scrape-off layer plasma profile between the last closed flux surface and the first wall with the spatial resolution of 1-2 mm measured with a laser displacement gauge. The profiles of the electron density n e and temperature T e were in good agreement with those obtained with a triple probe method. During the LH wave injection with good coupling to the core plasma, an increase in the local T e was observed in front of the LH launcher mouth. The local n e was (7-10)x10 16 m -3 , consistent values needed for the good coupling. copyright 1995 American Institute of Physics

  3. A novel mitochondria-targeted two-photon fluorescent probe for dynamic and reversible detection of the redox cycles between peroxynitrite and glutathione.

    Science.gov (United States)

    Sun, Chunlong; Du, Wen; Wang, Peng; Wu, Yang; Wang, Baoqin; Wang, Jun; Xie, Wenjun

    2017-12-16

    Redox homeostasis is important for maintenance of normal physiological functions within cells. Redox state of cells is primarily a consequence of precise balance between levels of reducing equivalents and reactive oxygen species. Redox homeostasis between peroxynitrite (ONOO - ) and glutathione (GSH) is closely associated with physiological and pathological processes, such as prolonged relaxation in vascular tissues and smooth muscle preparations, attenuation of hepatic necrosis, and activation of matrix metalloproteinase-2. We report a two-photon fluorescent probe (TP-Se) based on water-soluble carbazole-based compound, which integrates with organic selenium, to monitor changes in ONOO - /GSH levels in cells. This probe can reversibly respond to ONOO - and GSH and exhibits high selectivity, sensitivity, and mitochondrial targeting. The probe was successfully applied to visualize changes in redox cycles during ONOO - outbreak and antioxidant GSH repair in cells. The probe will lead to significant development on redox events involved in cellular redox regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Improving comparability between microarray probe signals by thermodynamic intensity correction

    DEFF Research Database (Denmark)

    Bruun, G. M.; Wernersson, Rasmus; Juncker, Agnieszka

    2007-01-01

    different probes. It is therefore of great interest to correct for the variation between probes. Much of this variation is sequence dependent. We demonstrate that a thermodynamic model for hybridization of either DNA or RNA to a DNA microarray, which takes the sequence-dependent probe affinities...... determination of transcription start sites for a subset of yeast genes. In another application, we identify present/absent calls for probes hybridized to the sequenced Escherichia coli strain O157:H7 EDL933. The model improves the correct calls from 85 to 95% relative to raw intensity measures. The model thus...... makes applications which depend on comparisons between probes aimed at different sections of the same target more reliable....

  5. Differential detection of type II methanotrophic bacteria in acidic peatlands using newly developed 16S rRNA-targeted fluorescent oligonucleotide probes.

    Science.gov (United States)

    Dedysh, Svetlana N; Dunfield, Peter F; Derakshani, Manigee; Stubner, Stephan; Heyer, Jürgen; Liesack, Werner

    2003-04-01

    Abstract Based on an extensive 16S rRNA sequence database for type II methanotrophic bacteria, a set of 16S rRNA-targeted oligonucleotide probes was developed for differential detection of specific phylogenetic groups of these bacteria by fluorescence in situ hybridisation (FISH). This set of oligonucleotides included a genus-specific probe for Methylocystis (Mcyst-1432) and three species-specific probes for Methylosinus sporium (Msins-647), Methylosinus trichosporium (Msint-1268) and the recently described acidophilic methanotroph Methylocapsa acidiphila (Mcaps-1032). These novel probes were applied to further characterise the type II methanotroph community that was detected in an acidic Sphagnum peat from West Siberia in a previous study (Dedysh et al. (2001) Appl. Environ. Microbiol. 67, 4850-4857). The largest detectable population of indigenous methanotrophs simultaneously hybridised with a group-specific probe targeting all currently known Methylosinus/Methylocystis spp. (M-450), with a genus-specific probe for Methylocystis spp. (Mcyst-1432), and with an additional probe (Mcyst-1261) that had been designed to target a defined phylogenetic subgroup of Methylocystis spp. The same subgroup of Methylocystis was also detected in acidic peat sampled from Sphagnum-dominated wetland in northern Germany. The population size of this peat-inhabiting Methylocystis subgroup was 2.0+/-0.1x10(6) cells g(-1) (wet weight) of peat from Siberia and 5.5+/-0.5x10(6) cells g(-1) of peat from northern Germany. This represented 60 and 95%, respectively, of the total number of methanotroph cells detected by FISH in these two wetland sites. Other major methanotroph populations were M. acidiphila and Methylocella palustris. Type I methanotrophs accounted for not more than 1% of total methanotroph cells. Neither M. trichosporium nor M. sporium were detected in acidic Sphagnum peat.

  6. Electrokinetic acceleration of DNA hybridization in microsystems.

    Science.gov (United States)

    Lei, Kin Fong; Wang, Yun-Hsiang; Chen, Huai-Yi; Sun, Jia-Hong; Cheng, Ji-Yen

    2015-06-01

    In this work, electrokinetic acceleration of DNA hybridization was investigated by different combinations of frequencies and amplitudes of actuating electric signals. Because the frequencies from low to high can induce different kinds of electrokinetic forces, i.e., electroosmotic to electrothermal forces, this work provides an in-depth investigation of electrokinetic enhanced hybridization. Concentric circular Cr/Au microelectrodes of 350 µm in diameter were fabricated on a glass substrate and probe DNA was immobilized on the electrode surface. Target DNA labeled with fluorescent dyes suspending in solution was then applied to the electrode. Different electrokinetic forces were induced by the application of different electric signals to the circular microelectrodes. Local microfluidic vortexes were generated to increase the collision efficiency between the target DNA suspending in solution and probe DNA immobilized on the electrode surface. DNA hybridization on the electrode surface could be accelerated by the electrokinetic forces. The level of hybridization was represented by the fluorescent signal intensity ratio. Results revealed that such 5-min dynamic hybridization increased 4.5 fold of signal intensity ratio as compared to a 1-h static hybridization. Moreover, dynamic hybridization was found to have better differentiation ability between specific and non-specific target DNA. This study provides a strategy to accelerate DNA hybridization in microsystems. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Implementation and verification of a four-probe motion error measurement system for a large-scale roll lathe used in hybrid manufacturing

    International Nuclear Information System (INIS)

    Chen, Yuan-Liu; Niu, Zengyuan; Matsuura, Daiki; Lee, Jung Chul; Shimizu, Yuki; Gao, Wei; Oh, Jeong Seok; Park, Chun Hong

    2017-01-01

    In this paper, a four-probe measurement system is implemented and verified for the carriage slide motion error measurement of a large-scale roll lathe used in hybrid manufacturing where a laser machining probe and a diamond cutting tool are placed on two sides of a roll workpiece for manufacturing. The motion error of the carriage slide of the roll lathe is composed of two straightness motion error components and two parallelism motion error components in the vertical and horizontal planes. Four displacement measurement probes, which are mounted on the carriage slide with respect to four opposing sides of the roll workpiece, are employed for the measurement. Firstly, based on the reversal technique, the four probes are moved by the carriage slide to scan the roll workpiece before and after a 180-degree rotation of the roll workpiece. Taking into consideration the fact that the machining accuracy of the lathe is influenced by not only the carriage slide motion error but also the gravity deformation of the large-scale roll workpiece due to its heavy weight, the vertical motion error is thus characterized relating to the deformed axis of the roll workpiece. The horizontal straightness motion error can also be synchronously obtained based on the reversal technique. In addition, based on an error separation algorithm, the vertical and horizontal parallelism motion error components are identified by scanning the rotating roll workpiece at the start and the end positions of the carriage slide, respectively. The feasibility and reliability of the proposed motion error measurement system are demonstrated by the experimental results and the measurement uncertainty analysis. (paper)

  8. Implementation and verification of a four-probe motion error measurement system for a large-scale roll lathe used in hybrid manufacturing

    Science.gov (United States)

    Chen, Yuan-Liu; Niu, Zengyuan; Matsuura, Daiki; Lee, Jung Chul; Shimizu, Yuki; Gao, Wei; Oh, Jeong Seok; Park, Chun Hong

    2017-10-01

    In this paper, a four-probe measurement system is implemented and verified for the carriage slide motion error measurement of a large-scale roll lathe used in hybrid manufacturing where a laser machining probe and a diamond cutting tool are placed on two sides of a roll workpiece for manufacturing. The motion error of the carriage slide of the roll lathe is composed of two straightness motion error components and two parallelism motion error components in the vertical and horizontal planes. Four displacement measurement probes, which are mounted on the carriage slide with respect to four opposing sides of the roll workpiece, are employed for the measurement. Firstly, based on the reversal technique, the four probes are moved by the carriage slide to scan the roll workpiece before and after a 180-degree rotation of the roll workpiece. Taking into consideration the fact that the machining accuracy of the lathe is influenced by not only the carriage slide motion error but also the gravity deformation of the large-scale roll workpiece due to its heavy weight, the vertical motion error is thus characterized relating to the deformed axis of the roll workpiece. The horizontal straightness motion error can also be synchronously obtained based on the reversal technique. In addition, based on an error separation algorithm, the vertical and horizontal parallelism motion error components are identified by scanning the rotating roll workpiece at the start and the end positions of the carriage slide, respectively. The feasibility and reliability of the proposed motion error measurement system are demonstrated by the experimental results and the measurement uncertainty analysis.

  9. Potential actionable targets in appendiceal cancer detected by immunohistochemistry, fluorescent in situ hybridization, and mutational analysis

    Science.gov (United States)

    Millis, Sherri Z.; Kimbrough, Jeffery; Doll, Nancy; Von Hoff, Daniel; Ramanathan, Ramesh K.

    2017-01-01

    Background Appendiceal cancers are rare and consist of carcinoid, mucocele, pseudomyxoma peritonei (PMP), goblet cell carcinoma, lymphoma, and adenocarcinoma histologies. Current treatment involves surgical resection or debulking, but no standard exists for adjuvant chemotherapy or treatment for metastatic disease. Methods Samples were identified from approximately 60,000 global tumors analyzed at a referral molecular profiling CLIA-certified laboratory. A total of 588 samples with appendix primary tumor sites were identified (male/female ratio of 2:3; mean age =55). Sixty-two percent of samples were adenocarcinomas (used for analysis); the rest consisted of 9% goblet cell, 15% mucinous; 6% pseudomyxoma, and less than 5% carcinoids and 2% neuroendocrine. Tests included sequencing [Sanger, next generation sequencing (NGS)], protein expression/immunohistochemistry (IHC), and gene amplification [fluorescent in situ hybridization (FISH) or CISH]. Results Profiling across all appendiceal cancer histological subtypes for IHC revealed: 97% BRCP, 81% MRP1, 81% COX-2, 71% MGMT, 56% TOPO1, 5% PTEN, 52% EGFR, 40% ERCC1, 38% SPARC, 35% PDGFR, 35% TOPO2A, 25% RRM1, 21% TS, 16% cKIT, and 12% for TLE3. NGS revealed mutations in the following genes: 50.4% KRAS, 21.9% P53, 17.6% GNAS, 16.5% SMAD4, 10% APC, 7.5% ATM, 5.5% PIK3CA, 5.0% FBXW7, and 1.8% BRAF. Conclusions Appendiceal cancers show considerable heterogeneity with high levels of drug resistance proteins (BCRP and MRP1), which highlight the difficulty in treating these tumors and suggest an individualized approach to treatment. The incidence of low TS (79%) could be used as a backbone of therapy (using inhibitors such as 5FU/capecitabine or newer agents). Therapeutic options includeTOPO1 inhibitors (irinotecan/topotecan), EGFR inhibitors (erlotinib, cetuximab), PDGFR antagonists (regorafenib, axitinib), MGMT (temozolomide). Clinical trials targeting pathways involving KRAS, p53, GNAS, SMAD4, APC, ATM, PIK3CA, FBXW7, and

  10. Clearance and Biodistribution of Liposomally Encapsulated Nitroxides: A Model for Targeted Delivery of Electron Paramagnetic Resonance Imaging Probes to Tumors

    Science.gov (United States)

    Burks, Scott R.; Legenzov, Eric A.; Rosen, Gerald M.

    2011-01-01

    Electron paramagnetic resonance (EPR) imaging using nitroxides as molecular probes is potentially a powerful tool for the detection and physiological characterization of micrometastatic lesions. Encapsulating nitroxides in anti-HER2 immunoliposomes at high concentrations to take advantage of the “self-quenching” phenomenon of nitroxides allows generation of robust EPR signals in HER2-overexpressing breast tumor cells with minimal background from indifferent tissues or circulating liposomes. We investigated the in vivo pharmacological properties of nitroxides encapsulated in sterically stabilized liposomes designed for long circulation times. We show that circulation times of nitroxides can be extended from hours to days; this increases the proportion of liposomes in circulation to enhance tumor targeting. Furthermore, nitroxides encapsulated in sterically stabilized anti-HER2 immunoliposomes can be delivered to HER2-overexpressing tumors at micromolar concentrations, which should be imageable by EPR. Lastly, after in vivo administration, liposomally encapsulated nitroxide signal also appears in the liver, spleen, and kidneys. Although these organs are spatially distinct and would not hinder tumor imaging in our model, understanding nitroxide signal retention in these organs is essential for further improvements in EPR imaging contrast between tumors and other tissues. These results lay the foundation to use liposomally delivered nitroxides and EPR imaging to visualize tumor cells in vivo. PMID:21737567

  11. Fluorescence-Guided Probes of Aptamer-Targeted Gold Nanoparticles with Computed Tomography Imaging Accesses for in Vivo Tumor Resection.

    Science.gov (United States)

    Li, Cheng-Hung; Kuo, Tsung-Rong; Su, Hsin-Jan; Lai, Wei-Yun; Yang, Pan-Chyr; Chen, Jinn-Shiun; Wang, Di-Yan; Wu, Yi-Chun; Chen, Chia-Chun

    2015-10-28

    Recent development of molecular imaging probes for fluorescence-guided surgery has shown great progresses for determining tumor margin to execute the tissue resection. Here we synthesize the fluorescent gold nanoparticles conjugated with diatrizoic acid and nucleolin-targeted AS1411 aptamer. The nanoparticle conjugates exhibit high water-solubility, good biocompatibility, visible fluorescence and strong X-ray attenuation for computed tomography (CT) contrast enhancement. The fluorescent nanoparticle conjugates are applied as a molecular contrast agent to reveal the tumor location in CL1-5 tumor-bearing mice by CT imaging. Furthermore, the orange-red fluorescence emitting from the conjugates in the CL1-5 tumor can be easily visualized by the naked eyes. After the resection, the IVIS measurements show that the fluorescence signal of the nanoparticle conjugates in the tumor is greatly enhanced in comparison to that in the controlled experiment. Our work has shown potential application of functionalized nanoparticles as a dual-function imaging agent in clinical fluorescence-guided surgery.

  12. Detection of supercoiled hepatitis B virus DNA and related forms by means of molecular hybridization to an oligonucleotide probe

    International Nuclear Information System (INIS)

    Lin, H.J.; Chung, H.T.; Lai, C.L.; Leong, S.; Tam, O.S.

    1989-01-01

    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

  13. mathFISH, a Web Tool That Uses Thermodynamics-Based Mathematical Models for In Silico Evaluation of Oligonucleotide Probes for Fluorescence In Situ Hybridization▿ †

    OpenAIRE

    Yilmaz, L. Safak; Parnerkar, Shreyas; Noguera, Daniel R.

    2010-01-01

    Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

  14. 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

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Mengjuan [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Chengquan [School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013 (China); Qian, Jing, E-mail: qianj@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kun, E-mail: wangkun@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China)

    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{sup 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{sup 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{sup 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{sup 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{sup 2+} quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg{sup 2+} detection. • The Hg{sup 2+} content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg{sup 2+} detection. • The ratiometric probe is of good simplicity, low toxicity, and

  15. Targeted and untargeted high resolution mass approach for a putative profiling of glycosylated simple phenols in hybrid grapes.

    Science.gov (United States)

    Barnaba, Chiara; Dellacassa, Eduardo; Nicolini, Giorgio; Giacomelli, Mattia; Roman Villegas, Tomas; Nardin, Tiziana; Larcher, Roberto

    2017-08-01

    Vitis vinifera is one of the most widespread grapevines around the world representing the raw material for high quality wine production. The availability of more resistant interspecific hybrid vine varieties, developed from crosses between Vitis vinifera and other Vitis species, has generated much interest, also due to the low environmental effect of production. However, hybrid grape wine composition and varietal differences between interspecific hybrids have not been well defined, particularly for the simple phenols profile. The dynamic of these phenols in wines, where the glycosylated forms can be transformed into the free ones during winemaking, also raises an increasing health interest by their role as antoxidants in wine consumers. In this work an on-line SPE clean-up device, to reduce matrix interference, was combined with ultra-high liquid chromatography-high resolution mass spectrometry in order to increase understanding of the phenolic composition of hybrid grape varieties. Specifically, the phenolic composition of 4 hybrid grape varieties (red, Cabernet Cantor and Prior; white, Muscaris and Solaris) and 2 European grape varieties (red, Merlot; white, Chardonnay) was investigated, focusing on free and glycosidically bound simple phenols and considering compound distribution in pulp, skin, seeds and wine. Using a targeted approach 53 free simple phenols and 7 glycosidic precursors were quantified with quantification limits ranging from 0.001 to 2mgKg -1 and calibration R 2 of 0.99 for over 86% of compounds. The untargeted approach made it possible to tentatively identify 79 glycosylated precursors of selected free simple phenols in the form of -hexoside (N=30), -pentoside (21), -hexoside-hexoside (17), -hexoside-pentoside (4), -pentoside-hexoside (5) and -pentoside-pentoside (2) derivatives on the basis of accurate mass, isotopic pattern and MS/MS fragmentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. High-resolution analysis of 16q22.1 in breast carcinoma using DNA amplifiable probes (multiplex amplifiable probe hybridization technique) and immunohistochemistry.

    Science.gov (United States)

    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

  17. Insulinoma imaging with glucagon-like peptide-1 receptor targeting probe (18)F-FBEM-Cys (39)-exendin-4.

    Science.gov (United States)

    Xu, Yuping; Pan, Donghui; Xu, Qing; Zhu, Chen; Wang, Lizhen; Chen, Fei; Yang, Runlin; Luo, Shineng; Yang, Min

    2014-09-01

    Glucagon-like peptide-1 receptor (GLP-1R) is a specific target for insulinomas imaging since it is overexpressed in the tumor. Exendin-4 exhibits high affinity for the GLP-1R. In this study, a novel (18)F-labeled exendin-4 analog, (18)F-FBEM-Cys(39)-exendin-4, was synthesized and its potentials for GLP-1R imaging were also evaluated. (18)F-FBEM was synthesized by coupling (18)F-fluorobenzoic acid ((18)F-FBA) with N-(2-aminoethyl) maleimide, and the reaction conditions were optimized. Cys(39)-exendin-4 was then conjugated with (18)F-FBEM to obtain (18)F-FBEM-Cys(39)-exendin-4. The GLP-1R targeting potential and pharmacokinetic profile of the tracer were analyzed in INS-1 insulinoma and MDA-MB-435 breast tumor model, respectively. Under the optimal conditions, the yield of radiolabeled (18)F-FBEM was 49.1 ± 2.0 % (based on (18)F-FBA, non-decay corrected). The yield of (18)F-FBEM-Cys(39)-exendin-4 was 35.1 ± 2.6 % (based on the starting (18)F-FBEM, non-decay corrected). The radiochemical purity of (18)F-FBEM-Cys(39)-exendin-4 is >95 %, and the specific activity was at least 35 GBq/μmol. The GLP-1R-positive INS-1 insulinoma xenograft was clearly visible with good contrast to background, whereas GLP-1R-negative MDA-MB435 breast tumor was barely visible. Low levels of radioactivity were also detected at pancreas and lungs due to few GLP-1R expressions. GLP-1R binding specificity was demonstrated by reduced INS-1 tumor uptake of the tracer after coinjection with an excess of unlabeled Cys(39)-exendin-4 at 1 h postinjection. The thiol-reactive reagent, (18)F-FBEM, was prepared with high yield and successfully conjugated to Cys(39)-exendin-4. Favorable preclinical data showing specific and effective tumor targeting by (18)F-FBEM-Cys(39)-exendin-4 suggest that the tracer may be a potential probe for insulinomas imaging.

  18. Sixty-five radiation hybrids for the short arm of human chromosome 6: their value as a mapping panel and as a source for rapid isolation of new probes using repeat element-mediated PCR

    International Nuclear Information System (INIS)

    Zoghbi, H.Y.; McCall, A.E.; LeBorgne-Demarquoy, F.

    1991-01-01

    We have used an irradiation and fusion procedure to generate somatic cell hybrids that retain fragments of the short arm of human chromosome 6 (6p). To identify hybrids retaining human material, we performed repeat element-mediated PCR on crude lysates of cells from individual clones. Sixty-five hybrids were shown to contain human material and fifty of those contained one or more 6p-specific probes. Detailed characterization of these hybrids identified a subset that divides 6p into ten mapping intervals. Using repeat element-mediated PCR, we were able to isolate and map 61 new DNA fragments from specific regions of 6p. Fifteen of these fragments were used to screen for restriction fragment length polymorphisms (RFLPs), and nine identified RFLPs with one or more enzymes. The radiation hybrids described in this study provide a valuable resource for high-resolution mapping of 6p and for the rapid isolation of region-specific markers

  19. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi; PCR associado a hibridizacao com sondas radioativas de DNA para a identificacao de infeccao subclinica causada por Leishmania Chagasi

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Moreno, Elizabeth Castro [Fundacao Nacional de Saude, Belo Horizonte, MG (Brazil). Coordenacao Regional de Minas Gerais; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Fernandes, Octavio [Fundacao Inst. Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical

    2002-07-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with {sup 32} P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with {sup 32} P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  20. Short Wavelength Electromagnetic Perturbations Excited Near the Solar Probe Plus Spacecraft in the Inner Heliosphere: 2.5D Hybrid Modeling

    Science.gov (United States)

    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.

  1. Synovitis in mice with inflammatory arthritis monitored with quantitative analysis of dynamic contrast-enhanced NIR fluorescence imaging using iRGD-targeted liposomes as fluorescence probes

    Directory of Open Access Journals (Sweden)

    Wu H

    2018-03-01

    Full Text Available Hao Wu,1,2,* Haohan Wu,1,2,* Yanni He,1 Zhen Gan,2 Zhili Xu,1,2 Meijun Zhou,1,2 Sai Liu,1,2 Hongmei Liu1 1Department of Ultrasonography, Guangdong Second Provincial General Hospital Affiliated to Southern Medical University, Guangzhou, China; 2Department of Ultrasonography, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China *These authors contributed equally to this work Background: Rheumatoid arthritis (RA is a common inflammatory disorder characterized primarily by synovitis and pannus formation in multiple joints, causing joints destruction and irreversible disability in most cases. Early diagnosis and effective therapy monitoring of RA are of importance for achieving the favorable prognosis. Methods: We first prepared the targeted fluorescence probes, and then explored the feasibility of near-infrared (NIR fluorescence molecular imaging to detect and evaluate the RA via the targeted fluorescence probes by quantitative analysis in this study. Results: The targeted fluorescence probes (indocyanine green-liposomes decorated with iRGD peptide [iLPs] was successfully prepared. The quantitative analysis found that strong fluorescence signal was detected in inflamed paws and the fluorescence signal in iLPs group was 3.03-fold higher than that in non-targeted (indocyanine green-liposomes decorated without iRGD peptide [LPs] group (P<0.01 at 15 min after injection, whereas the fluorescence signal from iLPs signal can almost not be observed in the non-inflamed paws, showing the high sensitivity and accuracy for arthritis by the NIR fluorescence imaging based on iLPs. Conclusion: The NIR fluorescence imaging by iLPs may facilitate improved arthritis diagnosis and early assessment of the disease progression by providing an in vivo characterization of angiogenesis in inflammatory joint diseases. Keywords: rheumatoid arthritis, synovitis, diagnosis, near-infrared fluorescence imaging, iRGD-targeted probes

  2. Interface-Targeting Strategy Enables Two-Photon Fluorescent Lipid Droplet Probes for High-Fidelity Imaging of Turbid Tissues and Detecting Fatty Liver.

