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Sample records for fret-based gfp probes

  1. Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes.

    Science.gov (United States)

    Auer, Alexander; Strauss, Maximilian T; Schlichthaerle, Thomas; Jungmann, Ralf

    2017-10-11

    DNA point accumulation in nanoscale topography (DNA-PAINT) enables super-resolution microscopy by harnessing the predictable, transient hybridization between short dye-labeled "imager" and complementary target-bound "docking" strands. DNA-PAINT microscopy allows sub-5 nm spatial resolution, spectrally unlimited multiplexing, and quantitative image analysis. However, these abilities come at the cost of nonfluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concentrations, which in turn is dictated by the necessity to facilitate the detection of single-molecule binding events over the background of unbound, freely diffusing probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, we here introduce FRET-based imaging probes, alleviating the concentration-limit of imager strands and speeding up image acquisition by several orders of magnitude. We assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resolution imaging on DNA origami structures in a few tens of seconds. Finally, we also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resolution imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.

  2. Genetically Encoded Fluorescent Probe for Imaging Apoptosis in Vivo with Spontaneous GFP Complementation.

    Science.gov (United States)

    Nasu, Yusuke; Asaoka, Yoichi; Namae, Misako; Nishina, Hiroshi; Yoshimura, Hideaki; Ozawa, Takeaki

    2016-01-05

    Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Dysfunction of apoptosis is involved in many fatal diseases such as cancer. Visualization of apoptosis in living animals is necessary to understand the mechanism of apoptosis-related diseases. Here, we describe a genetically encoded fluorescent probe for imaging apoptosis in living multicellular organisms, based on spontaneous complementation of two fragments of a green fluorescent protein (GFP) variant (GFP OPT). The probe is designed for detection of mitochondria-mediated apoptosis during which a mitochondrial protein of Smac is released into cytosol. The Smac is connected with a carboxy-terminal fragment of GFP OPT (GFP11), whereas the remainder of GFP OPT (GFP(1-10)) is located in the cytosol. Under an apoptotic condition, the Smac is released from mitochondria into cytosol, allowing complementation of the GFP-OPT fragments and the emission of fluorescence. Live-cell imaging demonstrates that the probe enables detection of apoptosis in living cells with a high signal-to-background ratio. We applied the probe to living zebrafish, in which apoptotic cells were visualized with fluorescence. The technique provides a useful tool for the study of apoptosis in living animals, facilitating elucidation of the mechanisms of apoptosis-related diseases.

  3. A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

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    Nidhi Mathur

    2008-01-01

    Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

  4. Biological properties of coral GFP-type proteins provide clues for engineering novel optical probes and biosensors

    Science.gov (United States)

    Salih, Anya; Larkum, Anthony W.; Cronin, Thomas W.; Wiedenmann, Joerg; Szymczak, Ron; Cox, Guy C.

    2004-06-01

    In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.

  5. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

    International Nuclear Information System (INIS)

    Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O.

    2010-01-01

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties

  6. Sniffing for gene-silencing efficiency of siRNAs in HeLa cells in comparison with that in HEK293T cells: correlation between knockdown efficiency and sustainability of sirnas revealed by FRET-based probing.

    Science.gov (United States)

    Shin, Seonmi; Kim, Yea Seul; Kim, Jisu; Kwon, Hyun-Mi; Kim, Dong-Eun; Hah, Sang Soo

    2013-04-01

    Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.

  7. 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.

  8. [FRET-based biosensors in cell migration research].

    Science.gov (United States)

    Lasota, Sławomir; Baster, Zbigniew; Witko, Tomasz; Zimoląg, Eliza; Sroka, Jolanta; Rajfur, Zenon; Madeja, Zbigniew

    2017-01-01

    Cell migration is a complicated process, which is crucial for functioning of multicellular organisms. Multiple signalling pathways are deeply involved in the precise control of consecutive cell migration stages based on remodelling of the actin cytoskeleton. Small Rho GTPases (RhoA, Rac1 and Cdc42) as well as multiple protein and lipid kinases, calcium ions and mechanosensors are crucial components in this process. Exploration of those complicated correlations is possible with constant advancement of fluorescence microscopy. A significant progress in this field has been achieved since discovery of fluorescent proteins and subsequently FRET-based biosensors. Such protein constructs react with a change of FRET efficiency in response to the particular protein activity change. Properly designed and regularly improved biosensors offer the possibility of real-time imaging of signalling pathways dynamics in migrating cells. The perception of Rho GTPases involvement and some other signalling pathways connected with cell migration have been clarified with multiple experiments already carried out with such FRET-based biosensors.

  9. FRET-based detection of isozyme-specific activities of transglutaminases.

    Science.gov (United States)

    Tatsukawa, Hideki; Liu, Hong Hong; Oba, Shota; Kamiya, Noriho; Nakanishi, Yoichi; Hitomi, Kiyotaka

    2017-03-01

    Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

  10. Probing microhydration effect on the electronic structure of the GFP chromophore anion: Photoelectron spectroscopy and theoretical investigations

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskaran-Nair, Kiran; Shelton, William A. [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803 (United States); Center for Computation and Technology, Louisiana State University, Baton Rouge, Louisiana 70803 (United States); Valiev, Marat; Kowalski, Karol, E-mail: karol.kowalski@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, K8-91, P.O. Box 999, Richland, Washington 99352 (United States); Deng, S. H. M.; Wang, Xue-Bin, E-mail: xuebin.wang@pnnl.gov [Physical Sciences Division, Pacific Northwest National Laboratory, K8-88, P.O. Box 999, Richland, Washington 99352 (United States)

    2015-12-14

    The photophysics of the Green Fluorescent Protein (GFP) chromophore is critically dependent on its local structure and on its environment. Despite extensive experimental and computational studies, there remain many open questions regarding the key fundamental variables that govern this process. One outstanding problem is the role of autoionization as a possible relaxation pathway of the excited state under different environmental conditions. This issue is considered in our work through combined experimental and theoretical studies of microsolvated clusters of the deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI{sup −}), an analog of the GFP chromophore. Through selective generation of microsolvated structures of predetermined size and subsequent analysis of experimental photoelectron spectra by high level ab initio methods, we are able to precisely identify the structure of the system, establish the accuracy of theoretical data, and provide reliable description of auto-ionization process as a function of hydrogen-bonding environment. Our study clearly illustrates the first few water molecules progressively stabilize the excited state of the chromophore anion against the autodetached neutral state, which should be an important trait for crystallographic water molecules in GFPs that has not been fully explored to date.

  11. Probing microhydration effect on the electronic structure of the GFP chromophore anion. Photoelectron spectroscopy and theoretical investigations

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskaran-Nair, Kiran; Valiev, Marat; Deng, Shihu; Shelton, William A.; Kowalski, Karol; Wang, Xue B.

    2015-12-14

    The photophysics of Green Fluorescent Protein (GFP) chromophore is critically dependent on its local structure and on its environment. Despite extensive experimental and computational studies, there remain many open questions regarding the key fundamental variables that govern this process. One outstanding problem is the role of autoionization as a possible relaxation pathway of the excited state under different environmental conditions. This issue is considered in our work through combined experimental and theoretical studies of microsolvated clusters of the deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI⁻), an analog of GFP chromophore. Through selective generation of microsolvated structures of predetermined size and subsequent analysis of experimental photoelectron spectra by high level ab-initio methods we are able to precisely identify the structure of the system, establish the accuracy of theoretical data, and provide reliable description of auto-ionization process as a function of hydrogen-bonding environment. Our study clearly illustrates the first few water molecules progressively stabilize the excited state of the chromophore anion against the autodetached neutral state, which should be an important trait for crystallographic water molecules in GFPs that has not been fully explored to date.

  12. Probing microhydration effect on the electronic structure of the GFP chromophore anion: Photoelectron spectroscopy and theoretical investigations

    Science.gov (United States)

    Bhaskaran-Nair, Kiran; Valiev, Marat; Deng, S. H. M.; Shelton, William A.; Kowalski, Karol; Wang, Xue-Bin

    2015-12-01

    The photophysics of the Green Fluorescent Protein (GFP) chromophore is critically dependent on its local structure and on its environment. Despite extensive experimental and computational studies, there remain many open questions regarding the key fundamental variables that govern this process. One outstanding problem is the role of autoionization as a possible relaxation pathway of the excited state under different environmental conditions. This issue is considered in our work through combined experimental and theoretical studies of microsolvated clusters of the deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI-), an analog of the GFP chromophore. Through selective generation of microsolvated structures of predetermined size and subsequent analysis of experimental photoelectron spectra by high level ab initio methods, we are able to precisely identify the structure of the system, establish the accuracy of theoretical data, and provide reliable description of auto-ionization process as a function of hydrogen-bonding environment. Our study clearly illustrates the first few water molecules progressively stabilize the excited state of the chromophore anion against the autodetached neutral state, which should be an important trait for crystallographic water molecules in GFPs that has not been fully explored to date.