    Science.gov (United States)

    Guo, Lifang; Tian, Minggang; Feng, Ruiqing; Zhang, Ge; Zhang, Ruoyao; Li, Xuechen; Liu, Zhiqiang; He, Xiuquan; Sun, Jing Zhi; Yu, Xiaoqiang

    2018-04-04

    Lipid droplets (LDs) with unique interfacial architecture not only play crucial roles in protecting a cell from lipotoxicity and lipoapoptosis but also closely relate with many diseases such as fatty liver and diabetes. Thus, as one of the important applied biomaterials, fluorescent probes with ultrahigh selectivity for in situ and high-fidelity imaging of LDs in living cells and tissues are critical to elucidate relevant physiological and pathological events as well as detect related diseases. However, available probes only utilizing LDs' waterless neutral cores but ignoring the unique phospholipid monolayer interfaces exhibit low selectivity. They cannot differentiate neutral cores of LDs from intracellular other lipophilic microenvironments, which results in extensively cloud-like background noise and severely limited their bioapplications. Herein, to design LD probes with ultrahigh selectivity, the exceptional interfacial architecture of LDs is considered adequately and thus an interface-targeting strategy is proposed for the first time. According to the novel strategy, we have developed two amphipathic fluorescent probes (N-Cy and N-Py) by introducing different cations into a lipophilic fluorophore (nitrobenzoxadiazole (NBD)). Consequently, their cationic moiety precisely locates the interfaces through electrostatic interaction and simultaneously NBD entirely embeds into the waterless core via hydrophobic interaction. Thus, high-fidelity and background-free fluorescence imaging of LDs are expectably realized in living cells in situ. Moreover, LDs in turbid tissues like skeletal muscle slices have been clearly imaged (up to 82 μm depth) by a two-photon microscope. Importantly, using N-Cy, we not only intuitively monitored the variations of LDs in number, size, and morphology but also clearly revealed their abnormity in hepatic tissues resulting from fatty liver. Therefore, these unique probes provide excellent imaging tools for elucidating LD

  3. Two rhodamine lactam modulated lysosome-targetable fluorescence probes for sensitively and selectively monitoring subcellular organelle pH change

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongmei [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Wang, Cuiling [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, Xi' an 710069 (China); She, Mengyao; Zhu, Yuelu; Zhang, Jidong; Yang, Zheng [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Liu, Ping, E-mail: liuping@nwu.edu.cn [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Wang, Yaoyu [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Li, Jianli, E-mail: lijianli@nwu.edu.cn [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China)

    2015-11-05

    Be a powerful technique for convenient detection of pH change in living cells, especially at subcellular level, fluorescent probes has attracted more and more attention. In this work, we designed and synthesized three rhodamine lactam modulated fluorescent probes RS1, RS2 and RS3, which all respond sensitively toward weak acidity (pH range 4–6) via the photophysical property in buffer solution without interference from the other metal ions, and they also show ideal pKa values and excellent reversibility. Particularly, by changing the lone pair electrons distribution of lactam-N atom with different conjugations, RS2 and RS3 exhibit high quantum yield, negligible cytotoxicity and excellent permeability. They are suitable to stain selectively lysosomes of tumor cells and monitor its pH changes sensitively via optical molecular imaging. The above findings suggest that the probes we designed could act as ideal and easy method for investigating the pivotal role of H{sup +} in lysosomes and are potential pH detectors in disease diagnosis through direct intracellular imaging. - Highlights: • Two probes for sensitively and selectively monitoring weak acidic pH change. • The pKa of the probes was highly suitable for staining lysosomes in tumor cells. • The properties of those probes were changed by different conjugate system. • These probes have negligible cytotoxicity and good sensitivity in vivo.

  4. Two rhodamine lactam modulated lysosome-targetable fluorescence probes for sensitively and selectively monitoring subcellular organelle pH change

    International Nuclear Information System (INIS)

    Li, Hongmei; Wang, Cuiling; She, Mengyao; Zhu, Yuelu; Zhang, Jidong; Yang, Zheng; Liu, Ping; Wang, Yaoyu; Li, Jianli

    2015-01-01

    Be a powerful technique for convenient detection of pH change in living cells, especially at subcellular level, fluorescent probes has attracted more and more attention. In this work, we designed and synthesized three rhodamine lactam modulated fluorescent probes RS1, RS2 and RS3, which all respond sensitively toward weak acidity (pH range 4–6) via the photophysical property in buffer solution without interference from the other metal ions, and they also show ideal pKa values and excellent reversibility. Particularly, by changing the lone pair electrons distribution of lactam-N atom with different conjugations, RS2 and RS3 exhibit high quantum yield, negligible cytotoxicity and excellent permeability. They are suitable to stain selectively lysosomes of tumor cells and monitor its pH changes sensitively via optical molecular imaging. The above findings suggest that the probes we designed could act as ideal and easy method for investigating the pivotal role of H + in lysosomes and are potential pH detectors in disease diagnosis through direct intracellular imaging. - Highlights: • Two probes for sensitively and selectively monitoring weak acidic pH change. • The pKa of the probes was highly suitable for staining lysosomes in tumor cells. • The properties of those probes were changed by different conjugate system. • These probes have negligible cytotoxicity and good sensitivity in vivo.

  5. Sequencing of megabase plus DNA by hybridization: Method development ENT. Final technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    Crkvenjakov, R.; Drmanac, R.

    1991-01-31

    Sequencing by hybridization (SBH) is the only sequencing method based on the experimental determination of the content of oligonucleotide sequences. The data acquisition relies on the natural process of base pairing. It is possible to determine the content of complementary oligosequences in the target DNA by the process of hybridization with oligonucleotide probes of known sequences.

  6. In situ hybridization; principles and applications: review article

    Directory of Open Access Journals (Sweden)

    Zahra Nozhat

    2015-06-01

    Full Text Available In situ hybridization (ISH is a method that uses labeled complementary single strand DNA or RNA to localize specific DNA or RNA sequences in an intact cell or in a fixed tissue section. The main steps of ISH consist of: probe selection, tissue or sample preparation, pre-hybridization treatment, hybridization and washing, detection and control procedure. Probe selection is one of the important aspects of successful hybridization. ISH sensitivity and specificity can be influenced by: probe construct, efficiency of labeling, percentage of GC, probe length and signal detection systems. Different methods such as nick translation, random priming, end tailing and T4 DNA polymerase replacement are used for probe generation. Both radioactive and non-radioactive labels can be used in order to probe labeling. Nucleic acid maintenance in samples, prevention of morphological changes of samples and probe penetration into tissue section are the main aims of sample preparation step. Then, a small amount of solution containing probe, is added on slides containing tissue sections for hybridization process, then slides are incubated overnight. Next day, washes are carried out to remove the probes which are not bound to target DNA or RNA. Finally, in order to be sure that the observed labeling is specific to the target sequence, using several control procedures is very important. Various techniques based on ISH consist of: Fluorescence in situ hybridization (FISH, chromogenic in situ hybridization (CISH, genomic in situ hybridization (GISH, comparative genomic hybridization (CGH, spectral karyotyping (SKY and multiplex fluorescence in situ hybridization (MFISH. One of the most common techniques of ISH is fluorescence in situ hybridization. FISH can be used to: 1 detect small deletions and duplications that are not visible using microscope analysis, 2 detect how many chromosomes of a certain type are present in each cell and 3 confirm rearrangements that are

  7. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    Energy Technology Data Exchange (ETDEWEB)

    Fehm, Thomas Felix; Razansky, Daniel, E-mail: dr@tum.de [Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg (Germany); Faculty of Medicine, Technische Universität München, Munich (Germany); Deán-Ben, Xosé Luís [Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg (Germany)

    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.

  8. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    Science.gov (United States)

    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.

  9. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    International Nuclear Information System (INIS)

    Fehm, Thomas Felix; Razansky, Daniel; Deán-Ben, Xosé Luís

    2014-01-01

    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.

  10. Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

    DEFF Research Database (Denmark)

    Kushon, S A; Jordan, J P; Seifert, J L

    2001-01-01

    The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA...... structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic...

  11. Obtaining source current density related to irregularly structured electromagnetic target field inside human body using hybrid inverse/FDTD method.

    Science.gov (United States)

    Han, Jijun; Yang, Deqiang; Sun, Houjun; Xin, Sherman Xuegang

    2017-01-01

    Inverse method is inherently suitable for calculating the distribution of source current density related with an irregularly structured electromagnetic target field. However, the present form of inverse method cannot calculate complex field-tissue interactions. A novel hybrid inverse/finite-difference time domain (FDTD) method that can calculate the complex field-tissue interactions for the inverse design of source current density related with an irregularly structured electromagnetic target field is proposed. A Huygens' equivalent surface is established as a bridge to combine the inverse and FDTD method. Distribution of the radiofrequency (RF) magnetic field on the Huygens' equivalent surface is obtained using the FDTD method by considering the complex field-tissue interactions within the human body model. The obtained magnetic field distributed on the Huygens' equivalent surface is regarded as the next target. The current density on the designated source surface is derived using the inverse method. The homogeneity of target magnetic field and specific energy absorption rate are calculated to verify the proposed method.

  12. A novel SERRS sandwich-hybridization assay to detect specific DNA target.

    Directory of Open Access Journals (Sweden)

    Cécile Feuillie

    Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

  13. 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

    Energy Technology Data Exchange (ETDEWEB)

    Youngblom, J.J. [California State University-Stanislaus, Turlock, CA (United States); Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States)] [and others

    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.

  14. Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

    Science.gov (United States)

    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. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer

    Science.gov (United States)

    Yang, Zhe; Luo, Huiyan; Cao, Zhong; Chen, Ya; Gao, Jinbiao; Li, Yingqin; Jiang, Qing; Xu, Ruihua; Liu, Jie

    2016-06-01

    Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44.Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor

  16. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  17. Targeting plug-in hybrid electric vehicle policies to increase social benefits

    International Nuclear Information System (INIS)

    Skerlos, Steven J.; Winebrake, James J.

    2010-01-01

    In 2009 the U.S. federal government enacted tax credits aimed at encouraging consumers to purchase plug-in hybrid electric vehicles (PHEVs). These tax credits are available to all consumers equally and therefore do not account for the variability in social benefits associated with PHEV operation in different parts of the country. The tax credits also do not consider variability in consumer income. This paper discusses why the PHEV subsidy policy would have higher social benefits at equal or less cost if the tax credits were offered at different levels depending on consumer income and the location of purchase. Quantification of these higher social benefits and related policy proposals are left for future work.

  18. Surface engineering of macrophages with nanoparticles to generate a cell-nanoparticle hybrid vehicle for hypoxia-targeted drug delivery.

    Science.gov (United States)

    Holden, Christopher A; Yuan, Quan; Yeudall, W Andrew; Lebman, Deborah A; Yang, Hu

    2010-02-02

    Tumors frequently contain hypoxic regions that result from a shortage of oxygen due to poorly organized tumor vasculature. Cancer cells in these areas are resistant to radiation- and chemotherapy, limiting the treatment efficacy. Macrophages have inherent hypoxia-targeting ability and hold great advantages for targeted delivery of anticancer therapeutics to cancer cells in hypoxic areas. However, most anticancer drugs cannot be directly loaded into macrophages because of their toxicity. In this work, we designed a novel drug delivery vehicle by hybridizing macrophages with nanoparticles through cell surface modification. Nanoparticles immobilized on the cell surface provide numerous new sites for anticancer drug loading, hence potentially minimizing the toxic effect of anticancer drugs on the viability and hypoxia-targeting ability of the macrophage vehicles. In particular, quantum dots and 5-(aminoacetamido) fluorescein-labeled polyamidoamine dendrimer G4.5, both of which were coated with amine-derivatized polyethylene glycol, were immobilized to the sodium periodate-treated surface of RAW264.7 macrophages through a transient Schiff base linkage. Further, a reducing agent, sodium cyanoborohydride, was applied to reduce Schiff bases to stable secondary amine linkages. The distribution of nanoparticles on the cell surface was confirmed by fluorescence imaging, and it was found to be dependent on the stability of the linkages coupling nanoparticles to the cell surface.

  19. Targeted analysis with benchtop quadrupole–orbitrap hybrid mass spectrometer: Application to determination of synthetic hormones in animal urine

    International Nuclear Information System (INIS)

    Kumar, Praveen; Rúbies, Antoni; Centrich, Francesc; Granados, Mercè; Cortés-Francisco, Nuria; Caixach, Josep; Companyó, Ramon

    2013-01-01

    Graphical abstract: -- Highlights: •The quadrupole in Q Exactive acts as a powerful filter to reduce ion suppression. •Reducing mass range using quadrupole in targeted modes increases the S/N ratio. •Targeted SIM data dependent scan modes are the most suitable for residue analysis. •A HRMS confirmatory method for synthetic hormones in urine has been developed. •The Q Exactive provides similar sensitivity and enhanced selectivity compared to QqQ. -- Abstract: Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a challenge in the field of food safety control. In this study, various acquisition modes (Full MS/AIF, Full MS + tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a quadrupole–orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the main conclusions has been that, reducing the scan range for Full MS (using the quadrupole) and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS (tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones (trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol, taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire chromatograph peak. The method was validated according to EC/657/2002. CCα (decision limit) for the analytes ranged between 0.11 μg L −1 and 0.69 μg L −1 and CCβ (detection capability) ranged between 0.29 μg L −1 and 0.90 μg L −1

  20. Dual-Ligand Modified Polymer-Lipid Hybrid Nanoparticles for Docetaxel Targeting Delivery to Her2/neu Overexpressed Human Breast Cancer Cells.

    Science.gov (United States)

    Yang, Zhe; Tang, Wenxin; Luo, Xingen; Zhang, Xiaofang; Zhang, Chao; Li, Hao; Gao, Di; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2015-08-01

    In this study, a dual-ligand polymer-lipid hybrid nanoparticle drug delivery vehicle comprised of an anti-HER2/neu peptide (AHNP) mimic with a modified HIV-1 Tat (mTAT) was established for the targeted treatment of Her2/neu-overexpressing cells. The resultant dual-ligand hybrid nanoparticles (NPs) consisted of a poly(lactide-co-glycolide) core, a near 90% surface coverage of the lipid monolayer, and a 5.7 nm hydrated polyethylene glycol shell. Ligand density optimization study revealed that cellular uptake efficiency of the hybrid NPs could be manipulated by controlling the surface-ligand densities. Furthermore, the cell uptake kinetics and mechanism studies showed that the dual-ligand modifications of hybrid NPs altered the cellular uptake pathway from caveolae-mediated endocytosis (CvME) to the multiple endocytic pathways, which would significantly enhance the NP internalization. Upon the systemic investigation of the cellular uptake behavior of dual-ligand hybrid NPs, docetaxel (DTX), a hydrophobic anticancer drug, was successfully encapsulated into dual-ligand hybrid NPs with high drug loading for Her2/neu-overexpressing SK-BR-3 breast cancer cell treatment. The DTX-loaded dual-ligand hybrid NPs showed a decreased burst release and a more gradual sustained drug release property. Because of the synergistic effect of dual-ligand modification, DTX-loaded dual-ligand hybrid NPs exerted substantially better therapeutic potency against SK-BR-3 cancer cells than other NP formulations and free DTX drugs. These results demonstrate that the dual-ligand hybrid NPs could be a promising vehicle for targeted drug delivery to treat breast cancer.

  1. Assessment of the effect of parallel temperature gradients in the JET SOL on Te measured by divertor target Langmuir probes

    NARCIS (Netherlands)

    Ďuran, I.; Ješko, K.; Fuchs, V.; Groth, M.; Guillemaut, C.; Gunn, J.P.; Horáček, J.; Pitts, R.A.; Tskhakaya, D.

    2015-01-01

    Abstract Higher than expected electron temperatures (Te) are often measured by divertor Langmuir probes (LP) in high recycling and detached regimes in JET and other tokamaks. As a possible mechanism to explain this discrepancy, we investigate the effect of penetration of fast, almost collisionless

  2. Discovery and in Vivo Evaluation of Novel RGD-Modified Lipid-Polymer Hybrid Nanoparticles for Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    Yinbo Zhao

    2014-09-01

    Full Text Available In the current study, the lipid-shell and polymer-core hybrid nanoparticles (lpNPs modified by Arg–Gly–Asp(RGD peptide, loaded with curcumin (Cur, were developed by emulsification-solvent volatilization method. The RGD-modified hybrid nanoparticles (RGD–lpNPs could overcome the poor water solubility of Cur to meet the requirement of intravenous administration and tumor active targeting. The obtained optimal RGD-lpNPs, composed of PLGA (poly(lactic-co-glycolic acid–mPEG (methoxyl poly(ethylene- glycol, RGD–polyethylene glycol (PEG–cholesterol (Chol copolymers and lipids, had good entrapment efficiency, submicron size and negatively neutral surface charge. The core-shell structure of RGD–lpNPs was verified by TEM. Cytotoxicity analysis demonstrated that the RGD–lpNPs encapsulated Cur retained potent anti-tumor effects. Flow cytometry analysis revealed the cellular uptake of Cur encapsulated in the RGD–lpNPs was increased for human umbilical vein endothelial cells (HUVEC. Furthermore, Cur loaded RGD–lpNPs were more effective in inhibiting tumor growth in a subcutaneous B16 melanoma tumor model. The results of immunofluorescent and immunohistochemical studies by Cur loaded RGD–lpNPs therapies indicated that more apoptotic cells, fewer microvessels, and fewer proliferation-positive cells were observed. In conclusion, RGD–lpNPs encapsulating Cur were developed with enhanced anti-tumor activity in melanoma, and Cur loaded RGD–lpNPs represent an excellent tumor targeted formulation of Cur which might be an attractive candidate for cancer therapy.

  3. Discovery and in vivo evaluation of novel RGD-modified lipid-polymer hybrid nanoparticles for targeted drug delivery.

    Science.gov (United States)

    Zhao, Yinbo; Lin, Dayong; Wu, Fengbo; Guo, Li; He, Gu; Ouyang, Liang; Song, Xiangrong; Huang, Wei; Li, Xiang

    2014-09-29

    In the current study, the lipid-shell and polymer-core hybrid nanoparticles (lpNPs) modified by Arg-Gly-Asp(RGD) peptide, loaded with curcumin (Cur), were developed by emulsification-solvent volatilization method. The RGD-modified hybrid nanoparticles (RGD-lpNPs) could overcome the poor water solubility of Cur to meet the requirement of intravenous administration and tumor active targeting. The obtained optimal RGD-lpNPs, composed of PLGA (poly(lactic-co-glycolic acid))-mPEG (methoxyl poly(ethylene- glycol)), RGD-polyethylene glycol (PEG)-cholesterol (Chol) copolymers and lipids, had good entrapment efficiency, submicron size and negatively neutral surface charge. The core-shell structure of RGD-lpNPs was verified by TEM. Cytotoxicity analysis demonstrated that the RGD-lpNPs encapsulated Cur retained potent anti-tumor effects. Flow cytometry analysis revealed the cellular uptake of Cur encapsulated in the RGD-lpNPs was increased for human umbilical vein endothelial cells (HUVEC). Furthermore, Cur loaded RGD-lpNPs were more effective in inhibiting tumor growth in a subcutaneous B16 melanoma tumor model. The results of immunofluorescent and immunohistochemical studies by Cur loaded RGD-lpNPs therapies indicated that more apoptotic cells, fewer microvessels, and fewer proliferation-positive cells were observed. In conclusion, RGD-lpNPs encapsulating Cur were developed with enhanced anti-tumor activity in melanoma, and Cur loaded RGD-lpNPs represent an excellent tumor targeted formulation of Cur which might be an attractive candidate for cancer therapy.

  4. Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization.

    Science.gov (United States)

    Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

    2016-03-20

    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). 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. 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. 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.

  5. Lower Hybrid Frequency Range Waves Generated by Ion Polarization Drift Due to Electromagnetic Ion Cyclotron Waves: Analysis of an Event Observed by the Van Allen Probe B

    Science.gov (United States)

    Khazanov, G. V.; Boardsen, S.; Krivorutsky, E. N.; Engebretson, M. J.; Sibeck, D.; Chen, S.; Breneman, A.

    2017-01-01

    We analyze a wave event that occurred near noon between 07:03 and 07:08 UT on 23 February 2014 detected by the Van Allen Probes B spacecraft, where waves in the lower hybrid frequency range (LHFR) and electromagnetic ion cyclotron (EMIC) waves are observed to be highly correlated, with Pearson correlation coefficient of approximately 0.86. We assume that the correlation is the result of LHFR wave generation by the ions polarization drift in the electric field of the EMIC waves. To check this assumption the drift velocities of electrons and H+, He+, and O+ ions in the measured EMIC wave electric field were modeled. Then the LHFR wave linear instantaneous growth rates for plasma with these changing drift velocities and different plasma compositions were calculated. The time distribution of these growth rates, their frequency distribution, and the frequency dependence of the ratio of the LHFR wave power spectral density (PSD)parallel and perpendicular to the ambient magnetic eld to the total PSD were found. These characteristics of the growth rates were compared with the corresponding characteristics of the observed LHFR activity. Reasonable agreement between these features and the strong correlation between EMIC and LHFR energy densities support the assumption that the LHFR wave generation can be caused by the ions polarization drift in the electric field of an EMIC wave.

  6. Laser-pump/X-ray-probe experiments with electrons ejected from a Cu(111) target: space-charge acceleration.

    Science.gov (United States)

    Schiwietz, G; Kühn, D; Föhlisch, A; Holldack, K; Kachel, T; Pontius, N

    2016-09-01

    A comprehensive investigation of the emission characteristics for electrons induced by X-rays of a few hundred eV at grazing-incidence angles on an atomically clean Cu(111) sample during laser excitation is presented. Electron energy spectra due to intense infrared laser irradiation are investigated at the BESSY II slicing facility. Furthermore, the influence of the corresponding high degree of target excitation (high peak current of photoemission) on the properties of Auger and photoelectrons liberated by a probe X-ray beam is investigated in time-resolved pump and probe measurements. Strong electron energy shifts have been found and assigned to space-charge acceleration. The variation of the shift with laser power and electron energy is investigated and discussed on the basis of experimental as well as new theoretical results.

  7. Hybridization Capture Using RAD Probes (hyRAD, a New Tool for Performing Genomic Analyses on Collection Specimens.

    Directory of Open Access Journals (Sweden)

    Tomasz Suchan

    Full Text Available 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

  8. Hybrid indirect-drive/direct-drive target for inertial confinement fusion

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, Lindsay John

    2018-02-27

    A hybrid indirect-drive/direct drive for inertial confinement fusion utilizing laser beams from a first direction and laser beams from a second direction including a central fusion fuel component; a first portion of a shell surrounding said central fusion fuel component, said first portion of a shell having a first thickness; a second portion of a shell surrounding said fusion fuel component, said second portion of a shell having a second thickness that is greater than said thickness of said first portion of a shell; and a hohlraum containing at least a portion of said fusion fuel component and at least a portion of said first portion of a shell; wherein said hohlraum is in a position relative to said first laser beam and to receive said first laser beam and produce X-rays that are directed to said first portion of a shell and said fusion fuel component; and wherein said fusion fuel component and said second portion of a shell are in a position relative to said second laser beam such that said second portion of a shell and said fusion fuel component receive said second laser beam.