  13. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

    Directory of Open Access Journals (Sweden)

    Potzkei Janko

    2012-03-01

    Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.

  14. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

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    Zamaleeva, Alsu I.; Despras, Guillaume; Luccardini, Camilla; Collot, Mayeul; de Waard, Michel; Oheim, Martin; Mallet, Jean-Maurice; Feltz, Anne

    2015-01-01

    Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging. PMID:26404317

  15. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

    Directory of Open Access Journals (Sweden)

    Alsu I. Zamaleeva

    2015-09-01

    Full Text Available Semiconductor nanocrystals (NCs or quantum dots (QDs are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxyethane-N,N,N′,N′-tetra-acetic acid for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging.

  16. Optimization of input parameters of acoustic-transfection for the intracellular delivery of macromolecules using FRET-based biosensors

    Science.gov (United States)

    Yoon, Sangpil; Wang, Yingxiao; Shung, K. K.

    2016-03-01

    Acoustic-transfection technique has been developed for the first time. We have developed acoustic-transfection by integrating a high frequency ultrasonic transducer and a fluorescence microscope. High frequency ultrasound with the center frequency over 150 MHz can focus acoustic sound field into a confined area with the diameter of 10 μm or less. This focusing capability was used to perturb lipid bilayer of cell membrane to induce intracellular delivery of macromolecules. Single cell level imaging was performed to investigate the behavior of a targeted single-cell after acoustic-transfection. FRET-based Ca2+ biosensor was used to monitor intracellular concentration of Ca2+ after acoustic-transfection and the fluorescence intensity of propidium iodide (PI) was used to observe influx of PI molecules. We changed peak-to-peak voltages and pulse duration to optimize the input parameters of an acoustic pulse. Input parameters that can induce strong perturbations on cell membrane were found and size dependent intracellular delivery of macromolecules was explored. To increase the amount of delivered molecules by acoustic-transfection, we applied several acoustic pulses and the intensity of PI fluorescence increased step wise. Finally, optimized input parameters of acoustic-transfection system were used to deliver pMax-E2F1 plasmid and GFP expression 24 hours after the intracellular delivery was confirmed using HeLa cells.

  17. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

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    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  18. Highly sensitive fluorescence resonance energy transfer (FRET)-based nanosensor for rapid detection of clenbuterol

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    Nghia Nguyen, Duc; Ngo, Trinh Tung; Liem Nguyen, Quang

    2012-09-01

    In this study we investigate the fabrication of a fluorescence resonance energy transfer (FRET)-based nanosensor for the detection of clenbuterol. The nanosensor consists of CdTe quantum dots coated by clenbuterol recognizable agent naphthol and diazotized clenbuterol. Changes in maximal photoluminescent intensities of the nanosensor were utilized to measure clenbuterol concentrations. The maximal photoluminescent intensities of the nanosensor were found to decrease with increasing clenbuterol concentrations, following a linear correlation. We have successfully fabricated a nanosensor for detection of clenbuterol with sensitivity up to 10 pg ml-1.

  19. Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.

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    Woehler, Andrew

    2013-01-01

    We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+)], [cAMP], and PKA activity.

  20. Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.

    Directory of Open Access Journals (Sweden)

    Andrew Woehler

    Full Text Available We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+], [cAMP], and PKA activity.

  1. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H. [School of Chemistry, University of Edinburgh, The King' s Buildings, Edinburgh, EH9 3JJ (United Kingdom); Dryden, David T.F., E-mail: david.dryden@ed.ac.uk [School of Chemistry, University of Edinburgh, The King' s Buildings, Edinburgh, EH9 3JJ (United Kingdom)

    2010-07-23

    Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  2. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex.

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    Kubala, Marta H; Kovtun, Oleksiy; Alexandrov, Kirill; Collins, Brett M

    2010-12-01

    The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody. Copyright © 2010 The Protein Society.

  3. Recapture of GFP chromophore fluorescence in a protein host.

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    Baldridge, Anthony; Feng, Suihan; Chang, Young-Tae; Tolbert, Laren M

    2011-05-09

    When encapsulated by human serum albumin (HSA), certain derivatives of the green fluorescent protein (GFP) chromophore recover their fluorescence due to inhibition of torsional motion. These derivatives show remarkable sensitivity and selectivity as well as favorable spectroscopic properties toward HSA, thus providing selective probes for this and similar proteins and demonstrating the use of GFP chromophores as topological fluorophores.

  4. FRET-based modified graphene quantum dots for direct trypsin quantification in urine

    Energy Technology Data Exchange (ETDEWEB)

    Poon, Chung-Yan; Li, Qinghua [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Zhang, Jiali; Li, Zhongping [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Dong, Chuan [Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Lee, Albert Wai-Ming; Chan, Wing-Hong [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Li, Hung-Wing, E-mail: hwli@hkbu.edu.hk [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong)

    2016-04-21

    A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening. - Highlights: • A FRET-based biosensor was developed for direct quantification of trypsin. • Fast and sensitive screening of pancreatic disease was facilitated. • The direct quantification of trypsin in urine samples was demonstrated.

  5. Detection of gfp expression from gfp-labelled bacteria spot ...

    African Journals Online (AJOL)

    Green fluorescent protein (GFP) as a marker gene has facilitated biological research in plant-microbe interactions. However, there is one major limiting factor in the detection of GFP in living organisms whose cells emit background autofluorescence. In this study, Herbaspirillum sp. B501gfp1 bacterial cells were spot ...

  6. GFP as potential cellular viscosimeter

    NARCIS (Netherlands)

    Visser, Antonie; Westphal, A.H.; Skakun, V.V.; Borst, J.W.

    2016-01-01

    The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the

  7. GFP as potential cellular viscosimeter

    Science.gov (United States)

    Visser, Antonie J. W. G.; Westphal, Adrie H.; Skakun, Victor V.; Borst, Jan Willem

    2016-09-01

    The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the solvent to flow, which is known as macroscopic viscosity. GFP in water/glycerol mixtures senses this macroscopic viscosity, because the translational and rotational diffusion coefficients are proportional to the reciprocal value of the viscosity as predicted by the Stokes-Einstein equations. To test this hypothesis, we have performed time-resolved fluorescence anisotropy (reporting on rotational diffusion) and fluorescence correlation spectroscopy (reporting on translational diffusion) experiments of GFP in water/glycerol mixtures. When the solvent also contains macromolecules of similar or larger dimensions as GFP, the microscopic and macroscopic viscosities can be markedly different and the Stokes-Einstein relations must be adapted. It was established from previous dynamic fluorescence spectroscopy observations of diffusing proteins with dextran polysaccharides as co-solvents (Lavalette et al 2006 Eur. Biophys. J. 35 517-22), that rotation and translation sense a different microscopic viscosity, in which the one arising from rotation is always less than that from translation. A microscopic viscosity parameter is defined that depends on scaling factors between GFP and its immediate environment. The direct consequence is discussed for two reported diffusion coefficients of GFP in living cells.

  8. Real Time FRET Based Detection of Mechanical Stress in Cytoskeletal and Extracellular Matrix Proteins.

    Science.gov (United States)

    Meng, Fanjie; Suchyna, Thomas M; Lazakovitch, Elena; Gronostajski, Richard M; Sachs, Frederick