  9. Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

    International Nuclear Information System (INIS)

    Ueda, Masashi; Fukushima, Takahiro; Ogawa, Kei; Kimura, Hiroyuki; Ono, Masahiro; Yamaguchi, Takashi; Ikehara, Yuzuru; Saji, Hideo

    2014-01-01

    Highlights: • We developed a radioiodinated peptide probe targeting αvβ6 integrin ( 123 I-IFMDV2). • 123 I-IFMDV2 had a high affinity and selectivity for αvβ6 integrin. • 123 I-IFMDV2 showed a specific binding to αvβ6 integrin in vivo. • 123 I-IFMDV2 enabled clear visualization of the αvβ6-integrin-positive tumor. - Abstract: Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT). Methods: Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed. Results: Among the 4 probes examined in this study, 125 I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of 125 I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of 125 I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, 123 I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft. Conclusion: 123 I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC

  10. The study of concentration effects of target hybridization on cervical cancer detection using interdigitated electrodes (IDE)

    Science.gov (United States)

    Noriani, C.; Hashim, U.; Azizah, N.

    2016-07-01

    Human Papilloma Virus (HPV) is a virus from the Papilloma virus family that affects human skin and the moist membranes that line the body, such as the throat, mouth, feet, fingers, nails, anus and cervix [1]. There are over 100 types, of which 40 can affect the genital area. Most known HPV types cause no symptoms to humans. Some, however, can cause verrucae (warts), while a small number can increase the risk of developing several cancers, such as that of the cervix, penis, vagina, anus and oropharynx (oral part of the pharynx - throat cancer). HPV strand 16 and 18 are well known for causing the advanced of Cervical Cancer (CC). Currently, integrated electrodes (IDEs) are implemented in various sensing devices including surface acoustic wave (SAW) sensors, chemical sensors as well as current MEMS biosensors. IDEs have been optimized for a variety of sensing applications including biosensors sensors, acoustic sensors, and chemical sensors. However, optimization for cancer cell detection has yet to be reported. The output signal strength of IDEs is controlled through careful design of the active area, width, and spacing of the electrode fingers the efficiency of DNA nanochip depends mainly on the sequence of the capture probes and the way they are attached to the support [2]. This strategy presented a simple, rapid and sensitive platform for HPV detection and would become a powerful tool for pathogenic microorganisms screening in clinical diagnosis. The coupling procedure must be quick, covalent, and reproducible.

  11. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    Science.gov (United States)

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

  12. Automated design of genomic Southern blot probes

    Directory of Open Access Journals (Sweden)

    Komiyama Noboru H

    2010-01-01

    Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

  13. Mobile Game Probes

    DEFF Research Database (Denmark)

    Borup Lynggaard, Aviaja

    2006-01-01

    This paper will examine how probes can be useful for game designers in the preliminary phases of a design process. The work is based upon a case study concerning pervasive mobile phone games where Mobile Game Probes have emerged from the project. The new probes are aimed towards a specific target...... group and the goal is to specify the probes so they will cover the most relevant areas for our project. The Mobile Game Probes generated many interesting results and new issues occurred, since the probes came to be dynamic and favorable for the process in new ways....

  14. 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

    International Nuclear Information System (INIS)

    Voordouw, G.; Voordouw, J.K.; Karkhoff-Schweizer, R.R.; Fedorak, P.M.; Westlake, D.W.S.

    1991-01-01

    A novel method for 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

  15. Oxaliplatin immuno hybrid nanoparticles for active targeting: an approach for enhanced apoptotic activity and drug delivery to colorectal tumors.

    Science.gov (United States)

    Tummala, Shashank; Gowthamarajan, K; Satish Kumar, M N; Wadhwani, Ashish

    2016-06-01

    Tumor necrosis factor related apoptosis inducing ligand (TRAIL) proved to be a promising new target for colorectal cancer treatment. Elevated expression of TRAIL protein in tumor cells distinguishes it from healthy cells, thereby delivering the drug at the specific site. Here, we formulated oxaliplatin immunohybrid nanoparticles (OIHNPs) to deliver oxaliplatin and anti-TRAIL for colorectal cancer treatment in xenograft tumor models. The polymeric chitosan layer binds to the lipid film with the mixture of phospholipids by an ultra sound method followed by conjugating with thiolated antibody using DSPE-PEG-mal3400, resulting in the formation of OIHNPs. The polymer layer helps in more encapsulation of the drug (71 ± 0.09%) with appreciable particle size (95 ± 0.01 nm), and lipid layer prevents degradation of the drug in serum by preventing nanoparticle aggregation. OIHNPs have shown a 4-fold decrease in the IC50 value compared to oxaliplatin in HT-29 cells by the MTT assay. These immuno-nanoparticles represent the successful uptake and internalization of oxaliplatin in HT-29 cells rather than in MCF-7 cells determined by triple fluorescence method. Apoptotic activity in vitro of OIHNPs was determined by the change in the mitochondria membrane potential that further elevates its anti-tumor property. Furthermore, the conjugated nanoparticles can effectively deliver the drug to the tumor sites, which can be attributed to its ability in reducing tumor mass and tumor volume in xenograft tumor models in vivo along with sustaining its release in vitro. These findings indicated that the oxaliplatin immuno-hybrid nanoparticles would be a promising nano-sized active targeted formulation for colorectal-tumor targeted therapy.

  16. Targeted analysis with benchtop quadrupole–orbitrap hybrid mass spectrometer: Application to determination of synthetic hormones in animal urine

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Praveen [Departament de Química Analítica, Universitat de Barcelona, Barcelona (Spain); Rúbies, Antoni; Centrich, Francesc [Laboratori Agència Salut Pública de Barcelona, Barcelona (Spain); CIBER of Epidemiology and Public Health (CIBERESP), Madrid (Spain); Granados, Mercè [Departament de Química Analítica, Universitat de Barcelona, Barcelona (Spain); Cortés-Francisco, Nuria; Caixach, Josep [Mass Spectrometry Laboratory-Organic Pollutants, IDAEA-CSIC, Barcelona (Spain); Companyó, Ramon, E-mail: compano@ub.edu [Departament de Química Analítica, Universitat de Barcelona, Barcelona (Spain)

    2013-05-30

    Graphical abstract: -- Highlights: •The quadrupole in Q Exactive acts as a powerful filter to reduce ion suppression. •Reducing mass range using quadrupole in targeted modes increases the S/N ratio. •Targeted SIM data dependent scan modes are the most suitable for residue analysis. •A HRMS confirmatory method for synthetic hormones in urine has been developed. •The Q Exactive provides similar sensitivity and enhanced selectivity compared to QqQ. -- Abstract: Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a challenge in the field of food safety control. In this study, various acquisition modes (Full MS/AIF, Full MS + tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a quadrupole–orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the main conclusions has been that, reducing the scan range for Full MS (using the quadrupole) and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS (tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones (trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol, taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire chromatograph peak. The method was validated according to EC/657/2002. CCα (decision limit) for the analytes ranged between 0.11 μg L{sup −1} and 0.69 μg L{sup −1} and CCβ (detection capability) ranged between 0.29 μg L{sup −1} and 0.90 μg L{sup −1}.

  17. Fluorescence detection of natural RNA using rationally designed "clickable" oligonucleotide probes

    DEFF Research Database (Denmark)

    Okholm, Anders; Kjems, Jørgen; Astakhova, Kira

    2014-01-01

    Herein a reliable approach to the design of effective fluorescent probes for RNA detection is described. The fluorescence signalling of hybridization by internally positioned polyaromatic hydrocarbons and rhodamine dyes was achieved with a low fluorescence background signal, high fluorescence qua...... quantum yields at ambient and elevated temperature, high selectivity and signal specificity of the probes when binding to miR-7 and circRNA targets....

  18. Chemical proteomics approach reveals the direct targets and the heme-dependent activation mechanism of artemisinin in Plasmodium falciparum using an activity-based artemisinin probe

    Directory of Open Access Journals (Sweden)

    Jigang Wang

    2016-04-01

    Full Text Available Artemisinin and its analogues are currently the most effective anti-malarial drugs. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources. Once activated, artemisinins attack macromolecules through alkylation and propagate a series of damages, leading to parasite death. Even though several parasite proteins have been reported as artemisinin targets, the exact mechanism of action (MOA of artemisinin is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted for. Recently, we have developed an unbiased chemical proteomics approach to directly probe the MOA of artemisinin in P. falciparum. We synthesized an activity-based artemisinin probe with an alkyne tag, which can be coupled with biotin through click chemistry. This enabled selective purification and identification of 124 protein targets of artemisinin. Many of these targets are critical for the parasite survival. In vitro assays confirmed the specific artemisinin binding and inhibition of selected targets. We thus postulated that artemisinin kills the parasite through disrupting its biochemical landscape. In addition, we showed that artemisinin activation requires heme, rather than free ferrous iron, by monitoring the extent of protein binding using a fluorescent dye coupled with the alkyne-tagged artemisinin. The extremely high level of heme released from the hemoglobin digestion by the parasite makes artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages compared to parasites at early ring stage, which have low level of heme, possibly derived from endogenous synthesis. Such a unique activation mechanism also confers artemisinin with extremely high specificity against the parasites, while the healthy red blood cells are unaffected. Our results provide a sound explanation of the MOA of artemisinin and its specificity against malaria

  19. Mitochondria Targetable Time-Gated Luminescence Probe for Singlet Oxygen Based on a β-Diketonate-Europium Complex.

    Science.gov (United States)

    Sun, Jingyan; Song, Bo; Ye, Zhiqiang; Yuan, Jingli

    2015-12-21

    Singlet oxygen ((1)O2) plays a key role in the photodynamic therapy (PDT) technique of neoplastic diseases. In this work, by using a 9,10-dimethyl-2-anthryl-containing β-diketone, 1,1,1,2,2-pentafluoro-5-(9',10'-dimethyl-2'-anthryl)-3,5-pentanedione (Hpfdap), as a (1)O2-recognition ligand, a novel β-diketonate-europium(III) complex that can act as a luminescence probe for (1)O2, [Eu(pfdap)3(tpy)] (tpy = 2,2',2″-terpyridine), has been designed and synthesized for the time-gated luminescence detection of (1)O2 in living cells. The complex is weakly luminescent due to the quenching effect of 9,10-dimethyl-2-anthryl groups. After reaction with (1)O2, accompanied by the formation of endoperoxides of 9,10-dimethyl-2-anthryl groups, the luminescence quenching disappears, so that the long-lived luminescence of the europium(III) complex is switched on. The complex showed highly selective luminescence response to (1)O2 with a remarkable luminescence enhancement. Combined with the time-gated luminescence imaging technique, the complex was successfully used as a luminescent probe for the monitoring of the time-dependent generation of (1)O2 in 5-aminolevulinic acid (a PDT drug) loaded HepG2 cells during the photodynamic process. In addition, by coloading the complex and a mitochondrial indicator, Mito-Tracker Green, into HepG2 cells, the specific localization of [Eu(pfdap)3(tpy)] molecules in mitochondria of HepG2 cells was demonstrated by confocal fluorescence imaging measurements.

  20. Improving probe set selection for microbial community analysis by leveraging taxonomic information of training sequences

    Directory of Open Access Journals (Sweden)

    Jiang Tao

    2011-10-01

    Full Text Available Abstract Background Population levels of microbial phylotypes can be examined using a hybridization-based method that utilizes a small set of computationally-designed DNA probes targeted to a gene common to all. Our previous algorithm attempts to select a set of probes such that each training sequence manifests a unique theoretical hybridization pattern (a binary fingerprint to a probe set. It does so without taking into account similarity between training gene sequences or their putative taxonomic classifications, however. We present an improved algorithm for probe set selection that utilizes the available taxonomic information of training gene sequences and attempts to choose probes such that the resultant binary fingerprints cluster into real taxonomic groups. Results Gene sequences manifesting identical fingerprints with probes chosen by the new algorithm are more likely to be from the same taxonomic group than probes chosen by the previous algorithm. In cases where they are from different taxonomic groups, underlying DNA sequences of identical fingerprints are more similar to each other in probe sets made with the new versus the previous algorithm. Complete removal of large taxonomic groups from training data does not greatly decrease the ability of probe sets to distinguish those groups. Conclusions Probe sets made from the new algorithm create fingerprints that more reliably cluster into biologically meaningful groups. The method can readily distinguish microbial phylotypes that were excluded from the training sequences, suggesting novel microbes can also be detected.

  1. Improving probe set selection for microbial community analysis by leveraging taxonomic information of training sequences.

    Science.gov (United States)

    Ruegger, Paul M; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

    2011-10-10

    Population levels of microbial phylotypes can be examined using a hybridization-based method that utilizes a small set of computationally-designed DNA probes targeted to a gene common to all. Our previous algorithm attempts to select a set of probes such that each training sequence manifests a unique theoretical hybridization pattern (a binary fingerprint) to a probe set. It does so without taking into account similarity between training gene sequences or their putative taxonomic classifications, however. We present an improved algorithm for probe set selection that utilizes the available taxonomic information of training gene sequences and attempts to choose probes such that the resultant binary fingerprints cluster into real taxonomic groups. Gene sequences manifesting identical fingerprints with probes chosen by the new algorithm are more likely to be from the same taxonomic group than probes chosen by the previous algorithm. In cases where they are from different taxonomic groups, underlying DNA sequences of identical fingerprints are more similar to each other in probe sets made with the new versus the previous algorithm. Complete removal of large taxonomic groups from training data does not greatly decrease the ability of probe sets to distinguish those groups. Probe sets made from the new algorithm create fingerprints that more reliably cluster into biologically meaningful groups. The method can readily distinguish microbial phylotypes that were excluded from the training sequences, suggesting novel microbes can also be detected.

  2. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

    International Nuclear Information System (INIS)

    Jung, Kyung oh; Youn, Hyewon; Kim, Seung Hoo; Kim, Young-Hwa; Kang, Keon Wook; Chung, June-Key

    2016-01-01

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and "6"4Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.

  3. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Kyung oh [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of); Youn, Hyewon, E-mail: hwyoun@snu.ac.kr [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of); Cancer Imaging Center, Seoul National University Hospital, Seoul (Korea, Republic of); Kim, Seung Hoo [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Kim, Young-Hwa [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Kang, Keon Wook [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Chung, June-Key, E-mail: jkchung@snu.ac.kr [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of)

    2016-08-26

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.

  4. Targets Fishing and Identification of Calenduloside E as Hsp90AB1: Design, Synthesis, and Evaluation of Clickable Activity-Based Probe

    Science.gov (United States)

    Wang, Shan; Tian, Yu; Zhang, Jing-Yi; Xu, Hui-Bo; Zhou, Ping; Wang, Min; Lu, Sen-Bao; Luo, Yun; Wang, Min; Sun, Gui-Bo; Xu, Xu-Dong; Sun, Xiao-Bo

    2018-01-01

    Calenduloside E (CE), a natural triterpenoid compound isolated from Aralia elata, can protect against ox-LDL-induced human umbilical vein endothelial cell (HUVEC) injury in our previous reports. However, the exact targets and mechanisms of CE remain elusive. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. Based on the previous studies of the structure-activity relationship (SAR), we introduced an alkyne moiety at the C-28 carboxylic group of CE, which kept the protective and anti-apoptosis activity. Via proteomic approach, one of the potential proteins bound to CE-P was identified as Hsp90AB1, and further verification was performed by pure recombinant Hsp90AB1 and competitive assay. These results demonstrated that CE could bind to Hsp90AB1. We also found that CE could reverse the Hsp90AB1 decrease after ox-LDL treatment. To make our results more convincing, we performed SPR analysis and the affinity kinetic assay showed that CE/CE-P could bind to Hsp90AB1 in a dose-dependent manner. Taken together, our research showed CE could probably bind to Hsp90AB1 to protect the cell injury, which might provide the basis for the further exploration of its cardiovascular protective mechanisms. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. PMID:29875664

  5. Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae▿

    Science.gov (United States)

    Nakamura, Kohei ; Terada, Takeshi; Sekiguchi, Yuji; Shinzato, Naoya; Meng, Xian-Ying; Enoki, Miho; Kamagata, Yoichi

    2006-01-01

    In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H2-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe. PMID:16950902

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  7. An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

    Directory of Open Access Journals (Sweden)

    Laurenzi Ian J

    2009-12-01

    Full Text Available Abstract Background Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches. Results In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay. Conclusions By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

  8. Mesoscale hybrid calibration artifact

    Science.gov (United States)

    Tran, Hy D.; Claudet, Andre A.; Oliver, Andrew D.

    2010-09-07

    A mesoscale calibration artifact, also called a hybrid artifact, suitable for hybrid dimensional measurement and the method for make the artifact. The hybrid artifact has structural characteristics that make it suitable for dimensional measurement in both vision-based systems and touch-probe-based systems. The hybrid artifact employs the intersection of bulk-micromachined planes to fabricate edges that are sharp to the nanometer level and intersecting planes with crystal-lattice-defined angles.

  9. Probe-diverse ptychography

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, I., E-mail: isaac.russellpeterson@rmit.edu.au [ARC Centre of Excellence for Coherent X-ray Science, the University of Melbourne, School of Physics, Victoria 3010 (Australia); Harder, R. [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Robinson, I.K. [Research Complex at Harwell, Didcot, Oxfordshire OX11 0DE (United Kingdom); London Centre for Nanotechnology, University College London, London WC1H 0AH (United Kingdom)

    2016-12-15

    We propose an extension of ptychography where the target sample is scanned separately through several probes with distinct amplitude and phase profiles and a diffraction image is recorded for each probe and each sample translation. The resulting probe-diverse dataset is used to iteratively retrieve high-resolution images of the sample and all probes simultaneously. The method is shown to yield significant improvement in the reconstructed sample image compared to the image obtained using the standard single-probe ptychographic phase-retrieval scheme.

  10. A light-up probe targeting for Bcl-2 2345 G-quadruplex DNA with carbazole TO

    Science.gov (United States)

    Gu, Yingchun; Lin, Dayong; Tang, Yalin; Fei, Xuening; Wang, Cuihong; Zhang, Baolian; Zhou, Jianguo

    2018-02-01

    As its significant role, the selective recognition of G-quadruplex with specific structures and functions is important in biological and medicinal chemistry. Carbazole derivatives have been reported as a kind of fluorescent probe with many excellent optical properties. In the present study, the fluorescence of the dye (carbazole TO) increased almost 70 fold in the presence of bcl-2 2345 G4 compared to that alone in aqueous buffer condition with almost no fluorescence and 10-30 fold than those in the presence of other DNAs. The binding study results by activity inhibition of G4/Hemin peroxidase experiment, NMR titration and molecular docking simulation showed the high affinity and selectivity to bcl-2 2345 G4 arises from its end-stacking interaction with G-quartet. It is said that a facile approach with excellent sensitive, good selectivity and quick response for bcl-2 2345 G-quadruplex was developed and may be used for antitumor recognition or antitumor agents.

  11. Specific down-regulation of spermatogenesis genes targeted by 22G RNAs in hybrid sterile males associated with an X-Chromosome introgression.

    Science.gov (United States)

    Li, Runsheng; Ren, Xiaoliang; Bi, Yu; Ho, Vincy Wing Sze; Hsieh, Chia-Ling; Young, Amanda; Zhang, Zhihong; Lin, Tingting; Zhao, Yanmei; Miao, Long; Sarkies, Peter; Zhao, Zhongying

    2016-09-01

    Hybrid incompatibility (HI) prevents gene flow between species, thus lying at the heart of speciation genetics. One of the most common HIs is male sterility. Two superficially contradictory observations exist for hybrid male sterility. First, an introgression on the X Chromosome is more likely to produce male sterility than on autosome (so-called large-X theory); second, spermatogenesis genes are enriched on the autosomes but depleted on the X Chromosome (demasculinization of X Chromosome). Analysis of gene expression in Drosophila hybrids suggests a genetic interaction between the X Chromosome and autosomes that is essential for male fertility. However, the prevalence of such an interaction and its underlying mechanism remain largely unknown. Here we examine the interaction in nematode species by contrasting the expression of both coding genes and transposable elements (TEs) between hybrid sterile males and its parental nematode males. We use two lines of hybrid sterile males, each carrying an independent introgression fragment from Caenorhabditis briggsae X Chromosome in an otherwise Caenorhabditis nigoni background, which demonstrate similar defects in spermatogenesis. We observe a similar pattern of down-regulated genes that are specific for spermatogenesis between the two hybrids. Importantly, the down-regulated genes caused by the X Chromosome introgressions show a significant enrichment on the autosomes, supporting an epistatic interaction between the X Chromosome and autosomes. We investigate the underlying mechanism of the interaction by measuring small RNAs and find that a subset of 22G RNAs specifically targeting the down-regulated spermatogenesis genes is significantly up-regulated in hybrids, suggesting that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction. © 2016 Li et al.; Published by Cold Spring Harbor Laboratory Press.

  12. Study of the effect of low-power pulse laser on arc plasma and magnesium alloy target in hybrid welding by spectral diagnosis technique

    Science.gov (United States)

    Liu, Liming; Hao, Xinfeng

    2008-10-01

    In order to study the effect of laser pulses on arc plasma and target metal in the hybrid welding process, the spectra of the plasmas in the welding process of magnesium alloys are analysed in this paper. The acquisition system of plasma spectra is set up and the spectral lines of welding plasma are acquired. Compared with tungsten-inert gas (TIG) welding, the intensities of the spectral lines of magnesium increase sharply while those of Ar decrease for strong evaporation and ionization of magnesium alloys in low-power laser/arc hybrid welding. The electron temperature and density are estimated by the Boltzmann plot method and the Stark broadening effect. The result shows that the electron temperature of arc plasma in the hybrid welding process is much lower than that in TIG welding, especially in the laser beam-affected zone. In contrast, the electron density of the plasma is enhanced. The influences of laser parameters on electron temperature are also studied. The changes in electron temperature and density indicate that the effect of laser pulse on the target metal is the dominant factor influencing the electron temperature and density in low-power laser/arc hybrid welding.

  13. Use of 16S rRNA-targeted oligonucleotide probes to investigate the distribution of sulphate-reducing bacteria in estuarine sediments.

    Science.gov (United States)

    Purdy, K J.; Nedwell, D B.; Embley, T M.; Takii, S

    2001-07-01

    The distribution of sulphate-reducing bacteria (SRBs) in three anaerobic sediments, one predominantly freshwater and low sulphate and two predominantly marine and high sulphate, on the River Tama, Tokyo, Japan, was investigated using 16S rRNA-targeted oligonucleotide probes. Hybridisation results and sulphate reduction measurements indicated that SRBs are a minor part of the bacterial population in the freshwater sediments. Only Desulfobulbus and Desulfobacterium were detected, representing 1.6% of the general bacterial probe signal. In contrast, the SRB community detected at the two marine-dominated sites was larger and more diverse, representing 10-11.4% of the bacterial signal and with Desulfobacter, Desulfovibrio, Desulfobulbus and Desulfobacterium detected. In contrast to previous reports our results suggest that Desulfovibrio may not always be the most abundant SRB in anaerobic sediments. Acetate-utilising Desulfobacter were the dominant SRB in the marine-dominated sediments, and Desulfobulbus and Desulfobacterium were active in low-sulphate sediments, where they may utilise electron acceptors other than sulphate.

  14. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    Duvaa, Uffe; Ørngreen, Rikke; Weinkouff Mathiasen, Anne-Gitte

    2013-01-01

    Mobile probing is a method, developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time and space......). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings point...... to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face). The development...

  15. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    Duvaa, Uffe; Ørngreen, Rikke; Weinkouff, Anne-Gitte

    2012-01-01

    Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

  16. Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

    Science.gov (United States)

    Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

    2015-10-07

    Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

  17. Optimized Fast-FISH with a-satellite probes: acceleration by microwave activation

    Directory of Open Access Journals (Sweden)

    Durm M.