    2011-06-01

    A molecular force sensing cassette (stFRET) was incorporated into actinin, filamin, and spectrin in vascular endothelial cells (BAECs) and into collagen-19 in Caenorhabditis elegans. To estimate the stress sensitivity of stFRET in solution, we used DNA springs. A 60-mer loop of single stranded DNA was covalently linked to the external cysteines of the donor and acceptor. When the complementary DNA was added it formed double stranded DNA with higher persistence length, stretching the linker and substantially reducing FRET efficiency. The probe stFRET detected constitutive stress in all cytoskeletal proteins tested, and in migrating cells the stress was greater at the leading edge than the trailing edge. The stress in actinin, filamin and spectrin could be reduced by releasing focal attachments from the substrate with trypsin. Inhibitors of actin polymerization produced a modest increase in stress on the three proteins suggesting they are mechanically in parallel. Local shear stress applied to the cell with a perfusion pipette showed gradients of stress leading from the site of perfusion. Transgenic C. elegans labeled in collagen-19 produced a behaviorally and anatomically normal animal with constitutive stress in the cuticle. Stretching the worm visibly stretched the probe in collagen showing that we can trace the distribution of mean tissue stress in specific molecules. stFRET is a general purpose dynamic sensor of mechanical stress that can be expressed intracellularly and extracellularly in isolated proteins, cells, tissues, organs and animals.

  9. Molecularly imprinted fluorescent probe based on FRET for selective and sensitive detection of doxorubicin

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhifeng, E-mail: 897061147@qq.com [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China); Deng, Peihong; Li, Junhua [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China); Xu, Li [Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou 510642 (China); Tang, Siping [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China)

    2017-04-15

    Highlights: • FRET-based molecularly imprinted probe for detection of doxorubicin was prepared. • The detection limit of the probe was 13.8 nM for doxorubicin. • The FRET-based probe had a higher selectivity for the template than ordinary MIMs. - Abstract: In this work, a new type of fluorescent probe for detection of doxorubicin has been constructed by the combined use of fluorescence resonance energy transfer (FRET) technology and molecular imprinting technique (MIT). Using doxorubicin as the template, the molecularly imprinted polymer thin layer was fabricated on the surfaces of carbon dot (CD) modified silica by sol-gel polymerization. The excitation energy of the fluorescent donor (CDs) could be transferred to the fluorescent acceptor (doxorubicin). The FRET based fluorescent probe demonstrated high sensitivity and selectivity for doxorubicin. The detection limit was 13.8 nM. The fluorescent probe was successfully applied for detecting doxorubicin in doxorubicin-spiked plasmas with a recovery of 96.8–103.8%, a relative standard deviation (RSD) of 1.3–2.8%. The strategy for construction of FRET-based molecularly imprinted materials developed in this work is very promising for analytical applications.

  10. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    Science.gov (United States)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-09-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  11. Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity

    Directory of Open Access Journals (Sweden)

    Alberola-Ila José

    2005-07-01

    Full Text Available Abstract Background Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. Conclusion EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.

  12. FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics.

    Science.gov (United States)

    Krebs, Melanie; Held, Katrin; Binder, Andreas; Hashimoto, Kenji; Den Herder, Griet; Parniske, Martin; Kudla, Jörg; Schumacher, Karin

    2012-01-01

    Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  13. One-step synthesis of DNA functionalized cadmium-free quantum dots and its application in FRET-based protein sensing

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Cuiling, E-mail: clzhang@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Ding, Caiping [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Zhou, Guohua [School of Chemistry and Chemical Engineering, Lingnan Normal University, Zhanjiang, 524048 (China); Xue, Qin [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Xian, Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China)

    2017-03-08

    DNA functionalized quantum dots (QDs) are promising nanoprobes for the fluorescence resonance energy transfer (FRET)-based biosensing. Herein, cadmium-free DNA functionalized Mn-doped ZnS (DNA-ZnS:Mn{sup 2+}) QDs were successfully synthesized by one-step route. As-synthesized QDs show excellent photo-stability with the help of PAA and DNA. Then, we constructed a novel FRET model based on the QDs and WS{sub 2} nanosheets as the energy donor-acceptor pairs, which was successfully applied for the protein detection through the terminal protection of small molecule-linked DNA assay. This work not only explores the potential bioapplication of the DNA-ZnS:Mn{sup 2+} QDs, but also provides a platform for the investigation of small molecule-protein interaction. - Highlights: • The stable and cadmium-free DNA functionalized ZnS:Mn{sup 2+} QDs were successfully synthesized through a facile one-step route. • We constructed a novel FRET system based on one-step synthesized DNA-ZnS:Mn{sup 2+} QDs (donor) and WS{sub 2} nanosheets (acceptor). • The FRET-based strategy was applied for the detection of streptavidin and folate receptor by combining TPSMLD and Exo III.

  14. Green fluorescent protein (GFP): is seeing believing and is that enough?

    Science.gov (United States)

    Shorter, Susan A; Pettit, Marie W; Dyer, Paul D R; Coakley Youngs, Emma; Gorringe-Pattrick, Monique A M; El-Daher, Samer; Richardson, Simon

    Intracellular compartmentalisation is a significant barrier to the successful nucleocytosolic delivery of biologics. The endocytic system has been shown to be responsible for compartmentalisation, providing an entry point, and trigger(s) for the activation of drug delivery systems. Consequently, many of the technologies used to understand endocytosis have found utility within the field of drug delivery. The use of fluorescent proteins as markers denoting compartmentalisation within the endocytic system has become commonplace. Several of the limitations associated with the use of green fluorescent protein (GFP) within the context of drug delivery have been explored here by asking a series of related questions: (1) Are molecules that regulate fusion to a specific compartment (i.e. Rab- or SNARE-GFP fusions) a good choice of marker for that compartment? (2) How reliable was GFP-marker overexpression when used to define a given endocytic compartment? (3) Can glutathione-s-transferase (GST) fused in frame with GFP (GST-GFP) act as a fluid phase endocytic probe? (4) Was GFP fluorescence a robust indicator of (GFP) protein integrity? This study concluded that there are many appropriate and useful applications for GFP; however, thought and an understanding of the biological and physicochemical character of these markers are required for the generation of meaningful data.

  15. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.

    Science.gov (United States)

    Westphal, M; Jungbluth, A; Heidecker, M; Mühlbauer, B; Heizer, C; Schwartz, J M; Marriott, G; Gerisch, G

    1997-03-01

    The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum. Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent. Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

  16. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

    International Nuclear Information System (INIS)

    Hsu, Y.-Y.; Liu, Y.-N.; Wang Wenyen; Kao, Fu-Jen; Kung, S.-H.

    2007-01-01

    An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP 2 )-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A pro ) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A pro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research

  17. YGFP: a spectral variant of GFP

    DEFF Research Database (Denmark)

    Hansen, Flemming G.; Atlung, Tove

    2011-01-01

    We describe YGFP, a slow bleaching green fluorescent protein (GFP) with unique spectral properties. YGFP is derived from an Escherichia coli codon-optimized synthetic gfp, mutant 2 derivative. In addition to the GFP-mut 2 changes, it also carries S202F and T203I substitutions. YGFP can be used...... as a substitute for yellow fluorescent protein (YFP) in experiments in which two or more fluorescent proteins are fused to different cellular protein components, expanding the ability to study multiple labeled proteins in a cell at once....

  18. A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase

    Science.gov (United States)

    2009-10-22

    inMaterials andMethods. (C)Rapid kinetics ofC-BR(L)-Y folding (9) andunfolding (b) at the indicated CaCl2 concentrations was measured using FRET as...described inMaterials andMethods. Error bars represent standard errors (Ng 3). Representative fluorescence time course traces are shown inFigure S1 of...aContour length (lc) between the chromophores of the FPs, calculated as described inMaterials andMethods. bAverage end-to-end distance of a theoretical

  19. Development of a Fluorescence Resonance Energy Transfer (FRET-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

    Directory of Open Access Journals (Sweden)

    Noremylia Mohd Bakhori

    2013-12-01

    Full Text Available An optical DNA biosensor based on fluorescence resonance energy transfer (FRET utilizing synthesized quantum dot (QD has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

  20. GFP Bunny: a coelhinha transgênica

    Directory of Open Access Journals (Sweden)

    Eduardo Kac

    2008-11-01

    Full Text Available GFP Bunny é um trabalho de arte transgênica que compreende a criação de um coelho verde fluorescente por meio da proteí­na que lhe confere essa cor, o diálogo público gerado pelo projeto e a integração social da coelha. GFP Bunny foi realizado em 2000 e apresentado publicamente pela primeira vez em Avignon, França. Esse trabalho foi proposto como uma nova forma de arte decorrente do uso de engenharia genéticana transferência de genes naturais ou sintéticos para um organismo com o objetivo de criar seres vivos únicos. Um trabalho que requer o máximo de cuidado, de consciência do grau de complexidade das questões que ele provoca e, acima de tudo, de compromisso para respeitar, cuidar e amar a vida criada.Abstract: GPF Bunny is a trasgenic artwork comprises the creation of a green fluorescent rabbit, its social integration, and the ensuing public debate. GFP Bunny was realized in 2000 and first presented publicity in Avignon, France. This work was proposed as a newart form based on the use of genetic engineering to transfer natural or synthetic genes to an organism, to create unique living beings. This must be done with great care, with acknowledgment of the complex issues thus raised and, above all, with a commitment to respect, nurture, and love the life thus created.