    1997-01-01

    Full Text Available It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization technique is well suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation time and the number of subsequent washing steps were reduced considerably by omitting denaturing chemical agents (e.g., formamide. The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical conditions for hybridization permit automatization of the procedure, e.g., by a programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific a-satellite probe. Highly fluorescent major binding sites were obtained for 74oC hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 ± 1.59 binding sites were measured in metaphase spreads, and 2.69 ± 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed in a standard microwave oven. After denaturation for 20 s at 900 W, hybridization was performed for 4 min at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chromosome 1-specific a-satellite probe. In this case, denaturation was performed at 630 W for 60 s and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clearly discriminated by their brightness

  18. Probe Selection for DNA Microarrays using OligoWiz

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Juncker, Agnieszka; Nielsen, Henrik Bjørn

    2007-01-01

    Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client-server appl......Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client......-server application that offers a detailed graphical interface and real-time user interaction on the client side, and massive computer power and a large collection of species databases (400, summer 2007) on the server side. Probes are selected according to five weighted scores: cross-hybridization, deltaT(m), folding...... computer skills and can be executed from any Internet-connected computer. The probe selection procedure for a standard microarray design targeting all yeast transcripts can be completed in 1 h....

  19. Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

    Directory of Open Access Journals (Sweden)

    Knut Rudi

    2003-01-01

    Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

  20. Design and synthesis of a biotinylated chemical probe for detecting the molecular targets of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin.

    Science.gov (United States)

    Baker, Ysobel R; Galloway, Warren R J D; Hodgkinson, James T; Spring, David R

    2013-09-25

    Pseudomonas aeruginosa is a human pathogen associated with a variety of life-threatening nosocomial infections. This organism produces a range of virulence factors which actively cause damage to host tissues. One such virulence factor is pyocyanin, known to play a crucial role in the pathogenesis of P. aeruginosa infections. Previous studies had identified a novel compound capable of strongly inhibiting the production of pyocyanin. It was postulated that this inhibition results from modulation of an intercellular communication system termed quorum sensing, via direct binding of the compound with the LasR protein receptor. This raised the possibility that the compound could be an antagonist of quorum sensing in P. aeruginosa, which could have important implications as this intercellular signaling mechanism is known to regulate many additional facets of P. aeruginosa pathogenicity. However, there was no direct evidence for the binding of the active compound to LasR (or any other targets). Herein we describe the design and synthesis of a biotin-tagged version of the active compound. This could potentially be used as an affinity-based chemical probe to ascertain, in a direct fashion, the active compound's macromolecular biological targets, and thus better delineate the mechanism by which it reduces the level of pyocyanin production.

  1. Optimizing the specificity of nucleic acid hybridization.

    Science.gov (United States)

    Zhang, David Yu; Chen, Sherry Xi; Yin, Peng

    2012-01-22

    The specific hybridization of complementary sequences is an essential property of nucleic acids, enabling diverse biological and biotechnological reactions and functions. However, the specificity of nucleic acid hybridization is compromised for long strands, except near the melting temperature. Here, we analytically derived the thermodynamic properties of a hybridization probe that would enable near-optimal single-base discrimination and perform robustly across diverse temperature, salt and concentration conditions. We rationally designed 'toehold exchange' probes that approximate these properties, and comprehensively tested them against five different DNA targets and 55 spurious analogues with energetically representative single-base changes (replacements, deletions and insertions). These probes produced discrimination factors between 3 and 100+ (median, 26). Without retuning, our probes function robustly from 10 °C to 37 °C, from 1 mM Mg(2+) to 47 mM Mg(2+), and with nucleic acid concentrations from 1 nM to 5 µM. Experiments with RNA also showed effective single-base change discrimination.

  2. A novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors.

    Science.gov (United States)

    Santagata, Sara; Portella, Luigi; Napolitano, Maria; Greco, Adelaide; D'Alterio, Crescenzo; Barone, Maria Vittoria; Luciano, Antonio; Gramanzini, Matteo; Auletta, Luigi; Arra, Claudio; Zannetti, Antonella; Scala, Stefania

    2017-05-31

    C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

  3. Fluorine-labeled Dasatinib Nanoformulations as Targeted Molecular Imaging Probes in a PDGFB-driven Murine Glioblastoma Model

    Directory of Open Access Journals (Sweden)

    Miriam Benezra

    2012-12-01

    Full Text Available Dasatinib, a new-generation Src and platelet-derived growth factor receptor (PDGFR inhibitor, is currently under evaluation in high-grade glioma clinical trials. To achieve optimum physicochemical and/or biologic properties, alternative drug delivery vehicles may be needed. We used a novel fluorinated dasatinib derivative (F-SKI249380, in combination with nanocarrier vehicles and metabolic imaging tools (microPET to evaluate drug delivery and uptake in a platelet-derived growth factor B (PDGFB-driven genetically engineered mouse model (GEMM of high-grade glioma. We assessed dasatinib survival benefit on the basis of measured tumor volumes. Using brain tumor cells derived from PDGFB-driven gliomas, dose-dependent uptake and time-dependent inhibitory effects of F-SKI249380 on biologic activity were investigated and compared with the parent drug. PDGFR receptor status and tumor-specific targeting were non-invasively evaluated in vivo using 18F-SKI249380 and 18F-SKI249380-containing micellar and liposomal nanoformulations. A statistically significant survival benefit was found using dasatinib (95 mg/kg versus saline vehicle (P < .001 in tumor volume-matched GEMM pairs. Competitive binding and treatment assays revealed comparable biologic properties for F-SKI249380 and the parent drug. In vivo, Significantly higher tumor uptake was observed for 18F-SKI249380-containing micelle formulations [4.9 percentage of the injected dose per gram tissue (%ID/g; P = .002] compared to control values (1.6%ID/g. Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5%ID/g, consistent with in vivo binding specificity. Using 18F-SKI249380-containing micelles as radiotracers to estimate therapeutic dosing requirements, we calculated intratumoral drug concentrations (24–60 nM that were comparable to in vitro 50% inhibitory concentration values. 18F-SKI249380 is a PDGFR-selective tracer, which

  4. Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

    International Nuclear Information System (INIS)

    Liu, P.; Hun, X.; Qing, H.

    2011-01-01

    We report on a highly sensitive chemiluminescent (CL) biosensor for the sequence-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H 2 O 2 and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L -1 of target DNA. (author)

  5. Biocompatible and colloidally stabilized mPEG-PE/calcium phosphate hybrid nanoparticles loaded with siRNAs targeting tumors.

    Science.gov (United States)

    Gao, Pei; Zhang, Xiangyu; Wang, Hongzhi; Zhang, Qinghong; Li, He; Li, Yaogang; Duan, Yourong

    2016-01-19

    Calcium phosphate nanoparticles are safe and effective delivery vehicles for small interfering RNA (siRNA), as a result of their excellent biocompatibility. In this work, mPEG-PE (polyethylene glycol-L-α-phosphatidylethanolamine) was synthesized and used to prepare nanoparticles composed of mPEG-PE and calcium phosphate for siRNA delivery. Calcium phosphate and mPEG-PE formed the stable hybrid nanoparticles through self-assembly resulting from electrostatic interaction in water. The average size of the hybrid nanoparticles was approximately 53.2 nm with a negative charge of approximately -16.7 mV, which was confirmed by dynamic light scattering (DLS) measurements. The nanoparticles exhibited excellent stability in serum and could protect siRNA from ribonuclease (RNase) degradation. The cellular internalization of siRNA-loaded nanoparticles was evaluated in SMMC-7721 cells using a laser scanning confocal microscope (CLSM) and flow cytometry. The hybrid nanoparticles could efficiently deliver siRNA to cells compared with free siRNA. Moreover, the in vivo distribution of Cy5-siRNA-loaded hybrid nanoparticles was observed after being injected into tumor-bearing nude mice. The nanoparticles concentrated in the tumor regions through an enhanced permeability and retention (EPR) effect based on the fluorescence intensities of tissue distribution. A safety evaluation of the nanoparticles was performed both in vitro and in vivo demonstrating that the hybrid nanoparticle delivery system had almost no toxicity. These results indicated that the mPEG-PE/CaP hybrid nanoparticles could be a stable, safe and promising siRNA nanocarrier for anticancer therapy.

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

    Science.gov (United States)

    2013-01-01

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

  7. Evaluation of 18F-labeled targeted perfluorocarbon-filled albumin microbubbles as a probe for microUS and microPET in tumor-bearing mice.

    Science.gov (United States)

    Liao, Ai-Ho; Wu, Shih-Yen; Wang, Hsin-Ell; Weng, Chien-Hsiu; Wu, Ming-Fang; Li, Pai-Chi

    2013-02-01

    In this study, albumin-shelled, targeted MBs (tMBs) were first demonstrated with the expectation of visualization of biodistribution of albumin-shelled tMBs. The actual biodistribution of albumin-shelled tMBs is of vital importance either for molecular imaging or for drug delivery. Recently, albumin microbubbles (MBs) have been studied for drug and gene delivery in vitro and in vivo through cavitation. Targeted lipid-shelled MBs have been applied for ultrasound molecular imaging and conjugated with radiolabeled antibodies for whole-body biodistribution evaluations. The novelty of the work is that, in addition to the lipid tMBs, the albumin tMBs was also applied in biodistribution detection. Multimodality albumin-shelled, (18)F-SFB-labeled VEGFR2 tMBs were synthesized, and their characteristics in mice bearing MDA-MB-231 human breast cancer were investigated with micro-positron-emission tomography (microPET) and high-frequency ultrasound (microUS). Albumin-shelled MBs can be labeled with (18)F-SFB directly and conjugated with antibodies for dual molecular imaging. The albumin-shelled tMBs show a lifetime in 30min in the blood pool and a highly specific adherence to tumor vessels in mice bearing human breast cancer. From the evaluations of whole-body biodistribution, the potential of the dual molecular imaging probe for drug or gene delivery in animal experiments with albumin shelled MBs has been investigated. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. An hybrid methodology for RL-based behavior coordination in a target following mission with an AUV

    OpenAIRE

    Carreras Pérez, Marc; Yuh, Junku; Batlle i Grabulosa, Joan

    2001-01-01

    Proposes a behavior-based scheme for high-level control of autonomous underwater vehicles (AUVs). Two main characteristics can be highlighted in the control scheme. Behavior coordination is done through a hybrid methodology, which takes in advantages of the robustness and modularity in competitive approaches, as well as optimized trajectories

  9. Hydraulic Hybrid and Conventional Parcel Delivery Vehicles' Measured Laboratory Fuel Economy on Targeted Drive Cycles

    Energy Technology Data Exchange (ETDEWEB)

    Lammert, M. P.; Burton, J.; Sindler, P.; Duran, A.

    2014-10-01

    This research project compares laboratory-measured fuel economy of a medium-duty diesel powered hydraulic hybrid vehicle drivetrain to both a conventional diesel drivetrain and a conventional gasoline drivetrain in a typical commercial parcel delivery application. Vehicles in this study included a model year 2012 Freightliner P100H hybrid compared to a 2012 conventional gasoline P100 and a 2012 conventional diesel parcel delivery van of similar specifications. Drive cycle analysis of 484 days of hybrid parcel delivery van commercial operation from multiple vehicles was used to select three standard laboratory drive cycles as well as to create a custom representative cycle. These four cycles encompass and bracket the range of real world in-use data observed in Baltimore United Parcel Service operations. The NY Composite cycle, the City Suburban Heavy Vehicle Cycle cycle, and the California Air Resources Board Heavy Heavy-Duty Diesel Truck (HHDDT) cycle as well as a custom Baltimore parcel delivery cycle were tested at the National Renewable Energy Laboratory's Renewable Fuels and Lubricants Laboratory. Fuel consumption was measured and analyzed for all three vehicles. Vehicle laboratory results are compared on the basis of fuel economy. The hydraulic hybrid parcel delivery van demonstrated 19%-52% better fuel economy than the conventional diesel parcel delivery van and 30%-56% better fuel economy than the conventional gasoline parcel delivery van on cycles other than the highway-oriented HHDDT cycle.

  10. Peptide-micelle hybrids containing fasudil for targeted delivery to the pulmonary arteries and arterioles to treat pulmonary arterial hypertension.

    Science.gov (United States)

    Gupta, Nilesh; Ibrahim, Hany M; Ahsan, Fakhrul

    2014-11-01

    This study investigates the respirability and efficacy of peptide-micelle hybrid nanoparticles as carriers for inhalational therapy of pulmonary arterial hypertension (PAH). CARSKNKDC (CAR), a cell-penetrating and lung-homing peptide, conjugated polyethylene glycol-distearoyl-phosphoethanolamine micelles containing fasudil, an investigational anti-PAH drug, were prepared by solvent evaporation method and characterized for various physicochemical properties. The pharmacokinetics and pharmacological efficacy of hybrid particles containing fasudil were evaluated in healthy rats and monocrotaline-induced PAH rats. CAR micelles containing fasudil had an entrapment efficiency of approximately 58%, showed controlled release of the drug, and were monodispersed with an average size of approximately 14 nm. Nuclear magnetic resonance scan confirmed the drug's presence in the core of peptide-micelle hybrid particles. Compared with plain micelles, CAR peptide increased the cellular uptake by approximately 1.7-fold and extended the drug half-life by approximately fivefold. The formulations were more prone to accumulate in the pulmonary vasculature than in the peripheral blood, which is evident from the ratio of the extent of reduction of pulmonary and systemic arterial pressures. On the whole, this study demonstrates that peptide-polymer hybrid micelles can serve as inhalational carriers for PAH therapy. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  11. Combined scanning probe and light scattering characterization of multi-stage self-assembly of targeted liposome-based delivery systems

    International Nuclear Information System (INIS)

    Farkas, N; Dagata, J A; Yang, C; Rait, A; Pirollo, K F; Chang, E H

    2011-01-01

    The mean size and size distribution of a targeted nanoparticle delivery system (NDS) strongly influences the intrinsic stability and functionality of this molecular complex, affects its performance as a systemic drug delivery platform and ultimately determines its efficacy toward early detection and treatment of cancer. Since its components undergo significant reorganization during multiple stages of self-assembly, it is essential to monitor the size and stability of the complex throughout the NDS formulation in order to ensure its potency and manufacturability prior to entering clinical trials. This work combines scanning probe microscopy (SPM) and dynamic light scattering (DLS) techniques to obtain quantitative and reliable size measurements of the NDS, and to investigate how variations in the NDS formulation or self-assembly process impact the size, structure and functionality of the complex with various therapeutic and diagnostic agent payloads. These combined SPM and DLS methods, when implemented at an early stage of the NDS formulation, present a potential measurement approach to facilitate drug discovery and development, optimization and quality control during manufacturing of the NDS

  12. Applications of ribosomal in situ hybridization for the study of bacterial cells in the mouse intestine

    DEFF Research Database (Denmark)

    Licht, Tine Rask; Poulsen, Lars Kongsbak; Molin, Søren

    1997-01-01

    Localization of E. coli and S. typhimurium in the large and small intestine of streptomycin-treated mice was visualized by in situ hybridization with specific rRNA target probes and epi-fluorescence microscopy. Growth rates of E. coli BJ4 colonizing the large intestine of streptomycin-treated mic...

  13. Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Deborggraeve, Stijn; Büscher, Philippe

    2011-01-01

    )avidin-coated fluorescent beads, differing in size and color [green beads (1 µm) and red beads (5.9 µm)], thereby allowing distinct detection of each PNA probe by conventional fluorescence microscopy. These two PNA beads showed easily detectable co-localization when simultaneously hybridizing to a target nucleic acid...

  14. Probing into hybrid organic-molecule and InAs quantum-dots nanosystem with multistacked dots-in-a-well units

    DEFF Research Database (Denmark)

    Chen, Miaoxiang Max; Kobashi, Kazufumi

    2012-01-01

    Hybridizing air-stable organic-molecules with advanced III-V semiconductor quantum-dots (QDs) structures can be utilized to create a new generation of biochemical sensing devices. In order to enhance their optical performances, the active regions in these QDs structures commonly consist of multis......Hybridizing air-stable organic-molecules with advanced III-V semiconductor quantum-dots (QDs) structures can be utilized to create a new generation of biochemical sensing devices. In order to enhance their optical performances, the active regions in these QDs structures commonly consist...

  15. Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon-degrading Alcanivorax strains.

    Science.gov (United States)

    Syutsubo, K; Kishira, H; Harayama, S

    2001-06-01

    The genus Alcanivorax comprises diverse hydrocarbon-degrading marine bacteria. Novel 16S rRNA-targeted oligonucleotide DNA probes (ALV735 and ALV735-b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single-mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil-contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4',6-diamidino-2-phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon-degrading microbial consortia.

  16. DNA hybridization sensing for cytogenetic analysis

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dapra, Johannes; Brøgger, Anna Line

    2013-01-01

    are rearrangements between two chromosome arms that results in two derivative chromosomes having a mixed DNA sequence. The current detection method is a Fluorescent In situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the DNA sequences of two chromosomes involved...... in the translocation (Kwasny et al., 2012). We have developed a new double hybridization assay that allows for sorting of the DNA chromosomal fragments into separate compartment, moreover allowing for detection of the translocation. To detect the translocation it is necessary to determine that the two DNA sequences...... forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The first example of the translocation detection was presented on lab-on-a-disc using fluorescently labeled DNA fragments, representing the derivative chromosome (Brøgger et al., 2012). To allow...

  17. Targeting NF-kB signaling with polymeric hybrid micelles that co-deliver siRNA and dexamethasone for arthritis therapy.

    Science.gov (United States)

    Wang, Qin; Jiang, Hao; Li, Yan; Chen, Wenfei; Li, Hanmei; Peng, Ke; Zhang, Zhirong; Sun, Xun

    2017-04-01

    The transcription factor NF-kB plays a pivotal role in the pathogenesis of rheumatoid arthritis. Here we attempt to slow arthritis progression by co-delivering the glucocorticoid dexamethasone (Dex) and small-interfering RNA targeting NF-kB p65 using our previously developed polymeric hybrid micelle system. These micelles contain two similar amphiphilic copolymers: polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG). The hybrid micelles loaded with Dex and siRNA effectively inhibited NF-kB signaling in murine macrophages more efficiently than micelles containing either Dex or siRNA on their own. In addition, the co-delivery system was able to switch macrophages from the M1 to M2 state. Injecting hybrid micelles containing Dex and siRNA into mice with collagen-induced arthritis led the therapeutic agents to accumulate in inflamed joints and reduce inflammation, without damaging renal or liver function. Thus, blocking NF-kB activation in inflammatory tissue using micelle-based co-delivery may provide a new approach for treating inflammatory disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. GeneChip microarrays-signal intensities, RNA concentrations and probe sequences

    International Nuclear Information System (INIS)

    Binder, Hans; Preibisch, Stephan

    2006-01-01

    GeneChip microarrays consist of hundreds of thousands of oligonucleotide probes. The transformation of their signal intensities into RNA transcript concentrations requires the knowledge of the response function of the measuring device. We analysed the 'apparatus' function of perfect match (PM) and mismatched (MM) oligonucleotide probes of GeneChip microarrays after changes of the target concentration using the results of a spiked-in experiment. In agreement with previous studies we found that a competitive two-species Langmuir-adsorption model describes the probe intensities well. Each PM and MM probe is characterized by two hybridization constants which specify the propensity of the probe to bind specific and non-specific transcripts. The affinity for non-specific hybridization is on average equal for PM and MM. The purine-pyrimidine asymmetry of base pair interaction strengths, however, causes a characteristic PM-MM intensity difference, the sign of which depends on the middle base of the probe. The affinity for specific hybridization of the PM exceeds that of the MM on average by nearly one order of magnitude because the central mismatched base only weakly contributes to the stability of the probe/target duplexes. For the first time we differentiate between the free energy parameters related to the 64 possible middle-triples of DNA/RNA oligomer duplexes with a central Watson-Crick pairing and a central mismatched pairing. Both the PM and MM probes respond to the concentration of specific transcripts, which can be estimated from the PM and MM probe intensities using the Langmuir-model. The analysis of the PM-MM intensity difference provides at least no loss of accuracy and precision of the estimated concentration compared with the PM-only estimates which in turn outperform the MM-only estimates. The results show that the processing of the PM-MM intensity difference requires the consideration of a background term due to non-specific hybridization, which is

  19. Life in Ice: Microbial Growth Dynamics and Greenhouse Gas Production During Winter in a Thermokarst Bog Revealed by Stable Isotope Probing Targeted Metagenomics

    Science.gov (United States)

    Blazewicz, S.; White, R. A., III; Tas, N.; Euskirchen, E. S.; Mcfarland, J. W.; Jansson, J.; Waldrop, M. P.

    2016-12-01

    Permafrost contains a reservoir of frozen C estimated to be twice the size of the current atmospheric C pool. In response to changing climate, permafrost is rapidly warming which could result in widespread seasonal thawing. When permafrost thaws, soils that are rich in ice and C often transform into thermokarst wetlands with anaerobic conditions and significant production of atmospheric CH4. While most C flux research in recently thawed permafrost concentrates on the few summer months when seasonal thaw has occurred, there is mounting evidence that sizeable portions of annual CO2 and CH4 efflux occurs over winter or during a rapid burst of emissions associated with seasonal thaw. A potential mechanism for such efflux patterns is microbial activity in frozen soils over winter where gasses produced are partially trapped within ice until spring thaw. In order to better understand microbial transformation of soil C to greenhouse gas over winter, we applied stable isotope probing (SIP) targeted metagenomics combined with process measurements and field flux data to reveal activities of microbial communities in `frozen' soil from an Alaskan thermokarst bog. Field studies revealed build-up of CO2 and CH4 in frozen soils suggesting that microbial activity persisted throughout the winter in soils poised just below the freezing point. Laboratory incubations designed to simulate in-situ winter conditions (-1.5 °C and anaerobic) revealed continuous CH4 and CO2 production. Strikingly, the quantity of CH4 produced in 6 months in frozen soil was equivalent to approximately 80% of CH4 emitted during the 3 month summer `active' season. Heavy water SIP targeted iTag sequencing revealed growing bacteria and archaea in the frozen anaerobic soil. Growth was primarily observed in two bacterial phyla, Firmicutes and Bacteroidetes, suggesting that fermentation was likely the major C mineralization pathway. SIP targeted metagenomics facilitated characterization of the primary metabolic

  20. Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chen

    2011-03-01

    Conclusions: Prenatal diagnosis of major congenital malformations should alert one to the possibility of chromosomal abnormalities. Multiplex ligation-dependent probe amplification and aCGH have the advantage of rapid aneuploidy diagnosis of common aneuploidies in cases with major congenital malformations.

  1. Biocompatible and colloidally stabilized mPEG-PE/calcium phosphate hybrid nanoparticles loaded with siRNAs targeting tumors

    OpenAIRE

    Gao, Pei; Zhang, Xiangyu; Wang, Hongzhi; Zhang, Qinghong; Li, He; Li, Yaogang; Duan, Yourong

    2015-01-01

    Calcium phosphate nanoparticles are safe and effective delivery vehicles for small interfering RNA (siRNA), as a result of their excellent biocompatibility. In this work, mPEG-PE (polyethylene glycol-L-?-phosphatidylethanolamine) was synthesized and used to prepare nanoparticles composed of mPEG-PE and calcium phosphate for siRNA delivery. Calcium phosphate and mPEG-PE formed the stable hybrid nanoparticles through self-assembly resulting from electrostatic interaction in water. The average s...