  1. Variable expression of GFP in different populations of peripheral cholinergic neurons of ChATBAC-eGFP transgenic mice.

    Science.gov (United States)

    Brown, T Christopher; Bond, Cherie E; Hoover, Donald B

    2018-03-01

    Immunohistochemistry is used widely to identify cholinergic neurons, but this approach has some limitations. To address these problems, investigators developed transgenic mice that express enhanced green fluorescent protein (GFP) directed by the promoter for choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme. Although, it was reported that these mice express GFP in all cholinergic neurons and non-neuronal cholinergic cells, we could not detect GFP in cardiac cholinergic nerves in preliminary experiments. Our goals for this study were to confirm our initial observation and perform a qualitative screen of other representative autonomic structures for the presences of GFP in cholinergic innervation of effector tissues. We evaluated GFP fluorescence of intact, unfixed tissues and the cellular localization of GFP and vesicular acetylcholine transporter (VAChT), a specific cholinergic marker, in tissue sections and intestinal whole mounts. Our experiments identified two major tissues where cholinergic neurons and/or nerve fibers lacked GFP: 1) most cholinergic neurons of the intrinsic cardiac ganglia and all cholinergic nerve fibers in the heart and 2) most cholinergic nerve fibers innervating airway smooth muscle. Most cholinergic neurons in airway ganglia stained for GFP. Cholinergic systems in the bladder and intestines were fully delineated by GFP staining. GFP labeling of input to ganglia with long preganglionic projections (vagal) was sparse or weak, while that to ganglia with short preganglionic projections (spinal) was strong. Total absence of GFP might be due to splicing out of the GFP gene. Lack of GFP in nerve projections from GFP-positive cell bodies might reflect a transport deficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae).

    Science.gov (United States)

    Allen, Margaret L; Christensen, Bruce M

    2004-12-01

    A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A GFP marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus. Transformation was confirmed by Mendelian genetics. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the flight muscles.

  3. Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection.

    Science.gov (United States)

    Abbady, Abdul Qader; Twair, Aya; Ali, Bouthaina; Murad, Hossam

    2017-01-01

    Programed cell death is a critical and unavoidable part of life. One of the most widely used markers for dying cells, by apoptosis or pyroptosis, is the redistribution of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Annexin V protein is a sensitive and specific probe to mark this event because of its high affinity to the exposed PS. Beyond that, annexin V can bind to any PS-containing phospholipid bilayer of almost all tiny forms of membranous vesicles like blood platelets, exosomes, or even nanostructured liposomes. In this work, recombinant human annexin V was produced as a fusion with a highly fluorescent superfolder derivative of the green fluorescent protein ( sf GFP) in Escherichia coli . The fusion protein( sf GFP-ANXV, 64 kDa), annexin V (ANXV, 40 kDa), and sf GFP (27 kDa) were separately produced after cloning their encoding genes in pRSET plasmid, and all proteins were expressed in a soluble form, then purified in high yields because of their N -terminal 6× His tag (~150 mg of pure protein per 1 L culture). Superiority of this fluorescent fusion protein over fluorescein-conjugated annexin V was demonstrated in binding to phospholipids (and their liposomes), prepared from natural sources (soya bean and egg yolk) that have different content of PS, by using different methods including ELISA, dot-blotting, surface plasmon resonance, and flow cytometry. We also applied fluorescent annexin V in the detection of apoptotic cells by flow cytometry and fluorescent microscopy. Interestingly, sf GFP-ANXV fusion was more sensitive to early apoptotic stressed HeLa cells than fluorescein-conjugated-ANXV. This highly expressed and functional sf GFP-ANXV fusion protein provides a promising ready-to-use molecular tool for quantifying liposomes (or similarly exosomes) and detecting apoptosis in cells.