  2. Hybrid Vlasov–Fokker–Planck–Maxwell simulations of fast electron transport and the time dependance of K-shell excitation in a mid-Z metallic target

    International Nuclear Information System (INIS)

    Thomas, A G R; Sherlock, M; Ridgers, C P; Kuranz, C; Drake, R P

    2013-01-01

    Using a hybrid Vlasov–Fokker–Planck–Maxwell code coupled to calculations using a modified relativistic binary-encounter Bethe model of ionizing collisions we study the time and space dependence of K α photon generation by a fast electron beam injected into a solid density copper plasma target. The electron beam is chosen to be representative of that expected to be generated by a picosecond duration, ∼10 19 W cm −2 intensity laser. K α photons are produced as electrons reflux laterally across the target and are slowed and thermalized by collisions with, and Ohmic heating of, the background fluid over a ∼10 ps timescale, which dictates the timescale for K α emission. The results show reasonable agreement with recent experimental results in terms of both the yield and time dependance. We show how lateral expansion of the electrons can be imaged in the K α radiation. (paper)

  3. Hybrid simulation using mixed reality for interventional ultrasound imaging training.

    Science.gov (United States)

    Freschi, C; Parrini, S; Dinelli, N; Ferrari, M; Ferrari, V

    2015-07-01

    Ultrasound (US) imaging offers advantages over other imaging modalities and has become the most widespread modality for many diagnostic and interventional procedures. However, traditional 2D US requires a long training period, especially to learn how to manipulate the probe. A hybrid interactive system based on mixed reality was designed, implemented and tested for hand-eye coordination training in diagnostic and interventional US. A hybrid simulator was developed integrating a physical US phantom and a software application with a 3D virtual scene. In this scene, a 3D model of the probe with its relative scan plane is coherently displayed with a 3D representation of the phantom internal structures. An evaluation study of the diagnostic module was performed by recruiting thirty-six novices and four experts. The performances of the hybrid (HG) versus physical (PG) simulator were compared. After the training session, each novice was required to visualize a particular target structure. The four experts completed a 5-point Likert scale questionnaire. Seventy-eight percentage of the HG novices successfully visualized the target structure, whereas only 45% of the PG reached this goal. The mean scores from the questionnaires were 5.00 for usefulness, 4.25 for ease of use, 4.75 for 3D perception, and 3.25 for phantom realism. The hybrid US training simulator provides ease of use and is effective as a hand-eye coordination teaching tool. Mixed reality can improve US probe manipulation training.

  4. Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Macíčková-Cahová, Hana; Pivoňková, Hana; Špaček, Jan; Havran, Luděk; Hocek, Michal; Fojta, Miroslav

    2011-01-01

    Roč. 9, č. 5 (2011), s. 1366-1371 ISSN 1477-0520 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk(CZ) LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : DNA tail- labelling * protein-DNA binding * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 3.696, year: 2011

  5. Design, validation and annotation of transcriptome-wide oligonucleotide probes for the oligochaete annelid Eisenia fetida.

    Directory of Open Access Journals (Sweden)

    Ping Gong

    Full Text Available High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs. Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59% probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1 does not require a major genomics core facility; (2 allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3 is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4 if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is

  6. Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization

    DEFF Research Database (Denmark)

    Eklund, Aron Charles; Friis, Pia; Wernersson, Rasmus

    2010-01-01

    BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequence. We found that the 10% spike induced 2-fold intensity changes in 3% of the probes, two......-third of which were decreases in intensity likely caused by bulk-hybridization. These changes were correlated with similarity between the spike and probe sequences. Interestingly, even very weak similarities tended to induce a change in probe intensity with the 10% spike. Using this data, we optimized the BLASTN...... substitution matrix to more accurately identify probes susceptible to non-specific hybridization with the spike. Relative to the default substitution matrix, the optimized matrix features a decreased score for A–T base pairs relative to G–C base pairs, resulting in a 5–15% increase in area under the ROC curve...

  7. Hybrid epidemic spreading - from Internet worms to HIV infection

    OpenAIRE

    Zhang, C.

    2015-01-01

    Epidemic phenomena are ubiquitous, ranging from infectious diseases, computer viruses, to information dissemination. Epidemics have traditionally been studied as a single spreading process, either in a fully mixed population or on a network. Many epidemics, however, are hybrid, employing more than one spreading mechanism. For example, the Internet worm Conficker spreads locally targeting neighbouring computers in local networks as well as globally by randomly probing any computer on the Inter...

  8. PSMA-Targeted Nano-Conjugates as Dual-Modality (MRI/PET) Imaging Probes for the Non-Invasive Detection of Prostate Cancer

    National Research Council Canada - National Science Library

    Sun, Xiankai

    2008-01-01

    The goal of this project is to develop dual modality imaging probes for the detection of prostate cancer by doping radioisotopes to iron oxide nanoparticles, so that the sensitivity and specificity...

  9. Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

    Science.gov (United States)

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

    2016-01-01

    In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

  10. DNA probe for lactobacillus delbrueckii

    Energy Technology Data Exchange (ETDEWEB)

    Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  11. DNA probe for lactobacillus delbrueckii

    International Nuclear Information System (INIS)

    Delley, M.; Mollet, B.; Hottinger, H.

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α- 32 P-labeled probe

  12. Cantilevered probe detector with piezoelectric element

    Science.gov (United States)

    Adams, Jesse D; Sulchek, Todd A; Feigin, Stuart C

    2013-04-30

    A disclosed chemical detection system for detecting a target material, such as an explosive material, can include a cantilevered probe, a probe heater coupled to the cantilevered probe, and a piezoelectric element disposed on the cantilevered probe. The piezoelectric element can be configured as a detector and/or an actuator. Detection can include, for example, detecting a movement of the cantilevered probe or a property of the cantilevered probe. The movement or a change in the property of the cantilevered probe can occur, for example, by adsorption of the target material, desorption of the target material, reaction of the target material and/or phase change of the target material. Examples of detectable movements and properties include temperature shifts, impedance shifts, and resonant frequency shifts of the cantilevered probe. The overall chemical detection system can be incorporated, for example, into a handheld explosive material detection system.

  13. Microcantilver-based DNA hybridization sensors for Salmonella identification

    Directory of Open Access Journals (Sweden)

    Carlo Ricciardi

    2012-02-01

    Full Text Available The detection of pathogenic microorganisms in foods remains a challenging since the safety of foodstuffs has to be ensured by the food producing companies. Conventional methods for the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. Biomolecular methods, are commonly used as a support for traditional techniques, thanks to their high sensitivity, specificity and not excessive costs. However, new methods like biosensors for example, can be an exciting alternative to the more traditional tecniques for the detection of pathogens in food. In this study we report Salmonella enterica serotype Enteritidis DNA detection through a novel class of label-free biosensors: microcantilevers (MCs. In general, MCs can operate as a microbalance and is used to detect the mass of the entities anchored to the cantilever surface using the decrease in the resonant frequency. We use DNA hybridization as model reaction system and for this reason, specific single stranded probe DNA of the pathogen and three different DNA targets (single-stranded complementary DNA, PCR product and serial dilutions of DNA extracted from S. Enteritidis strains were applied. Two protocols were reported in order to allow the probe immobilization on cantilever surface: i MC surface was functionalized with 3-aminopropyltriethoxysilane and glutaraldehyde and an amino-modified DNA probe was used; ii gold-coated sensors and thiolated DNA probes were used in order to generate a covalent bonding (Th-Au. For the first one, measures after hybridization with the PCR product showed related frequency shift 10 times higher than hybridization with complementary probe and detectable signals were obtained at the concentrations of 103 and 106 cfu/mL after hybridization with bacterial DNA. There are currently optimizations of the second protocol, where preliminary results have shown to be more uniform and therefore more precise within each of the

  14. Probing into hybrid organic-molecule and InAs quantum-dots nanosystem with multistacked dots-in-a-well units

    Science.gov (United States)

    Chen, Miaoxiang; Kobashi, Kazufumi

    2012-09-01

    Hybridizing air-stable organic-molecules with advanced III-V semiconductor quantum-dots (QDs) structures can be utilized to create a new generation of biochemical sensing devices. In order to enhance their optical performances, the active regions in these QDs structures commonly consist of multistacked dots-in-a-well (DWELL) units. The effects of grafted molecules on the performances of the QDs structures with multistacked DWELLs, however, still remain unclear. Here, we show the significant improvements in the optical properties of InAs QDs in a hybrid nanosystem obtained by grafting biocompatible diazonium salt compound (amine donor) atop InAs QDs structure. Since its interface between the QDs structure and molecular monolayer retains an uncontaminated and non-oxidized condition, the nanosystem is an ideal platform to study the intrinsic properties of charge-carrier transport inside the system. Because of the complexity of the energy-levels in the QDs structure due to the existing surface QDs and DWELLs, selective excitation wavelengths (400, 633, and 885 nm, respectively) with different photo-energies are used to exactly analyze the complete charging mechanism in these QDs. A clear view of charge-carrier transfer inside the nanosystem is revealed by employing photoluminescence technique under selective-wavelength excitations. The present work provides new quantitative evidences for exploiting inorganic QDs applications in complex biological systems.

  15. Probing into hybrid organic-molecule and InAs quantum-dots nanosystem with multistacked dots-in-a-well units

    Energy Technology Data Exchange (ETDEWEB)

    Chen Miaoxiang [Department of Micro- and Nano technology, Technical University of Denmark, Orsteds Plads, 2800 Kgs. Lyngby (Denmark); Kobashi, Kazufumi [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565 (Japan)

    2012-09-15

    Hybridizing air-stable organic-molecules with advanced III-V semiconductor quantum-dots (QDs) structures can be utilized to create a new generation of biochemical sensing devices. In order to enhance their optical performances, the active regions in these QDs structures commonly consist of multistacked dots-in-a-well (DWELL) units. The effects of grafted molecules on the performances of the QDs structures with multistacked DWELLs, however, still remain unclear. Here, we show the significant improvements in the optical properties of InAs QDs in a hybrid nanosystem obtained by grafting biocompatible diazonium salt compound (amine donor) atop InAs QDs structure. Since its interface between the QDs structure and molecular monolayer retains an uncontaminated and non-oxidized condition, the nanosystem is an ideal platform to study the intrinsic properties of charge-carrier transport inside the system. Because of the complexity of the energy-levels in the QDs structure due to the existing surface QDs and DWELLs, selective excitation wavelengths (400, 633, and 885 nm, respectively) with different photo-energies are used to exactly analyze the complete charging mechanism in these QDs. A clear view of charge-carrier transfer inside the nanosystem is revealed by employing photoluminescence technique under selective-wavelength excitations. The present work provides new quantitative evidences for exploiting inorganic QDs applications in complex biological systems.

  16. Hybrid fluorescent nanoparticles fabricated from pyridine-functionalized polyfluorene-based conjugated polymer as reversible pH probes over a broad range of acidity-alkalinity

    International Nuclear Information System (INIS)

    Cui, Haijun; Chen, Ying; Li, Lianshan; Tang, Zhiyong; Wu, Yishi; Fu, Hongbing; Tian, Zhiyuan

    2014-01-01

    Conjugated polymer nanoparticles (CPNs) were developed based on a polyfluorene-based conjugated polymer with thiophene units carrying pyridyl moieties incorporated in the backbone of polymer chains (PFPyT). Hybrid CPNs fabricated from PFPyT and an amphiphilic polymer (NP1) displayed pH-sensitive fluorescence emission features in the range from pH 4.8 to 13, which makes them an attractive nanomaterial for wide range optical sensing of pH values. The fluorescence of hybrid CPNs based on chemically close polyfluorene derivatives without pyridyl moieties (NP3), in contrast, remains virtually unperturbed by pH values in the same range. The fluorescence emission features of NP1 underwent fully reversible changes upon alternating acidification/basification of aqueous dispersions of the CPNs and also displayed excellent repeatability. The observed pH sensing properties of NP1 are attributed to protonation/deprotonation of the nitrogen atoms of the pyridine moieties. This, in turn, leads to the redistribution of electron density of pyridine moieties and their participation in the π-conjugation within the polymer main chains. The optically transparent amphiphilic polymers also exerted significant influence on the pH sensing features of the CPNs, likely by acting as proton sponge and/or acid chaperone. (author)

  17. G-stack modulated probe intensities on expression arrays - sequence corrections and signal calibration

    Directory of Open Access Journals (Sweden)

    Fasold Mario

    2010-04-01

    Full Text Available Abstract Background The brightness of the probe spots on expression microarrays intends to measure the abundance of specific mRNA targets. Probes with runs of at least three guanines (G in their sequence show abnormal high intensities which reflect rather probe effects than target concentrations. This G-bias requires correction prior to downstream expression analysis. Results Longer runs of three or more consecutive G along the probe sequence and in particular triple degenerated G at its solution end ((GGG1-effect are associated with exceptionally large probe intensities on GeneChip expression arrays. This intensity bias is related to non-specific hybridization and affects both perfect match and mismatch probes. The (GGG1-effect tends to increase gradually for microarrays of later GeneChip generations. It was found for DNA/RNA as well as for DNA/DNA probe/target-hybridization chemistries. Amplification of sample RNA using T7-primers is associated with strong positive amplitudes of the G-bias whereas alternative amplification protocols using random primers give rise to much smaller and partly even negative amplitudes. We applied positional dependent sensitivity models to analyze the specifics of probe intensities in the context of all possible short sequence motifs of one to four adjacent nucleotides along the 25meric probe sequence. Most of the longer motifs are adequately described using a nearest-neighbor (NN model. In contrast, runs of degenerated guanines require explicit consideration of next nearest neighbors (GGG terms. Preprocessing methods such as vsn, RMA, dChip, MAS5 and gcRMA only insufficiently remove the G-bias from data. Conclusions Positional and motif dependent sensitivity models accounts for sequence effects of oligonucleotide probe intensities. We propose a positional dependent NN+GGG hybrid model to correct the intensity bias associated with probes containing poly-G motifs. It is implemented as a single-chip based calibration

  18. Hybrid mimics and hybrid vigor in Arabidopsis

    Science.gov (United States)

    Wang, Li; Greaves, Ian K.; Groszmann, Michael; Wu, Li Min; Dennis, Elizabeth S.; Peacock, W. James

    2015-01-01

    F1 hybrids can outperform their parents in yield and vegetative biomass, features of hybrid vigor that form the basis of the hybrid seed industry. The yield advantage of the F1 is lost in the F2 and subsequent generations. In Arabidopsis, from F2 plants that have a F1-like phenotype, we have by recurrent selection produced pure breeding F5/F6 lines, hybrid mimics, in which the characteristics of the F1 hybrid are stabilized. These hybrid mimic lines, like the F1 hybrid, have larger leaves than the parent plant, and the leaves have increased photosynthetic cell numbers, and in some lines, increased size of cells, suggesting an increased supply of photosynthate. A comparison of the differentially expressed genes in the F1 hybrid with those of eight hybrid mimic lines identified metabolic pathways altered in both; these pathways include down-regulation of defense response pathways and altered abiotic response pathways. F6 hybrid mimic lines are mostly homozygous at each locus in the genome and yet retain the large F1-like phenotype. Many alleles in the F6 plants, when they are homozygous, have expression levels different to the level in the parent. We consider this altered expression to be a consequence of transregulation of genes from one parent by genes from the other parent. Transregulation could also arise from epigenetic modifications in the F1. The pure breeding hybrid mimics have been valuable in probing the mechanisms of hybrid vigor and may also prove to be useful hybrid vigor equivalents in agriculture. PMID:26283378

  19. Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

    Directory of Open Access Journals (Sweden)

    Li J

    2017-09-01

    Full Text Available Jun Li,1,* Wenqing Xu,2,* Xiaoli Yuan,3,* Huaiwen Chen,3 Hao Song,1,4 Bingquan Wang,5 Jun Han5 1College of Pharmacy, Liaocheng University, Liaocheng, Shandong, 2Railway Police College, Zhengzhou, 3Department of Cadre Health Care, Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu, 4Centre for Stem Cell & Regenerative Medicine, Liaocheng People’s Hospital, 5Institute of Biopharmaceutical Research, Liaocheng University, Liaocheng, Shandong, China *These authors contributed equally to this work Purpose: Breast cancer stem cells (CSCs are responsible for the initiation, recurrence, and metastasis of breast cancer. Sufficient evidence has established that breast cancer cells can spontaneously turn into breast CSCs. Thus, it is essential to simultaneously target breast CSCs and cancer cells to maximize the efficacy of breast cancer therapy. HER2 has been found to be overexpressed in both breast CSCs and cancer cells. We developed salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles (Sali-NP-HER2 to target both HER2-positive breast CSCs and cancer cells.Methods: The antitumor activity of Sali-NP-HER2 constructed by conjugating anti-HER2 antibodies to polymer–lipid salinomycin nanoparticles was evaluated in vitro and in vivo.Results: Sali-NP-HER2 efficiently bound to HER2-positive breast CSCs and cancer cells, resulting in enhanced cytotoxic effects compared with non-targeted nanoparticles or salinomycin. In mice bearing breast cancer xenografts, administration of Sali-NP-HER2 exhibited superior efficacy in inhibiting tumor growth. Sali-NP-HER2 reduced the breast tumorsphere formation rate and the proportion of breast CSCs more effectively than non-targeted nanoparticles or salinomycin alone.Conclusion: Sali-NP-HER2 represents a promising approach in treating HER2-positive breast cancer by targeting both breast CSCs and cancer cells. Keywords: nanoparticles, breast cancer, cancer stem cells, salinomycin, HER2

  20. Design and docking of novel series of hybrid xanthones as anti-cancer agent to target human DNA topoisomerase 2-alpha

    Directory of Open Access Journals (Sweden)

    Lalit Mohan Nainwal

    2014-06-01

    Full Text Available Topoisomerase (topo IIα is a homodimeric protein catalyzes topological vicissitudes by adding or by soothing super coiling transpiration, occurs in human DNA during DNA replication as an outcome chromosome segregation and condensation occurs during meiosis I and recombination. To prevent the cleavage and religation activity we administered novel hybrid substituted Xanthone series of drugs. The toxicity prediction showed outstanding results which impetus to study its anticancer activities by targeting topoisomerase (topo IIα. We developed the homology model of the topoisomerase (topo IIα due to the unavailability of 3D structure in the Protein Data Bank. Structural assessment of the modeled protein and confirmed the quality of the model. The ligands were docked using Autodock4.2 software and binding energy was reported. The compound XM9, XN2, XM7, XLNU and XNS scored lowest binding energy and highest binding affinity. The interaction sites and the hydrogen bond were observed.

  1. A boundary integral method for numerical computation of radar cross section of 3D targets using hybrid BEM/FEM with edge elements

    Science.gov (United States)

    Dodig, H.

    2017-11-01

    This contribution presents the boundary integral formulation for numerical computation of time-harmonic radar cross section for 3D targets. Method relies on hybrid edge element BEM/FEM to compute near field edge element coefficients that are associated with near electric and magnetic fields at the boundary of the computational domain. Special boundary integral formulation is presented that computes radar cross section directly from these edge element coefficients. Consequently, there is no need for near-to-far field transformation (NTFFT) which is common step in RCS computations. By the end of the paper it is demonstrated that the formulation yields accurate results for canonical models such as spheres, cubes, cones and pyramids. Method has demonstrated accuracy even in the case of dielectrically coated PEC sphere at interior resonance frequency which is common problem for computational electromagnetic codes.

  2. iRGD-modified lipid–polymer hybrid nanoparticles loaded with isoliquiritigenin to enhance anti-breast cancer effect and tumor-targeting ability

    Directory of Open Access Journals (Sweden)

    Gao F

    2017-06-01

    Full Text Available Fei Gao,1–3 Jinming Zhang,3 Chaomei Fu,3 Xiaoming Xie,4 Fu Peng,1–3 Jieshu You,1,2 Hailin Tang,1,2,4 Zhiyu Wang,5 Peng Li,6 Jianping Chen1–3 1School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, 2Shenzhen Institute of Research and Innovation, University of Hong Kong, Shenzhen, 3College of Pharmacy, Chengdu University of Chinese Medicine, Chengdu, 4Department of Breast Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, 5Department of Mammary Disease, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, 6State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, People’s Republic of China Abstract: Isoliquiritigenin (ISL, a natural anti-breast cancer dietary compound, has poor delivery characteristics and low bioavailability. In order to promote the therapeutic outcome of ISL, a tumor-targeting lipid–polymer hybrid nanoparticle (NP system modified by tumor-homing iRGD peptides has been developed. The hybrid NPs were prepared by a modified single-step nanoprecipitation method to encapsulate ISL. iRGD peptides were anchored on the surface by a postinsertion method (ISL-iRGD NPs. The stable lipid–polymer structure of ISL-iRGD NPs, with high encapsulation and loading efficiency, was confirmed. Compared to free ISL and non-iRGD-modified counterparts, ISL-iRGD NPs showed higher cytotoxicity and cell apoptosis against the different type of breast cancer cells. This was attributable to higher cellular accumulation mediated by the iRGD-integrin recognition and the nanoscale effect. More importantly, based on the active tumor-tissue accumulation by iRGD peptides and the prolonged in vivo circulation by the stealth nanostructure, ISL-iRGD NPs displayed higher tumor-growth inhibition efficiency in 4T1-bearing breast-tumor mouse

  3. Structural disorder and electronic hybridization in NicMg1-cO solid solutions probed by XANES at the oxygen K edge

    International Nuclear Information System (INIS)

    Chen Dongliang; Zhong Jun; Chu Wangsheng; Wu Ziyu; Kuzmin, Alexei; Mironova-Ulmane, Nina; Marcelli, Augusto

    2007-01-01

    A series of Ni c Mg 1-c O solid solutions has been studied for the first time looking at the structural disorder by means of x-ray absorption near-edge-structure (XANES) spectroscopy at the oxygen K edge. The experimental XANES signals were analysed within the full multiple scattering formalism and were interpreted taking into account clusters of up to 15 coordination shells around an absorbing oxygen atom. The substitution of nickel atoms by magnesium atoms results in a dramatic decrease of the empty density of states in the conduction band close to the Fermi level due to an exchange of the 3d(Ni)-2p(O) interaction with 3p(Mg)-2p(O). Besides, a simultaneous small decrease of the 3d(Ni)-2p(O) hybridization is also induced by the lattice expansion, determined by the difference in ionic radii between nickel and magnesium ions

  4. Structural disorder and electronic hybridization in Ni{sub c}Mg{sub 1-c}O solid solutions probed by XANES at the oxygen K edge

    Energy Technology Data Exchange (ETDEWEB)

    Chen Dongliang [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Zhong Jun [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Chu Wangsheng [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Wu Ziyu [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Kuzmin, Alexei [Institute of Solid State Physics, University of Latvia, Kengaraga 8, LV-1063 Riga (Latvia); Mironova-Ulmane, Nina [Institute of Solid State Physics, University of Latvia, Kengaraga 8, LV-1063 Riga (Latvia); Marcelli, Augusto [Laboratori Nazionali di Frascati, Istituto Nazionale di Fisica Nucleare, PO Box 13, 00044 Frascati (Italy)

    2007-09-05

    A series of Ni{sub c}Mg{sub 1-c}O solid solutions has been studied for the first time looking at the structural disorder by means of x-ray absorption near-edge-structure (XANES) spectroscopy at the oxygen K edge. The experimental XANES signals were analysed within the full multiple scattering formalism and were interpreted taking into account clusters of up to 15 coordination shells around an absorbing oxygen atom. The substitution of nickel atoms by magnesium atoms results in a dramatic decrease of the empty density of states in the conduction band close to the Fermi level due to an exchange of the 3d(Ni)-2p(O) interaction with 3p(Mg)-2p(O). Besides, a simultaneous small decrease of the 3d(Ni)-2p(O) hybridization is also induced by the lattice expansion, determined by the difference in ionic radii between nickel and magnesium ions.

  5. Detection of iron-depositing Pedomicrobium species in native biofilms from the Odertal National Park by a new, specific FISH probe.

    Science.gov (United States)

    Braun, Burga; Richert, Inga; Szewzyk, Ulrich

    2009-10-01

    Iron-depositing bacteria play an important role in technical water systems (water wells, distribution systems) due to their intense deposition of iron oxides and resulting clogging effects. Pedomicrobium is known as iron- and manganese-oxidizing and accumulating bacterium. The ability to detect and quantify members of this species in biofilm communities is therefore desirable. In this study the fluorescence in situ hybridization (FISH) method was used to detect Pedomicrobium in iron and manganese incrusted biofilms. Based on comparative sequence analysis, we designed and evaluated a specific oligonucleotide probe (Pedo 1250) complementary to the hypervariable region 8 of the 16S rRNA gene for Pedomicrobium. Probe specificities were tested against 3 different strains of Pedomicrobium and Sphingobium yanoikuyae as non-target organism. Using optimized conditions the probe hybridized with all tested strains of Pedomicrobium with an efficiency of 80%. The non-target organism showed no hybridization signals. The new FISH probe was applied successfully for the in situ detection of Pedomicrobium in different native, iron-depositing biofilms. The hybridization results of native bioflims using probe Pedo_1250 agreed with the results of the morphological structure of Pedomicrobium bioflims based on scanning electron microscopy.

  6. Preparation of dual-responsive hybrid fluorescent nano probe based on graphene oxide and boronic acid/BODIPY-conjugated polymer for cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Khoerunnisa [Department of IT Convergence, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); Kang, Eun Bi [Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); Mazrad, Zihnil Adha Islamy [Department of IT Convergence, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); Lee, Gibaek [Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); In, Insik [Department of IT Convergence, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); Department of Polymer Science and Engineering, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); Park, Sung Young, E-mail: parkchem@ut.ac.kr [Department of IT Convergence, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of); Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380–702 (Korea, Republic of)

    2017-02-01

    Here, we report a pH- and thermo-responsive fluorescent nanomaterial of functionalized reduced graphene oxide (rGO) with cross-linked polymer produced via catechol-boronate diol binding mechanism. When conjugated with the hydrophobic dye boron dipyrromethane (BODIPY), this material can act as a dual-responsive nanoplatform for cells imaging. 2-Chloro-3′,4′-dihydroxyacetophenone (CCDP)-quaternized-poly(dimethylaminoethyl methacrylate-co-N-isopropylacrylamide) [C-PDN] was cross-linked with BODIPY and 4-chlorophenyl boronic acid (BA)-quaternized-poly(ethylene glycol)-g-poly(dimethylaminoethyl methacrylate-co-N-isopropylacrylamide) [BB-PPDN]. The GO was then reduced by the catechol group in the cross-linked polymer to synthesize rGO nanoparticles, which able to stabilize the quenching mechanism. This nanoplatform exhibits intense fluorescence at acidic pH and low fluorescence at physiological pH. Confocal laser scanning microscopy (CLSM) images shows bright fluorescence at lysosomal pH and total quench at physiological pH. Therefore, we have successfully developed a promising sensitive bio-imaging probe for identifying cancer cells. - Graphical abstract: [BB-PPDN]-[C-PDN]/rGO nanoparticles with boronic acid-catechol cis-diol binding mechanism toward change in pH demonstrated good biocompatibility and effective quenching for cancer cell detection. - Highlights: • Dual responsive (pH- and thermo) fluorescent nano probe was proposed for cells imaging. • The mechanism was based on cis-diol binding mechanism of boronic acid and catechol. • Reduced graphene oxide was used as quencher on nano-platform. • Detection was controlled dependent on pH based on diol compound of boron chemistry.

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

    Directory of Open Access Journals (Sweden)

    Zhang Wei

    2009-09-01

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

  8. Hybridization interactions between probesets in short oligo microarrays lead to spurious correlations

    Directory of Open Access Journals (Sweden)

    Miller Crispin J

    2006-06-01

    Full Text Available Abstract Background Microarrays measure the binding of nucleotide sequences to a set of sequence specific probes. This information is combined with annotation specifying the relationship between probes and targets and used to make inferences about transcript- and, ultimately, gene expression. In some situations, a probe is capable of hybridizing to more than one transcript, in others, multiple probes can target a single sequence. These 'multiply targeted' probes can result in non-independence between measured expression levels. Results An analysis of these relationships for Affymetrix arrays considered both the extent and influence of exact matches between probe and transcript sequences. For the popular HGU133A array, approximately half of the probesets were found to interact in this way. Both real and simulated expression datasets were used to examine how these effects influenced the expression signal. It was found not only to lead to increased signal strength for the affected probesets, but the major effect is to significantly increase their correlation, even in situations when only a single probe from a probeset was involved. By building a network of probe-probeset-transcript relationships, it is possible to identify families of interacting probesets. More than 10% of the families contain members annotated to different genes or even different Unigene clusters. Within a family, a mixture of genuine biological and artefactual correlations can occur. Conclusion Multiple targeting is not only prevalent, but also significant. The ability of probesets to hybridize to more than one gene product can lead to false positives when analysing gene expression. Comprehensive annotation describing multiple targeting is required when interpreting array data.

  9. Study on the Detection of Moving Target in the Mining Method Based on Hybrid Algorithm for Sports Video Analysis

    Directory of Open Access Journals (Sweden)

    Huang Tian

    2014-10-01

    Full Text Available Moving object detection and tracking is the computer vision and image processing is a hot research direction, based on the analysis of the moving target detection and tracking algorithm in common use, focus on the sports video target tracking non rigid body. In sports video, non rigid athletes often have physical deformation in the process of movement, and may be associated with the occurrence of moving target under cover. Media data is surging to fast search and query causes more difficulties in data. However, the majority of users want to be able to quickly from the multimedia data to extract the interested content and implicit knowledge (concepts, rules, rules, models and correlation, retrieval and query quickly to take advantage of them, but also can provide the decision support problem solving hierarchy. Based on the motion in sport video object as the object of study, conducts the system research from the theoretical level and technical framework and so on, from the layer by layer mining between low level motion features to high-level semantic motion video, not only provides support for users to find information quickly, but also can provide decision support for the user to solve the problem.

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

    Science.gov (United States)

    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

  11. A mitochondrial targeted two-photon iridium(III) phosphorescent probe for selective detection of hypochlorite in live cells and in vivo.

    Science.gov (United States)

    Li, Guanying; Lin, Qian; Sun, Lingli; Feng, Changsheng; Zhang, Pingyu; Yu, Bole; Chen, Yu; Wen, Ya; Wang, Hui; Ji, Liangnian; Chao, Hui

    2015-01-01

    Endogenous hypochlorite ion (ClO(-)) is a highly reactive oxygen species (ROS) that is produced from hydrogen peroxide and chloride ions catalyzed by myeloperoxidase (MPO). And mitochondrion is one of the major sources of ROS including ClO(-). In the present work, a two-photon phosphorescent probe for ClO(-) in mitochondria was developed. An iridium(III) complex bearing a diaminomaleonitrile group as ClO(-) reactive moiety specifically responded to ClO(-) over other ions and ROSs. When the probe was reacted with ClO(-) to form an oxidized carboxylate product, a significant enhancement in phosphorescence intensity was observed under one-photon (402 nm) and two-photon (750 nm) excitation, with a two-photon absorption cross-section of 78.1 GM at 750 nm. More importantly, ICP-MS results and cellular images co-stained with Mito-tracker Green demonstrated that this probe possessed high specificity for mitochondria. This probe was applied in the one- and two-photon imaging of ClO(-) in vitro and in vivo. The results suggested endotoxin lipopolysaccharide (LPS) induced ClO(-) mostly generated in the liver of zebrafish. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. A Single Electrochemical Probe Used for Analysis of Multiple Nucleic Acid Sequences

    Science.gov (United States)

    Mills, Dawn M.; Calvo-Marzal, Percy; Pinzon, Jeffer M.; Armas, Stephanie; Kolpashchikov, Dmitry M.; Chumbimuni-Torres, Karin Y.

    2017-01-01

    Electrochemical hybridization sensors have been explored extensively for analysis of specific nucleic acids. However, commercialization of the platform is hindered by the need for attachment of separate oligonucleotide probes complementary to a RNA or DNA target to an electrode’s surface. Here we demonstrate that a single probe can be used to analyze several nucleic acid targets with high selectivity and low cost. The universal electrochemical four-way junction (4J)-forming (UE4J) sensor consists of a universal DNA stem-loop (USL) probe attached to the electrode’s surface and two adaptor strands (m and f) which hybridize to the USL probe and the analyte to form a 4J associate. The m adaptor strand was conjugated with a methylene blue redox marker for signal ON sensing and monitored using square wave voltammetry. We demonstrated that a single sensor can be used for detection of several different DNA/RNA sequences and can be regenerated in 30 seconds by a simple water rinse. The UE4J sensor enables a high selectivity by recognition of a single base substitution, even at room temperature. The UE4J sensor opens a venue for a re-useable universal platform that can be adopted at low cost for the analysis of DNA or RNA targets. PMID:29371782

  13. Probe Storage

    NARCIS (Netherlands)

    Gemelli, Marcellino; Abelmann, Leon; Engelen, Johannes Bernardus Charles; Khatib, M.G.; Koelmans, W.W.; Zaboronski, Olog; Campardo, Giovanni; Tiziani, Federico; Laculo, Massimo

    2011-01-01

    This chapter gives an overview of probe-based data storage research over the last three decades, encompassing all aspects of a probe recording system. Following the division found in all mechanically addressed storage systems, the different subsystems (media, read/write heads, positioning, data

  14. Cultural probes

    DEFF Research Database (Denmark)

    Madsen, Jacob Østergaard

    The aim of this study was thus to explore cultural probes (Gaver, Boucher et al. 2004), as a possible methodical approach, supporting knowledge production on situated and contextual aspects of occupation.......The aim of this study was thus to explore cultural probes (Gaver, Boucher et al. 2004), as a possible methodical approach, supporting knowledge production on situated and contextual aspects of occupation....

  15. Photochemical reactions in biological systems: probing the effect of the environment by means of hybrid quantum chemistry/molecular mechanics simulations.

    Science.gov (United States)

    Boggio-Pasqua, Martial; Burmeister, Carl F; Robb, Michael A; Groenhof, Gerrit

    2012-06-14

    Organisms have evolved a wide variety of mechanisms to utilize and respond to light. In many cases, the biological response is mediated by structural changes that follow photon absorption in a protein complex. The initial step in such cases is normally the photoisomerization of a highly conjugated prosthetic group. To understand better the factors controlling the isomerization, we perform atomistic molecular dynamics simulations. In this perspective article we briefly review the key theoretical concepts of photochemical reactions and present a practical simulation scheme for simulating photochemical reactions in biomolecular systems. In our scheme, a multi-configurational quantum mechanical description is used to model the electronic rearrangement for those parts of the system that are involved in the photon absorption. For the remainder, typically consisting of the apo-protein and the solvent, a simple force field model is used. The interactions in the systems are thus computed within a hybrid quantum/classical framework. Forces are calculated on-the-fly, and a diabatic surface hopping procedure is used to model the excited-state decay. To demonstrate how this method is used we review our studies on photoactivation of the photoactive yellow protein, a bacterial photoreceptor. We will show what information can be obtained from the simulations, and, by comparing to recent experimental findings, what the limitations of our simulations are.

  16. Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

    International Nuclear Information System (INIS)

    Sousa, Josane F; Espreafico, Enilza M

    2008-01-01

    Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of αvβ3-integrin and low levels of RHOC. Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a

  17. Biotin-Pt (IV)-indomethacin hybrid: A targeting anticancer prodrug providing enhanced cancer cellular uptake and reversing cisplatin resistance.

    Science.gov (United States)

    Hu, Weiwei; Fang, Lei; Hua, Wuyang; Gou, Shaohua

    2017-10-01

    A Pt(IV) prodrug (2) composed of cancer-targeting biotin and nonsteroidal anti-inflammatory drug indomethacin in the axial positions of the six-coordinated octahedral geometry derived from cisplatin was developed, which could be highly accumulated in cancer cells more than normal ones and activated by endogenous reducing molecules to release cisplatin and indomethacin moieties simultaneously to inhibit tumor progression synergistically. In vitro assays revealed that 2 exhibited significantly selective inhibition to the tested cancer cell lines and sensitivity to cisplatin resistant cancer cells. Moreover, 2 presented cyclooxygenases inhibition properties to reduce tumor-associated inflammation, reduced the invasiveness of the highly aggressive PC-3 cells, and disrupted capillary-like tube formation in EA.hy926 cells. In all, this study offers a new strategy to enhance sensitivity and reduce toxicity of cisplatin. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Diagnosis of Lower Hybrid on MST

    International Nuclear Information System (INIS)

    Burke, D. R.; Goetz, J. A.; Kaufman, M. C.; Almagri, A. F.; Anderson, J. K.; Forest, C. B.; Prager, S. C.

    2007-01-01

    RF driven current has never been demonstrated in a Reversed Field Pinch. Recently the lower hybrid system on the Madison Symmetric Torus reached a new operating regime. This upgrade allows RF powers of up to 5% of the Ohmic input power to be injected. It is therefore anticipated that the lower hybrid system is on the threshold of producing meaningful changes to the RFP equilibrium. A diagnostic set is under development to facilitate the study of such changes and lay the foundation for near megawatt operations. Many measurements are being studied for viability. These include electron cyclotron emission, examinations of bulk ion and electron heating, surface perturbation pickup coils, magnetic probe measurements, and Langmuir probe measurements. In addition, several x-ray diagnostics are in operation: pulse height analysis is performed on detector arrays to determine the 5-200 keV spectrum. An insertable target probe is available to create x-rays from fast electrons. Tomographic inversion of 2-D Soft x-ray detectors yields equilibrium information through island structure. Results from experiments with source power up to 225 kW will be presented. Preliminary results from CQL3D Fokker-Planck simulations will also be presented

  19. Mobile probes

    DEFF Research Database (Denmark)

    Ørngreen, Rikke; Jørgensen, Anna Neustrup; Noesgaard, Signe Schack

    2016-01-01

    A project investigating the effectiveness of a collection of online resources for teachers' professional development used mobile probes as a data collection method. Teachers received questions and tasks on their mobile in a dialogic manner while in their everyday context as opposed...... to in an interview. This method provided valuable insight into the contextual use, i.e. how did the online resource transfer to the work practice. However, the research team also found that mobile probes may provide the scaffolding necessary for individual and peer learning at a very local (intra-school) community...... level. This paper is an initial investigation of how the mobile probes process proved to engage teachers in their efforts to improve teaching. It also highlights some of the barriers emerging when applying mobile probes as a scaffold for learning....

  20. Optical probe

    International Nuclear Information System (INIS)

    Denis, J.; Decaudin, J.M.

    1984-01-01

    The probe includes optical means of refractive index n, refracting an incident light beam from a medium with a refractive index n1>n and reflecting an incident light beam from a medium with a refractive index n2 [fr

  1. Counting probe

    International Nuclear Information System (INIS)

    Matsumoto, Haruya; Kaya, Nobuyuki; Yuasa, Kazuhiro; Hayashi, Tomoaki

    1976-01-01

    Electron counting method has been devised and experimented for the purpose of measuring electron temperature and density, the most fundamental quantities to represent plasma conditions. Electron counting is a method to count the electrons in plasma directly by equipping a probe with the secondary electron multiplier. It has three advantages of adjustable sensitivity, high sensitivity of the secondary electron multiplier, and directional property. Sensitivity adjustment is performed by changing the size of collecting hole (pin hole) on the incident front of the multiplier. The probe is usable as a direct reading thermometer of electron temperature because it requires to collect very small amount of electrons, thus it doesn't disturb the surrounding plasma, and the narrow sweep width of the probe voltage is enough. Therefore it can measure anisotropy more sensitively than a Langmuir probe, and it can be used for very low density plasma. Though many problems remain on anisotropy, computer simulation has been carried out. Also it is planned to provide a Helmholtz coil in the vacuum chamber to eliminate the effect of earth magnetic field. In practical experiments, the measurement with a Langmuir probe and an emission probe mounted to the movable structure, the comparison with the results obtained in reverse magnetic field by using a Helmholtz coil, and the measurement of ionic sound wave are scheduled. (Wakatsuki, Y.)

  2. Photonics at the frontiers. Generation of few-cycle light pulses via NOPCPA and real-time probing of charge transfer in hybrid photovoltaics

    Energy Technology Data Exchange (ETDEWEB)

    Herrmann, Daniel

    2011-11-11

    this thesis for the first time succeeded to resolve the photoinduced charge-transfer in the conjugate polymer polythiophene and in hybrid polythiophene/silicon solar cells in real time. Thereby a controverse debate about the nature of the primary photoexcitation in organic semiconductors is resolved: Excitons dissociate with 140 fs time constant to polarons (charge carriers). Deciding parameters (for instance structural order, charge-carrier mobility) for the efficiency of the generation and extraction of free charge carriers can be determined. Further ultrashort-time experiments at novel organic solar cells have here been begun and indicated.

  3. Novel (Phenylethynyl)pyrene-LNA Constructs for Fluorescence SNP Sensing in Polymorphic Nucleic Acid Targets

    DEFF Research Database (Denmark)

    Astakhova, Irina Kira; Samokhina, Evgeniya; Babu, B Ravindra

    2012-01-01

    We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single-nucleotide polymorphisms in natural nucleic acids. High-affinity hybridization......DNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti-HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges...

  4. Evaluation of the Gen-Probe DNA probe for the detection of legionellae in culture

    International Nuclear Information System (INIS)

    Edelstein, P.H.

    1986-01-01

    A commercial DNA probe kit designed to detect rRNA from legionellae was evaluated for its ability to correctly discriminate between legionellae and non-legionellae taken from culture plates. The probe kit, made by the Gen-Probe Corp. (San Diego, Calif.), was radiolabeled with 125 I, and probe bacterial RNA hybridization, detected in a simple one-tube system hybridization assay, was quantitated with a gamma counter. A total of 156 Legionella sp. strains were tested, of which 125 were Legionella pneumophila and the remainder were strains from 21 other Legionella spp. A total of 106 gram-negative non-legionellae, isolated from human respiratory tract (81%) and other body site (19%) specimens, were also tested; 14 genera and 28 species were represented. The probe easily distinguished all of the legionellae from the non-legionellae. The average legionellae/non-legionellae hybridization ratio was 42:1, and the lowest ratio was 2:1; a minor modification in the procedure increased the lowest ratio to 5:1. In addition to correctly identifying all Legionella species, the probe was able to separate some of the various species of Legionella. L. pneumophila strains hybridized more completely to the probe than did the other Legionella spp.; L. wadsworthii and L. oakridgensis hybridized only about 25% of the probe relative to L. pneumophila. Some strains of phenotypically identified L. pneumophila had much lower hybridization to the probe than other members of the species and may represent a new Legionella species. The simplicity of the technique and specificity of the probe make it a good candidate for confirming the identity of legionellae in culture

  5. Experimental estimation of tungsten impurity sputtering due to Type i ELMs in JET-ITER-like wall using pedestal electron cyclotron emission and target Langmuir probe measurements

    Czech Academy of Sciences Publication Activity Database

    Guillemaut, C.; Jardin, A.; Horáček, Jan; Borodkina, I.; Autricque, A.; Arnoux, G.; Boom, J.; Brezinsek, S.; Coenen, J.W.; De La Luna, E.; Devaux, S.; Eich, T.; Harting, D.; Kirschner, A.; Lipschultz, B.; Matthews, G. F.; Meigs, A.; Moulton, D.; O'Mullane, M.; Stamp, M.

    T167, February (2016), s. 014005 ISSN 0031-8949. [International Conference on Plasma-Facing Materials and Components for Fusion Applications, PFMC 2015/15./. Aix-en-Provence, 18.05.2015-22.05.2015] R&D Projects: GA MŠk LG14002 EU Projects: European Commission(XE) 633053 - EUROfusion Institutional support: RVO:61389021 Keywords : tungsten sputtering * edge localized mode * magnetic confinement fusion * ITER * H-mode * ELMs * Langmuir Probes (LP) Subject RIV: BL - Plasma and Gas Discharge Physics OBOR OECD: 1.3 Physical sciences Impact factor: 1.280, year: 2016 http://iopscience.iop.org/issue/1402-4896/2016/T167

  6. Preparation and in vitro evaluation of folate-receptor-targeted SPION–polymer micelle hybrids for MRI contrast enhancement in cancer imaging

    International Nuclear Information System (INIS)

    Mahajan, Shveta; Choudhary, Veena; Koul, Veena; Shishodia, Gauri; Bharti, Alok C

    2013-01-01

    Polymer–SPION hybrids were investigated for receptor-mediated localization in tumour tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared by high-temperature decomposition of iron acetylacetonate were monodisperse (9.27 ± 3.37 nm), with high saturation magnetization of 76.8 emu g −1 . Amphiphilic copolymers prepared from methyl methacrylate and PEG methacrylate by atom transfer radical polymerization were conjugated with folic acid (for folate-receptor specificity). The folate-conjugated polymer had a low critical micellar concentration (0.4 mg l −1 ), indicating stability of the micellar formulation. SPION–polymeric micelle clusters were prepared by desolvation of the SPION dispersion/polymer solution in water. Magnetic resonance imaging of the formulation revealed very good contrast enhancement, with transverse (T 2 ) relaxivity of 260.4 mM −1 s −1 . The biological evaluation of the SPION micelles included cellular viability assay (MTT) and uptake in HeLa cells. These studies demonstrated the potential use of these nanoplatforms for imaging and targeting. (paper)

  7. Preparation and in vitro evaluation of folate-receptor-targeted SPION-polymer micelle hybrids for MRI contrast enhancement in cancer imaging

    Science.gov (United States)

    Mahajan, Shveta; Koul, Veena; Choudhary, Veena; Shishodia, Gauri; Bharti, Alok C.

    2013-01-01

    Polymer-SPION hybrids were investigated for receptor-mediated localization in tumour tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared by high-temperature decomposition of iron acetylacetonate were monodisperse (9.27 ± 3.37 nm), with high saturation magnetization of 76.8 emu g-1. Amphiphilic copolymers prepared from methyl methacrylate and PEG methacrylate by atom transfer radical polymerization were conjugated with folic acid (for folate-receptor specificity). The folate-conjugated polymer had a low critical micellar concentration (0.4 mg l-1), indicating stability of the micellar formulation. SPION-polymeric micelle clusters were prepared by desolvation of the SPION dispersion/polymer solution in water. Magnetic resonance imaging of the formulation revealed very good contrast enhancement, with transverse (T2) relaxivity of 260.4 mM-1 s-1. The biological evaluation of the SPION micelles included cellular viability assay (MTT) and uptake in HeLa cells. These studies demonstrated the potential use of these nanoplatforms for imaging and targeting.

  8. Dual pH/redox responsive and CD44 receptor targeting hybrid nano-chrysalis based on new oligosaccharides of hyaluronan conjugates.

    Science.gov (United States)

    Chen, Daquan; Dong, Xue; Qi, Mengjiao; Song, Xiaoyan; Sun, Jingfang

    2017-02-10

    A smart hybrid microenvironment-mediated dual pH/redox-responsive polymeric nanoparticles combined with inorganic calcium phosphate (CaP) was fabricated, which we term as armored nano-chrysalis inspired by butterfly pupa. The nano-chrysalis has an inner core composed of specially designed oligosaccharides of hyaluronan (oHA) targeting CD44 receptor. The inner core has two functions, i.e., the dual pH/redox responsive polymeric conjugate and the fluorescent curcumin-prodrug function. The prepared nano-chrysalis possessed a smaller size (102.5±4.6nm) than the unarmored nano-chrysalis (122.5±6.6nm). Interestingly, while the nano-chrysalis were stable under pH 7.4, when incubated under the tumor acidic conditions (pH 6.5) the outer CaP armor would dissolve in a pH-dependent, sustained manner. Moreover, nano-chrysalis was demonstrated to present the most effective antitumor efficacy than other formulations. This study provides a promising smart nano-carrier platform to enhance the stability, decrease the side effects, and improve the therapeutic efficacy of anticancer drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

    International Nuclear Information System (INIS)

    Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping

    2013-01-01

    Graphical abstract: -- Highlights: •A new signal-on metallobioassay was developed for detection of nucleic acids. •Target-triggered long-range self-assembled DNA nanostructures are used for amplification of electronic signal. •Hybridization chain reaction is utilized for construction of long-range DNA nanostructures. -- Abstract: Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling

  10. Proximity hybridization-mediated isothermal exponential amplification for ultrasensitive electrochemical protein detection

    Directory of Open Access Journals (Sweden)

    Yu Y

    2017-08-01

    Full Text Available Yanyan Yu, Gaoxing Su, Hongyan Zhu, Qing Zhu, Yong Chen, Bohui Xu, Yuqin Li, Wei Zhang School of Pharmacy, Nantong University, Nantong, People’s Republic of China Abstract: In this study, we fabricated a novel electrochemical biosensing platform on the basis of target-triggered proximity hybridization-mediated isothermal exponential amplification reaction (EXPAR for ultrasensitive protein analysis. Through rational design, the aptamers for protein recognition were integrated within two DNA probes. Via proximity hybridization principle, the affinity protein-binding event was converted into DNA assembly process. The recognition of protein by aptamers can trigger the strand displacement through the increase of the local concentrations of the involved probes. As a consequence, the output DNA was displaced, which can hybridize with the duplex probes immobilized on the electrode surface subsequently, leading to the initiation of the EXPAR as well as the cleavage of duplex probes. Each cleavage will release the gold nanoparticles (AuNPs binding sequence. With the modification of G-quadruplex sequence, electrochemical signals were yielded by the AuNPs through oxidizing 3,3',5,5'-tetramethylbenzidine in the presence of H2O2. The study we proposed exhibited high sensitivity toward platelet-derived growth factor BB (PDGF-BB with the detection limit of 52 fM. And, this method also showed great selectivity among the PDGF isoforms and performed well in spiked human serum samples. Keywords: electrochemical biosensor, proximity hybridization, PDGF-BB, isothermal exponential amplification, G-quadruplex 

  11. Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

    Science.gov (United States)

    Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

    2007-06-01

    To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the nonfluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

  12. Measurement of H2O2 within Living Drosophila during Aging Using a Ratiometric Mass Spectrometry Probe Targeted to the Mitochondrial Matrix

    Science.gov (United States)

    Cochemé, Helena M.; Quin, Caroline; McQuaker, Stephen J.; Cabreiro, Filipe; Logan, Angela; Prime, Tracy A.; Abakumova, Irina; Patel, Jigna V.; Fearnley, Ian M.; James, Andrew M.; Porteous, Carolyn M.; Smith, Robin A.J.; Saeed, Saima; Carré, Jane E.; Singer, Mervyn; Gems, David; Hartley, Richard C.; Partridge, Linda; Murphy, Michael P.

    2011-01-01

    Summary Hydrogen peroxide (H2O2) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H2O2 in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative. PMID:21356523

  13. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.

    1992-01-01

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  14. DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Castelino, J

    1993-12-31

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with {sup 32}P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism`s genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens 10 figs, 2 tabs

  15. Multiplexed interfacial transduction of nucleic acid hybridization using a single color of immobilized quantum dot donor and two acceptors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Algar, W Russ; Krull, Ulrich J

    2010-01-01

    A multiplexed solid-phase assay for the detection of nucleic acid hybridization was developed on the basis of a single color of immobilized CdSe/ZnS quantum dot (QD) as a donor in fluorescence resonance energy transfer (FRET). This work demonstrated that two channels of detection did not necessitate two different QD donors. Two probe oligonucleotides were coimmobilized on optical fibers modified with QDs, and a sandwich assay was used to associate the acceptor dyes with interfacial hybridization events without target labeling. FRET-sensitized acceptor emission provided an analytical signal that was concentration dependent down to 10 nM. Changes in the ratio of coimmobilized probe oligonucleotides were found to yield linear changes in the relative amounts of acceptor emission. These changes were compared to previous studies that used mixed films of two QD donors for two detection channels. The analysis indicated that probe dilution effects were primarily driven by changes in acceptor number density and that QD dilution effects or changes in mean donor-acceptor distance were secondary. Hybridization kinetics were found to be consistent between different ratios of coimmobilized probes, suggesting that hybridization in this type of system occurred via the accepted model for solid-phase hybridization, where adsorption and then diffusion at the solid interface drove hybridization.

  16. Conductivity Probe

    Science.gov (United States)

    2008-01-01

    The Thermal and Electrical Conductivity Probe (TECP) for NASA's Phoenix Mars Lander took measurements in Martian soil and in the air. The needles on the end of the instrument were inserted into the Martian soil, allowing TECP to measure the propagation of both thermal and electrical energy. TECP also measured the humidity in the surrounding air. The needles on the probe are 15 millimeters (0.6 inch) long. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  17. An aggregation-induced emission (AIE) active probe for multiple targets: a fluorescent sensor for Zn(2+) and Al(3+) & a colorimetric sensor for Cu(2+) and F(-).

    Science.gov (United States)

    Samanta, Soham; Manna, Utsab; Ray, Turjya; Das, Gopal

    2015-11-21

    A rationally designed probe L, which consists of both cation and anion binding sites, is capable of displaying interesting aggregation induced emission (AIE) properties. L not only can sense Al(3+) and Zn(2+) through selective turn-on fluorescence responses in 9 : 1 methanol-HEPES buffer (5 mM, pH 7.3; 9 : 1, v/v) medium due to metal ion triggered AIE activity, but also can distinguish them through individual emission signals. L can also detect Cu(2+) in mixed buffer medium and F(-) in acetonitrile through sharp colorimetric responses. All the sensing processes are conspicuous through the naked eye. A theoretical study strongly backed the proposed sensing mechanisms.

  18. Advances in hybrid optics physical sensors for extreme environments

    Science.gov (United States)

    Riza, Nabeel A.

    2010-04-01

    Highlighted are novel innovations in hybrid optical design physical sensors for extreme environments. Various hybrid design compositions are proposed that are suited for a particular sensor application. Examples includes combining freespace (wireless) and fiber-optics (wired) for gas turbine sensing and combining single crystal and sintered Silicon Carbide (SiC) materials for robust extreme environment Coefficent of Thermal Expansion (CTE) matched frontend probe design. Sensor signal processing also includes the hybrid theme where for example Black-Body radiation thermometry (pyrometry) is combined with laser interferometry to provide extreme temperature measurements. The hybrid theme also operates on the optical device level where a digital optical device such as a Digital Micromirror Device (DMD) is combined with an analog optical device such as an Electronically Controlled Variable Focal Length Lens (ECVFL) to deliver a smart and compressive Three Dimensional (3-D) imaging sensor for remote scene and object shape capture including both ambient light (passive) mode and active laser targeting and receive processing. Within a device level, the hybrid theme also operates via combined analog and digital control such as within a wavelength-coded variable optical delay line. These powerful hybrid design optical sensors have numerous applications in engineering and science applications from the military to the commercial/industrial sectors.

  19. DNA Probe for Lactobacillus delbrueckii

    Science.gov (United States)

    Delley, Michèle; Mollet, Beat; Hottinger, Herbert

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

  20. Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

    Directory of Open Access Journals (Sweden)

    Ceren Sengiz

    2015-09-01

    Full Text Available In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL modified pencil graphite electrode (IL-PGEs was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC. The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC or mismatched DNA sequence (MM, or and in the presence of mixture of DNA target: NC (1:1 and DNA target: MM (1:1.

  1. Probe specificity

    International Nuclear Information System (INIS)

    Laget, J.M.

    1986-11-01

    Specificity and complementarity of hadron and electron probes must be systematically developed to answer three questions currently asked in intermediate energy nuclear physics: what is nucleus structure at short distances, what is nature of short range correlations, what is three body force nature [fr

  2. Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

    Science.gov (United States)

    Xie, Xilei; Tang, Fuyan; Shangguan, Xiaoyan; Che, Shiyi; Niu, Jinye; Xiao, Yongsheng; Wang, Xu; Tang, Bo

    2017-06-13

    Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.

  3. Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes.

    Science.gov (United States)

    Ye, Y W; Ling, N; Han, Y J; Wu, Q P

    2014-11-01

    Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10(2)cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28 °C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  4. A Novel Method for Rapid Hybridization of DNA to a Solid Support

    Science.gov (United States)

    Pettersson, Erik; Ahmadian, Afshin; Ståhl, Patrik L.

    2013-01-01

    Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself. PMID:23950946

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

    Science.gov (United States)

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

    2011-05-01

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

  6. Experimental conical-head abutment screws on the microbial leakage through the implant-abutment interface: an in vitro analysis using target-specific DNA probes.

    Science.gov (United States)

    Pita, Murillo S; do Nascimento, Cássio; Dos Santos, Carla G P; Pires, Isabela M; Pedrazzi, Vinícius

    2017-07-01

    The aim of this in vitro study was to identify and quantify up to 38 microbial species from human saliva penetrating through the implant-abutment interface in two different implant connections, external hexagon and tri-channel internal connection, both with conventional flat-head or experimental conical-head abutment screws. Forty-eight two-part implants with external hexagon (EH; n = 24) or tri-channel internal (TI; n = 24) connections were investigated. Abutments were attached to implants with conventional flat-head or experimental conical-head screws. After saliva incubation, Checkerboard DNA-DNA hybridization was used to identify and quantify up to 38 bacterial colonizing the internal parts of the implants. Kruskal-Wallis test followed by Bonferroni's post-tests for multiple comparisons was used for statistical analysis. Twenty-four of thirty-eight species, including putative periodontal pathogens, were found colonizing the inner surfaces of both EH and TI implants. Peptostreptococcus anaerobios (P = 0.003), Prevotella melaninogenica (P abutment screws have impacted the microbial leakage through the implant-abutment interface. Implants attached with experimental conical-head abutment screws showed lower counts of microorganisms when compared with conventional flat-head screws. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. DNA Probe for Lactobacillus delbrueckii

    OpenAIRE

    Delley, Michèle; Mollet, Beat; Hottinger, Herbert

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-l...

  8. Targeted sequencing of large genomic regions with CATCH-Seq.

    Directory of Open Access Journals (Sweden)

    Kenneth Day

    Full Text Available Current target enrichment systems for large-scale next-generation sequencing typically require synthetic oligonucleotides used as capture reagents to isolate sequences of interest. The majority of target enrichment reagents are focused on gene coding regions or promoters en masse. Here we introduce development of a customizable targeted capture system using biotinylated RNA probe baits transcribed from sheared bacterial artificial chromosome clone templates that enables capture of large, contiguous blocks of the genome for sequencing applications. This clone adapted template capture hybridization sequencing (CATCH-Seq procedure can be used to capture both coding and non-coding regions of a gene, and resolve the boundaries of copy number variations within a genomic target site. Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing. We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb. Our approach provides a simple and cost effective alternative to other capture platforms because of template-based, enzymatic probe synthesis and the lack of oligonucleotide design costs. Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.

  9. Synthesis and detection of 3'-OH terminal biotin-labeled DNA probes

    International Nuclear Information System (INIS)

    Brakel, C.L.; Engelhardt, D.L.

    1985-01-01

    Nick translation has been used to prepare biotin-dUTP-containing DNA probes. These stable DNA probes have been identified, following hybridization to target DNA, by fluorescence using antibiotin antibodies or by enzyme reactions in which the enzyme has been linked to avidin or streptavidin. It is probable that this technology will be applicable to certain diagnostic determinations and that, with sufficient sensitivity, this technology might provide a system for obtaining rapid and specific diagnoses in situations presently requiring time-consuming growth assays. The sensitivity of this assay can be increased in two ways: (1) by increasing the amount of biotin contained in the DNA probes, and (2) by increasing the response to individual biotin molecules in the DNA probes. This report demonstrates that terminal deoxynucleotide transferase can be employed to increase the biotin content of DNA probes. We also introduce a new streptavidin-linked enzyme system that produces a greater response to biotinylated DNA probes than does streptavidin-linked horseradish peroxidase

  10. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  11. Morphoproteomics, E6/E7 in-situ hybridization, and biomedical analytics define the etiopathogenesis of HPV-associated oropharyngeal carcinoma and provide targeted therapeutic options.

    Science.gov (United States)

    Brown, Robert E; Naqvi, Syed; McGuire, Mary F; Buryanek, Jamie; Karni, Ron J

    2017-08-17

    Human papillomavirus (HPV) has been identified as an etiopathogenetic factor in oropharyngeal squamous cell carcinoma. The HPV E6 and E7 oncogenes are instrumental in promoting proliferation and blocking differentiation leading to tumorigenesis. Although surgical intervention can remove such tumors, the potential for an etiologic field effect with recurrent disease is real. A downstream effector of E7 oncoprotein, enhancer of zeste homolog 2 (EZH2), is known to promote proliferation and to pose a block in differentiation and in turn, could lead to HPV-induced malignant transformation. However, the EZH2 pathway is amenable to low toxicity therapies designed to promote differentiation to a more benign state and prevent recurrent disease by inhibiting the incorporation of HPV into the genome. This is the first study using clinical specimens to demonstrate EZH2 protein expression in oropharyngeal carcinoma (OPC). The study included eight patients with oropharyngeal carcinoma, confirmed p16INK4a- positive by immunohistochemistry (IHC). The tissue expression of E6/E7 messenger RNA (mRNA) was measured by RNAscope® in-situ hybridization technology. Expression of EZH2, Ki-67, and mitotic indices were assessed by morphoproteomic analysis. Biomedical analytics expanded the results with data from Ingenuity Pathway Analysis (IPA) and KEGG databases to construct a molecular network pathway for further insights. Expression of E6 and E7 oncogenes in p16INK4a- positive oropharyngeal carcinoma was confirmed. EZH2 and its correlates, including elevated proliferation index (Ki-67) and mitotic progression were also present. Biomedical analytics validated the relationship between HPV- E6 and E7 and the expression of the EZH2 pathway. There is morphoproteomic and mRNA evidence of the association of p16INK4a-HPV infection with the E6 and E7 oncogenes and the expression of EZH2, Ki-67 and mitotic progression in oropharyngeal carcinoma. The molecular network biology was confirmed by

  12. Hybrid system concepts

    International Nuclear Information System (INIS)

    Landeyro, P.A.

    1995-01-01

    Hybrid systems studied for fissile material production, were reconsidered for minor actinide and long-lived fission product destruction as alternative to the traditional final disposal of nuclear waste. Now there are attempts to extend the use of the concepts developed for minor actinide incineration to plutonium burning. The most promising hybrid system concept considers fuel and target both as liquids. From the results obtained, the possibility to adopt composite targets seems the most promising solution, but still there remains the problem of Pu production, not acceptable in a burning system. This kind of targets can be mainly used for fissile material production, while for accelerator driven burners it is most convenient to use a liquid lead target. The most suitable solvent is heavy water for minor actinide annihilation in the blanket of a hybrid system. Due to the criticality conditions and the necessity of electric energy production, the blanket using plutonium dissolved in molten salts is the most convenient one. (author)

  13. Five Performance Enhancements for Hybrid Hash Join

    National Research Council Canada - National Science Library

    Graefe, Goetz

    1992-01-01

    .... We discuss five performance enhancements for hybrid hash join algorithms, namely data compression, large cluster sizes and multi-level recursion, role reversal of build and probe inputs, histogram...

  14. Triplex in-situ hybridization

    Science.gov (United States)

    Fresco, Jacques R.; Johnson, Marion D.

    2002-01-01

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

  15. Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

    Science.gov (United States)

    Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

    2001-01-01

    We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

  16. Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline-Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays.

    Science.gov (United States)

    Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

    2016-06-01

    Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular

  17. Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays*

    Science.gov (United States)

    Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

    2016-01-01

    Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular

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

    Science.gov (United States)

    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

  19. Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions.

    Science.gov (United States)

    Valle, Edith R; Henderson, Gemma; Janssen, Peter H; Cox, Faith; Alexander, Trevor W; McAllister, Tim A

    2015-06-01

    In this study, methanogen-specific coenzyme F420 autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

  20. Spatial Exploration and Characterization of Endozoicomonas spp. Bacteria in Stylophora pistillata Using Fluorescence In Situ Hybridization

    KAUST Repository

    Alsheikh-­Hussain, Areej

    2011-12-12

    Studies of coral-­associated bacterial communities have repeatedly demonstrated that the microbial assemblages of the coral host are highly specific and complex. In particular, bacterial community surveys of scleractinian and soft corals from geographically diverse reefs continually uncover a high abundance of sequences affiliated with the Gammaproteobacteria genus Endozoicomonas. The role of these bacteria within the complex coral holobiont is currently unknown. In order to localize these cells and gain an understanding of their potential interactions within the coral, we developed a fluorescence in situ hybridization(FISH) approach for reef-­building coral tissues. Using a custom small-­subunit ribosomal RNA gene database, we developed two Endozoicomonas-­specific probes that cover almost all known coral-­associated Endozoicomonas sequences. Probe hybridization conditions were quantitatively evaluated against target and non-­target bacterial cultures using fluorescence microscopy. Using these experimentally tested conditions, probes were then hybridized to the branching coral Stylophora pistillata, obtained from the Red Sea, using whole mount and paraffin embedding techniques. This study allowed preliminary spatial exploration and characterization of Endozoicomonas in coral, which has provided insight into their functional role and interactions within the coral holobiont.

  1. PEGylated Self-Assembled Nano-Bacitracin A: Probing the Antibacterial Mechanism and Real-Time Tracing of Target Delivery in Vivo.

    Science.gov (United States)

    Hong, Wei; Zhao, Yining; Guo, Yuru; Huang, Chengcheng; Qiu, Peng; Zhu, Jia; Chu, Chun; Shi, Hong; Liu, Mingchun

    2018-04-04

    Although nano-self-assemblies of hydrophobic-modified bacitracin A with poly(d,l-lactic- co-glycolic acid) (PLGA) (nano-BA PLGA ) have demonstrated promising antibacterial activities, the application of nano-BA PLGA was severely compromised by low water solubility. In this study, a series of PEGylated PLGA copolymers were selected to conjugate with the N-terminus of bacitracin A to construct PEGylated self-assembled nano-BAs and to further develop nano-self-assemblies of bacitracin A with strong antibacterial potency and high solubility. Compared with nano-BA PLGA , all PEGylated nano-BAs, except nano-BA 5k , exhibited strong antibacterial efficiency against both Gram-positive and Gram-negative bacteria by inducing loss of cytoplasmic membrane potential, membrane permeabilization, and leakage of calcein from artificial cell membranes. Studies elucidating the underlying mechanism of PEGylated nano-BAs against Gram-negative bacteria indicated that the strong hydrophobic and van der Waals interactions between PLGA and lipopolysaccharide (LPS) could bind, neutralize, and disassociate LPS, facilitating cellular uptake of the nanoparticles, which could destabilize the membrane, resulting in cell death. Moreover, PEGylated nano-BAs (nano-BA 12k ) with a longer PLGA block were expected to occupy a higher local density of BA mass on the surface and result in stronger hydrophobic and van der Waals interactions with LPS, which were responsible for the enhanced antibacterial activity against Gram-positive and emerging antibacterial activity against Gram-negative bacteria, respectively. In vivo imaging verified that PEGylated nano-BAs exhibited higher inflammatory tissue distribution and longer circulation time than nano-BA PLGA . Therefore, although PEGylation did not affect antibacterial activity, it is necessary for target delivery and resistance to clearance of the observed PEGylated nano-BAs. In vivo, nano-BA 12k also showed the highest therapeutic index against infection

  2. Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere

    NARCIS (Netherlands)

    Rosado, A.S.; Seldin, L.; Wolters, A.; Elsas, van J.D.

    1996-01-01

    A molecular method for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere was developed. The system consisted of polymerase chain reaction (PCR) amplification of part of the variable V1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridization with a specific

  3. Femtomolar detection of single mismatches by discriminant analysis of DNA hybridization events using gold nanoparticles.

    Science.gov (United States)

    Ma, Xingyi; Sim, Sang Jun

    2013-03-21

    Even though DNA-based nanosensors have been demonstrated for quantitative detection of analytes and diseases, hybridization events have never been numerically investigated for further understanding of DNA mediated interactions. Here, we developed a nanoscale platform with well-designed capture and detection gold nanoprobes to precisely evaluate the hybridization events. The capture gold nanoprobes were mono-laid on glass and the detection probes were fabricated via a novel competitive conjugation method. The two kinds of probes combined in a suitable orientation following the hybridization with the target. We found that hybridization efficiency was markedly dependent on electrostatic interactions between DNA strands, which can be tailored by adjusting the salt concentration of the incubation solution. Due to the much lower stability of the double helix formed by mismatches, the hybridization efficiencies of single mismatched (MMT) and perfectly matched DNA (PMT) were different. Therefore, we obtained an optimized salt concentration that allowed for discrimination of MMT from PMT without stringent control of temperature or pH. The results indicated this to be an ultrasensitive and precise nanosensor for the diagnosis of genetic diseases.

  4. Hybrid strategies in nanolithography

    Energy Technology Data Exchange (ETDEWEB)

    Saavedra, Hector M; Mullen, Thomas J; Zhang Pengpeng; Dewey, Daniel C; Claridge, Shelley A; Weiss, Paul S [Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 (United States)], E-mail: psw@cnsi.ucla.edu

    2010-03-15

    Hybrid nanoscale patterning strategies combine the registration and addressability of conventional lithographic techniques with the chemical and physical functionality enabled by intermolecular, electrostatic and/or biological interactions. This review aims to highlight and to provide a comprehensive description of recent developments in hybrid nanoscale patterning strategies that enhance existing lithographic techniques or can be used to fabricate functional chemical patterns that interact with their environment. These functional structures create new capabilities, such as the fabrication of physicochemical surfaces that can recognize and capture analytes from complex liquid or gaseous mixtures. The nanolithographic techniques we describe can be classified into three general areas: traditional lithography, soft lithography and scanning-probe lithography. The strengths and limitations of each hybrid patterning technique will be discussed, along with the current and potential applications of the resulting patterned, functional surfaces.

  5. Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

    International Nuclear Information System (INIS)

    Ruijter, Jan M.; Hoff, Maurice J. B. van den; Lorenz, Peter; Tuomi, Jari M.; Hecker, Michael

    2014-01-01

    The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of −1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. (author)

  6. Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Leite, Marina; Cappoen, Davie

    2016-01-01

    ). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy. RESULTS: H. pylori SS1 strain infecting C57BL/6 mice was successfully detected...... by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy. CONCLUSIONS: In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo......INTRODUCTION: In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary...

  7. Simple and label-free electrochemical impedance Amelogenin gene hybridization biosensing based on reduced graphene oxide.

    Science.gov (United States)

    Benvidi, Ali; Rajabzadeh, Nooshin; Mazloum-Ardakani, Mohammad; Heidari, Mohammad Mehdi; Mulchandani, Ashok

    2014-08-15

    The increasing desire for sensitive, easy, low-cost, and label free methods for the detection of DNA sequences has become a vital matter in biomedical research. For the first time a novel label-free biosensor for sensitive detection of Amelogenin gene (AMEL) using reduced graphene oxide modified glassy carbon electrode (GCE/RGO) has been developed. In this work, detection of DNA hybridization of the target and probe DNA was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The optimum conditions were found for the immobilization of probe on RGO surface and its hybridization with the target DNA. CV and EIS carried out in an aqueous solution containing [Fe(CN)6](3-/4-) redox pair have been used for the biosensor characterization. The biosensor has a wide linear range from 1.0×10(-20) to 1.0×10(-14)M with the lower detection limit of 3.2×10(-21)M. Moreover, the present electrochemical detection offers some unique advantages such as ultrahigh sensitivity, simplicity, and feasibility for apparatus miniaturization in analytical tests. The excellent performance of the biosensor is attributed to large surface-to-volume ratio and high conductivity of RGO, which enhances the probe absorption and promotes direct electron transfer between probe and the electrode surface. This electrochemical DNA sensor could be used for the detection of specific ssDNA sequence in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Responsive hybrid inorganic-organic system derived from lanthanide luminescence

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Zhan [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Zheng, Yuhui, E-mail: yhzheng78@scnu.edu.cn [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Jiang, Lasheng; Yang, Jinglian [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Wang, Qianming, E-mail: qmwang@scnu.edu.cn [Key Laboratory of Theoretical Chemistry of Environment, Ministry of Education, School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Guangzhou Key Laboratory of Materials for Energy Conversion and Storage, Guangzhou 510006 (China)

    2016-05-15

    Highlights: • A novel covalent hybrid material was used to detect hemoglobin. • All the recognition experiments were performed in buffer solution. • Porous nano-structures was extensively studied for the recognition. - Abstract: Terbium ions were incorporated into new organic-inorganic matrices to achieve intense green emissions. Hemoglobin (HB) interactions lead to dramatic changes in the luminescence emission intensities. Infrared spectra, morphological studies and photoluminescence give information for the speciation and process of hemoglobin additions. The porous material has a large specific surface area of 351 cm{sup 2}/g and the detection limit for HB (0.7 μM) was much lower than its physical doped material (8 μM). This promising hybrid material will lead to the design of versatile optical probes that are efficiently responding to the external targets.

  9. DNA probe labeling with digoxigenin-dUTP and its application in gene diagnosis

    International Nuclear Information System (INIS)

    Liu Guoyang

    1992-01-01

    DNA probe labeling by the randomly primed incorporation of digoxigenin-dUTP is reported. The sensitivity of color reaction and hybridization were 32 fg and 200 fg, respectively, and both were specific for the target. Single-copy and multi-copy gene fragments among 2 μg human genomic DNA were detected by β IVS II, Fr 3-42 and 3'HVR labeled with digoxigenin-dUTP. The results were consistent with a radioactive control assay. This method has been successfully used in the gene diagnosis of adult polycystic kidney disease

  10. Diagnosis, prognosis and disease management using in situ hybridization

    International Nuclear Information System (INIS)

    Kucheria, K.; Talwar, R.

    2002-01-01

    cytogenetics were identified using ISH technique. The SRY region was detected in few cases of ambiguous genitalia that lacked the Y chromosome. Most leukemia patients who were in complete cytogenetic remission showed evidence of minimal residual disease when analyzed for bcr/abl and PML/RARα fusion genes. Aneuploidies and gene rearrangements could also be detected on interphase (non-dividing) cells obtained from peripheral blood and bone marrow. Further, many targets could be visualized in the same experiment using probes labeled with different coloured fluorescent dyes. To conclude, the present study stresses that for analysis of chromosomal rearrangements at the molecular level, conventional cytogenetics needs to be supplemented with highly sensitive molecular methods like In Situ Hybridization. Further, Interphase cytogenetics is a simple, rapid and efficient technique that can add to the diagnostic utility of conventional cytogenetics by decreasing the time interval between sampling and diagnosis

  11. Far Western: probing membranes.

    Science.gov (United States)

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

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

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura

    2017-01-01

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

  13. Modified beacon probe assisted dual signal amplification for visual detection of microRNA.

    Science.gov (United States)

    Sun, Xiuwei; Ying, Na; Ju, Chuanjing; Li, Zhongyi; Xu, Na; Qu, Guijuan; Liu, Wensen; Wan, Jiayu

    2018-04-21

    In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. The application of fluorescence in situ hybridization (FISH technique for studying the microbial communities in intestinal tissues of white shrimp (Penaeus vannamei

    Directory of Open Access Journals (Sweden)

    Supamattaya, K.

    2005-02-01

    Full Text Available Fluorescence in situ hybridization technique is very useful for the evaluation of microbial communities in various environments. It is possible to apply this technique to study the intestinal microflora in white shrimp (Penaeus vannamei. Different fixatives and storage temperature were tested in this technique. It was found that fixation with 10% buffered formalin for 12 hours and changed to 70% ethanol shown positive results when compared to the fixation with Davidson's fixative or RF fixative. The best signaling was obtainedfrom the samples which were stored in -20ºC. By using the DNA probe targeted to the Eubacteria domain (EUB338 probe, 5′-GCT GCC TCC CGT AGG AGT-3′ labeled with fluorescein as a hybridizing probe, it was found that most intestinal microflora were aggregated with the intestinal contents, or dispersed in the lumen. There was not evidence of the attachment of the microflora with the intestinal epithelium in this study.

  15. Chemo-enzymatic Synthesis of Propionyl-ester-linked Taxol-monosaccharide Conjugate and its Drug Delivery System Using Hybrid-Bio-nanocapsules Targeting Brain Glioma Cells

    Directory of Open Access Journals (Sweden)

    Hiroki Hamada

    2013-01-01

    Full Text Available Taxol is recognized as one of the most potent anticancer agents used in the treatment of breast and ovarian cancers, which are common cancers in women. To overcome its shortcomings, that is, its low water-solubility that reduces drug loading capacity of DDS carriers when incorporating taxol, chemo-enzymatic synthesis of ester-linked taxol-glucose conjugate, i.e., 7-propionyltaxol 2′- O -α-D-glucoside, as a water soluble taxol prodrug was achieved by using a-glucosidase as a glucosylation catalyst. The water-solubility of 7-propionyltaxol 2′- O -α-D-glucoside (25 mM was 63 fold higher than that of taxol (0.4 mM. The pre-S1 peptide which displays on the surface of bio-nanocapsules, which are nanoparticles composed of the hepatitis B virus surface antigen, was replaced with the antibody affinity motif of protein A. Conjugation of such bio-nanocapsules with anti-human epidermal growth factor receptor antibody gave hybrid bio-nanocapsules. The hybrid bio-nanocapsules were effective for delivering 7-propionyltaxol 2′- O -α-D-glucoside to human brain glioma cells. 7-Propionyltaxol 2′- O -α-D-glucoside was effectively hydrolyzed to give taxol in 95% by human glioma cells. The drug loading capacity of hybrid bio-nanocapsules incorporating 7-propionyltaxol 2′- O -α-D-glucoside was 120 times higher than that incorporating taxol itself.

  16. Detection of dengue group viruses by fluorescence in situ hybridization

    Directory of Open Access Journals (Sweden)

    Raquin Vincent

    2012-10-01

    Full Text Available Abstract Background Dengue fever (DF and dengue hemorrhagic fever (DHF represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus that comprises four distinct serotypes (DENV-1 to DENV-4. Fluorescence in situ hybridization (FISH has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae. The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use

  17. Proximal Probes Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Proximal Probes Facility consists of laboratories for microscopy, spectroscopy, and probing of nanostructured materials and their functional properties. At the...

  18. Probe Techniques. Introductory Remarks

    Energy Technology Data Exchange (ETDEWEB)

    Emeleus, K. G. [School of Physics and Applied Mathematics, Queen' s University, Belfast (United Kingdom)

    1968-04-15

    In this brief introduction to the session on probes, the history of theii development is first touched on briefly. Reference is then made to the significance of the work to be described by Medicus, for conductivity and recombination calculations, and by Lam and Su, for a wide range of medium and higher pressure plasmas. Finally, a number of other probe topics are mentioned, including multiple probes; probes in electronegative plasmas; resonance probes; probes in noisy discharges; probes as oscillation detectors; use of probes where space-charge is not negligible. (author)

  19. Silver ions-mediated conformational switch: facile design of structure-controllable nucleic acid probes.

    Science.gov (United States)

    Wang, Yongxiang; Li, Jishan; Wang, Hao; Jin, Jianyu; Liu, Jinhua; Wang, Kemin; Tan, Weihong; Yang, Ronghua

    2010-08-01

    Conformationally constraint nucleic acid probes were usually designed by forming an intramolecular duplex based on Watson-Crick hydrogen bonds. The disadvantages of these approaches are the inflexibility and instability in complex environment of the Watson-Crick-based duplex. We report that this hydrogen bonding pattern can be replaced by metal-ligation between specific metal ions and the natural bases. To demonstrate the feasibility of this principle, two linear oligonucleotides and silver ions were examined as models for DNA hybridization assay and adenosine triphosphate detection. The both nucleic acids contain target binding sequences in the middle and cytosine (C)-rich sequences at the lateral portions. The strong interaction between Ag(+) ions and cytosines forms stable C-Ag(+)-C structures, which promises the oligonucleotides to form conformationally constraint formations. In the presence of its target, interaction between the loop sequences and the target unfolds the C-Ag(+)-C structures, and the corresponding probes unfolding can be detected by a change in their fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using Ag(+) ion complexes instead of traditional Watson-Crick-based duplex. In particular, the intrinsic feature of the metal-ligation motif facilitates the design of functional nucleic acids probes by independently varying the concentration of Ag(+) ions in the medium.

  20. A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer.

    Science.gov (United States)

    Zhou, Feng; Noor, M Omair; Krull, Ulrich J

    2015-09-24

    Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.

  1. Microfluidic Arrayed Lab-On-A-Chip for Electrochemical Capacitive Detection of DNA Hybridization Events.

    Science.gov (United States)

    Ben-Yoav, Hadar; Dykstra, Peter H; Bentley, William E; Ghodssi, Reza

    2017-01-01

    A microfluidic electrochemical lab-on-a-chip (LOC) device for DNA hybridization detection has been developed. The device comprises a 3 × 3 array of microelectrodes integrated with a dual layer microfluidic valved manipulation system that provides controlled and automated capabilities for high throughput analysis of microliter volume samples. The surface of the microelectrodes is functionalized with single-stranded DNA (ssDNA) probes which enable specific detection of complementary ssDNA targets. These targets are detected by a capacitive technique which measures dielectric variation at the microelectrode-electrolyte interface due to DNA hybridization events. A quantitative analysis of the hybridization events is carried out based on a sensing modeling that includes detailed analysis of energy storage and dissipation components. By calculating these components during hybridization events the device is able to demonstrate specific and dose response sensing characteristics. The developed microfluidic LOC for DNA hybridization detection offers a technology for real-time and label-free assessment of genetic markers outside of laboratory settings, such as at the point-of-care or in-field environmental monitoring.

  2. Development of DNA probes for Candida albicans

    International Nuclear Information System (INIS)

    Cheung, L.L.; Hudson, J.B.

    1988-01-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both 32 P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis

  3. Development of DNA probes for Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

  4. Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization

    Science.gov (United States)

    Jezbera, Jan; Jezberová, Jitka; Kasalický, Vojtěch; Šimek, Karel; Hahn, Martin W.

    2013-01-01

    Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S–23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria

  5. Patterns of Limnohabitans microdiversity across a large set of freshwater habitats as revealed by Reverse Line Blot Hybridization.

    Directory of Open Access Journals (Sweden)

    Jan Jezbera

    Full Text Available Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S-23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of

  6. A novel fluorescent in situ hybridization technique for detection of Rickettsia spp. in archival samples

    DEFF Research Database (Denmark)

    Svendsen, Claus Bo; Boye, Mette; Struve, Carsten

    2009-01-01

    A novel, sensitive and specific method for detecting Rickettsia spp. in archival samples is described. The method involves the use of fluorescently marked oligonucleotide probes for in situ hybridization. Specific hybridization of Ricekttsia was found without problems of cross-reactions with bact......A novel, sensitive and specific method for detecting Rickettsia spp. in archival samples is described. The method involves the use of fluorescently marked oligonucleotide probes for in situ hybridization. Specific hybridization of Ricekttsia was found without problems of cross...

  7. Interfacial transduction of nucleic acid hybridization using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Algar, W Russ; Krull, Ulrich J

    2009-01-06

    Fluorescence resonance energy transfer (FRET) using immobilized quantum dots (QDs) as energy donors was explored as a transduction method for the detection of nucleic acid hybridization at an interface. This research was motivated by the success of the QD-FRET-based transduction of nucleic acid hybridization in solution-phase assays. This new work represents a fundamental step toward the assembly of a biosensor, where immobilization of the selective chemistry on a surface is desired. After immobilizing QD-probe oligonucleotide conjugates on optical fibers, a demonstration of the retention of selectivity was achieved by the introduction of acceptor (Cy3)-labeled single-stranded target oligonucleotides. Hybridization generated the proximity required for FRET, and the resulting fluorescence spectra provided an analytical signal proportional to the amount of target. This research provides an important framework for the future development of nucleic acid biosensors based on QDs and FRET. The most important findings of this work are that (1) a QD-FRET solid-phase hybridization assay is viable and (2) a passivating layer of denatured bovine serum albumin alleviates nonspecific adsorption, ultimately resulting in (3) the potential for a reusable assay format and mismatch discrimination. In this, the first incarnation of a solid-phase QD-FRET hybridization assay, the limit of detection was found to be 5 nM, and the dynamic range was almost 2 orders of magnitude. Selective discrimination of the target was shown using a three-base-pairs mismatch from a fully complementary sequence. Despite a gradual loss of signal, reuse of the optical fibers over multiple cycles of hybridization and dehybridization was possible. Directions for further improvement of the analytical performance by optimizing the design of the QD-probe oligonucleotide interface are identified.

  8. Spaser as a biological probe

    Science.gov (United States)

    Galanzha, Ekaterina I.; Weingold, Robert; Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Nolan, Jacqueline; Harrington, Walter; Kuchyanov, Alexander S.; Parkhomenko, Roman G.; Watanabe, Fumiya; Nima, Zeid; Biris, Alexandru S.; Plekhanov, Alexander I.; Stockman, Mark I.; Zharov, Vladimir P.

    2017-06-01

    Understanding cell biology greatly benefits from the development of advanced diagnostic probes. Here we introduce a 22-nm spaser (plasmonic nanolaser) with the ability to serve as a super-bright, water-soluble, biocompatible probe capable of generating stimulated emission directly inside living cells and animal tissues. We have demonstrated a lasing regime associated with the formation of a dynamic vapour nanobubble around the spaser that leads to giant spasing with emission intensity and spectral width >100 times brighter and 30-fold narrower, respectively, than for quantum dots. The absorption losses in the spaser enhance its multifunctionality, allowing for nanobubble-amplified photothermal and photoacoustic imaging and therapy. Furthermore, the silica spaser surface has been covalently functionalized with folic acid for molecular targeting of cancer cells. All these properties make a nanobubble spaser a promising multimodal, super-contrast, ultrafast cellular probe with a single-pulse nanosecond excitation for a variety of in vitro and in vivo biomedical applications.

  9. Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

    Science.gov (United States)

    Rio, Donald C

    2015-03-02

    In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

  10. Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

    Directory of Open Access Journals (Sweden)

    Jouventin Pierre

    2010-05-01

    Full Text Available Abstract Background Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. Results We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. Conclusions MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.

  11. Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample.

    Science.gov (United States)

    Ootsubo, M; Shimizu, T; Tanaka, R; Sawabe, T; Tajima, K; Ezura, Y

    2003-01-01

    A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.

  12. Human serum albumin supported lipid patterns for the targeted recognition of microspheres coated by membrane based on ss-DNA hybridization

    International Nuclear Information System (INIS)

    Zhang Xiaoming; He Qiang; Cui Yue; Duan Li; Li Junbai

    2006-01-01

    Human serum albumin (HSA) patterns have been successfully fabricated for the deposition of lipid bilayer, 1,2-dimyristoyl-sglycerophosphate (DMPA), by making use of the micro-contact printing (μCP) technique and liposome fusion. Confocal laser scanning microscopy (CLSM) results indicate that lipid bilayer has been assembled in HSA patterns with a good stability. Such well-defined lipid patterns formed on HSA surface create possibility to incorporate specific components like channels or receptors for specific recognition. In view of this, microspheres coated with lipid membranes were immobilized in HSA-supported lipid patterns via the hybridization of complementary ss-DNAs. This procedure enables to transfer solid materials to a soft surface through a specific recognition

  13. Detection of c-myc amplification in formalin-fixed paraffin-embedded tumor tissue by chromogenic in situ hybridization (CISH).

    Science.gov (United States)

    Todorović-Raković, Nataša

    2013-01-01

    In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40 × magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology and the slides can be stored for a long time.

  14. New hybrid systems

    International Nuclear Information System (INIS)

    Bernardin, B.

    2001-01-01

    New hybrid systems are made up of a subcritical core, a spallation target and a proton accelerator. The neutrons that are produced in the target by the flux of protons are necessary to maintain the chain reaction of fission. Some parameters that are important for a classical nuclear reactor like doppler coefficient or delayed neutron fraction do not matter in a hybrid system. In a PWR-type reactor or in a fast reactor the concentration of actinides has a bad impact on these 2 parameters, so it is justified to study hybrid systems as actinide transmuters. The hybrid system, because of its external source of neutrons can put aside an important reactivity margin. This reactivity margin can be used to design safer nuclear reactors (particularly in some situations of reactivity accidents) or to irradiate fuel elements containing high concentrations of minor actinides that could not be allowed in a classical reactor. This article reviews various ways of integrating hybrid systems in a population of already existing nuclear reactors in order to manage quantities of plutonium, of minor actinides or of long-life fission products. (A.C.)

  15. Systems for hybrid cars

    Science.gov (United States)

    Bitsche, Otmar; Gutmann, Guenter

    Not only sharp competition but also legislation are pushing development of hybrid drive trains. Based on conventional internal combustion engine (ICE) vehicles, these drive trains offer a wide range of benefits from reduced fuel consumption and emission to multifaceted performance improvements. Hybrid electric drive trains may also facilitate the introduction of fuel cells (FC). The battery is the key component for all hybrid drive trains, as it dominates cost and performance issues. The selection of the right battery technology for the specific automotive application is an important task with an impact on costs of development and use. Safety, power, and high cycle life are a must for all hybrid applications. The greatest pressure to reduce cost is in soft hybrids, where lead-acid embedded in a considerate management presents the cheapest solution, with a considerable improvement in performance needed. From mild to full hybridization, an improvement in specific power makes higher costs more acceptable, provided that the battery's service life is equivalent to the vehicle's lifetime. Today, this is proven for the nickel-metal hydride system. Lithium ion batteries, which make use of a multiple safety concept, and with some development anticipated, provide even better prospects in terms of performance and costs. Also, their scalability permits their application in battery electric vehicles—the basis for better performance and enhanced user acceptance. Development targets for the batteries are discussed with a focus on system aspects such as electrical and thermal management and safety.

  16. Colorimetric detection of Ehrlichia canis via nucleic acid hybridization in gold nano-colloids.

    Science.gov (United States)

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

    2014-08-08

    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.

  17. Detection of DNA hybridization based on SnO2 nanomaterial enhanced fluorescence

    International Nuclear Information System (INIS)

    Gu Cuiping; Huang Jiarui; Ni Ning; Li Minqiang; Liu Jinhuai

    2008-01-01

    In this paper, enhanced fluorescence emissions were firstly investigated based on SnO 2 nanomaterial, and its application in the detection of DNA hybridization was also demonstrated. The microarray of SnO 2 nanomaterial was fabricated by the vapour phase transport method catalyzed by patterned Au nanoparticles on a silicon substrate. A probe DNA was immobilized on the substrate with patterned SnO 2 nanomaterial, respectively, by covalent and non-covalent linking schemes. When a fluorophore labelled target DNA was hybridized with a probe DNA on the substrate, fluorescence emissions were only observed on the surface of SnO 2 nanomaterial, which indicated the property of enhancing fluorescence signals from the SnO 2 nanomaterial. By comparing the different fluorescence images from covalent and non-covalent linking schemes, the covalent method was confirmed to be more effective for immobilizing a probe DNA. With the combined use of SnO 2 nanomaterial and the covalent linking scheme, the target DNA could be detected at a very low concentration of 10 fM. And the stability of SnO 2 nanomaterial under the experimental conditions was also compared with silicon nanowires. The findings strongly suggested that SnO 2 nanomaterial could be extensively applied in detections of biological samples with enhancing fluorescence property and high stability

  18. A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [¹⁸F]FB-ML5, as a molecular probe for non-invasive MMP/ADAM-targeted imaging.

    Science.gov (United States)

    Matusiak, Nathalie; Castelli, Riccardo; Tuin, Adriaan W; Overkleeft, Herman S; Wisastra, Rosalina; Dekker, Frank J; Prély, Laurette M; Bischoff, Rainer; Bischoff, Rainer P M; van Waarde, Aren; Dierckx, Rudi A J O; Elsinga, Philip H

    2015-01-01

    Numerous clinical studies have shown a correlation between increased matrix metalloproteinase (MMP)/a disintegrin and metalloproteinase (ADAM) activity and poor outcome of cancer. Various MMP inhibitors (MMPIs) have been developed for therapeutic purposes in oncology. In addition, molecular imaging of MMP/ADAM levels in vivo would allow the diagnosis of tumors. We selected the dual inhibitor of MMPs and ADAMs, ML5, which is a hydroxamate-based inhibitor with affinities for many MMPs and ADAMs. ML5 was radiolabelled with (18)F and the newly obtained radiolabelled inhibitor was evaluated in vitro and in vivo. ML5 was radiolabelled by direct acylation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) for PET (positron emission tomography). The resulting radiotracer [(18)F]FB-ML5 was evaluated in vitro in human bronchial epithelium 16HBE cells and breast cancer MCF-7 cells. The non-radioactive probe FB-ML5 and native ML5 were tested in a fluorogenic inhibition assay against MMP-2, -9, -12 and ADAM-17. The in vivo kinetics of [(18)F]FB-ML5 were examined in a HT1080 tumor-bearing mouse model. Specificity of probe binding was examined by co-injection of 0 or 2.5mg/kg ML5. ML5 and FB-ML5 showed high affinity for MMP-2, -9, -12 and ADAM-17; indeed IC50 values were respectively 7.4 ± 2.0, 19.5 ± 2.8, 2.0 ± 0.2 and 5.7 ± 2.2 nM and 12.5 ± 3.1, 31.5 ± 13.7, 138.0 ± 10.9 and 24.7 ± 2.8 nM. Radiochemical yield of HPLC-purified [(18)F]FB-ML5 was 13-16% (corrected for decay). Cellular binding of [(18)F]FB-ML5 was reduced by 36.6% and 27.5% in MCF-7 and 16 HBE cells, respectively, after co-incubation with 10 μM of ML5. In microPET scans, HT1080 tumors exhibited a low and homogeneous uptake of the tracer. Tumors of mice injected with [(18)F]FB-ML5 showed a SUVmean of 0.145 ± 0.064 (n=6) which decreased to 0.041 ± 0.027 (n=6) after target blocking (pFB-ML5 demonstrated rather low binding in ADAM-17 overexpressing cell lines. [(18)F]FB-ML5 uptake showed significant

  19. Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

    Science.gov (United States)

    Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

    2018-04-03

    A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

  20. A generic approach for expanding homolog-targeted residue screening of sulfonamides using a fast matrix separation and class-specific fragmentation-dependent acquisition with a hybrid quadrupole-linear ion trap mass spectrometer

    International Nuclear Information System (INIS)

    Huang Chunlin; Guo Bin; Wang Xiaoying; Li Jie; Zhu Weitao; Chen Bo; Ouyang Shan; Yao Shouzhuo

    2012-01-01

    Highlights: ► Generic homolog-targeted screening approach for multi-residual sulfonamide analogs. ► Single-tube extraction/partitioning-multifunction adsorption cleanup for direct injection. ► Class-specific fragmentation for expanding coverage of N 4 -acetyl and N-OH metabolites. ► PreS–IDA–EPI in LC–QqLIT for simultaneous screening and confirmation of real samples. - Abstract: A generic and efficient homolog-targeted approach was used to expand screening and detection of target class of sulfonamides and structural analogs, based on a fast single-tube extraction/partitioning-multifunction adsorption cleanup (SEP/MAC) for class-specific fragmentation-dependent acquisition with a liquid chromatography–hybrid triple-quadrupole linear ion trap mass spectrometer (LC–QqLIT). By combining the two-stage process conducted in a single tube as one-pot protocol, the straightforward SEP/MAC procedure was optimized to offer clean extracts with reasonable recovery (71–109% with RSDs 4 -acetyl and hydroxylamine metabolites plus their possible dimers. Moreover, the PreS-triggered automatically enhanced product ion spectral acquisition enabled simultaneous screening, profiling and confirmation of an unlimited number of analytes belonging to the sulfonamide class within a single analysis. The validation and application results of the generic SEP/MAC-based LC–QqLIT strategy consistently demonstrated favorable performances with acceptable accuracy (67–116%), precision (RSDs −1 ) to meet the acceptance criteria for all the s