  4. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    Science.gov (United States)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2008-12-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  5. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    International Nuclear Information System (INIS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yamamoto, Kenji; Yasuhara, Masato; Kondo, Akihiko

    2008-01-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  6. Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining

    Directory of Open Access Journals (Sweden)

    Rohr Karl

    2007-12-01

    Full Text Available Abstract Background GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH. Direct comparisons of these detection methods are scarce, however. Results We provide a quantitative comparison of all three approaches. We make use of a cell line that contains a transgene array of lac operator repeats which are detected by GFP-lac repressor fusion proteins. Thus we can detect the same structure in individual cells by GFP fluorescence, by antibodies against GFP and by FISH with a probe against the transgene array. Anti-GFP antibody detection was repeated after FISH. Our results show that while all four signals obtained from a transgene array generally showed qualitative and quantitative similarity, they also differed in details. Conclusion Each of the tested methods revealed particular strengths and weaknesses, which should be considered when interpreting respective experimental results. Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.

  7. A two-photon fluorescent turn-on probe for imaging of SO2 derivatives in living cells and tissues

    International Nuclear Information System (INIS)

    Zhu, Xiaoyan; Zhu, Longming; Liu, Hong-wen; Hu, Xiaoxiao; Peng, Rui-zi; Zhang, Jing; Zhang, Xiao-Bing; Tan, Weihong

    2016-01-01

    SO 2 and its derivatives (bisulfite/sulfite) play crucial roles in several physiological processes. Therefore, development of reliable analytical methods for monitoring SO 2 and its derivatives in biological systems is very significant. In this paper, a FRET-based two-photon fluorescent turn-on probe, A-HCy, was proposed for specific detection of SO 2 derivatives through the bisulfite/sulfite-promoted Michael addition reaction. In this FRET system, an acedan (2-acetyl-6-dialkylaminonaphthalene) moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite. A-HCy exhibited excellent selectivity and rapid response to HSO 3 − with a detection limit of 0.24 μM. More importantly, probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO 2 derivatives in living cells and tissues, suggesting its great potential for practical application in biological systems. - Highlights: • A FRET-based two-photon fluorescent probe for detecting SO 2 derivatives was proposed. • The probe shows high sensitivity, excellent selectivity, rapid response as well as low cytotoxicity. • The probe was capable of detecting SO 2 derivatives up to 170 μm depth in tissues.

  8. Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

    Science.gov (United States)

    Balic, Anamaria; Mina, Mina

    2011-01-01

    Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

  9. Energy profile of nanobody–GFP complex under force

    International Nuclear Information System (INIS)

    Klamecka, Kamila; Severin, Philip M; Milles, Lukas F; Gaub, Hermann E; Leonhardt, Heinrich

    2015-01-01

    Nanobodies (Nbs)—the smallest known fully functional and naturally occuring antigen-binding fragments—have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb–green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that—despite identical epitopes—the Nb binds stronger (41–56 pN) to enhanced GFP than to wild-type GFP (28–45 pN). Measured forces make the Nb–GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo. (paper)

  10. Energy profile of nanobody-GFP complex under force

    Science.gov (United States)

    Klamecka, Kamila; Severin, Philip M.; Milles, Lukas F.; Gaub, Hermann E.; Leonhardt, Heinrich

    2015-10-01

    Nanobodies (Nbs)—the smallest known fully functional and naturally occuring antigen-binding fragments—have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb-green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that—despite identical epitopes—the Nb binds stronger (41-56 pN) to enhanced GFP than to wild-type GFP (28-45 pN). Measured forces make the Nb-GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo.

  11. GFP fluorescence imaging with laser confocal scanning microscope

    Science.gov (United States)

    Yu, Yanhua; Xing, Da; Shi, Qiaojuan; Zhou, Junchu

    1999-09-01

    With gene marking technique, green fluorescent protein (GFP) and nodule bacteria gene has been linked together to form a single fusion gene expression vector. Then the vector is transferred into the nodule bacteria and the astragalus sinicus is invaded by the vector. With laser confocal scanning microscope, some important morphological information in plant nitrogen fixation research about the invading of nodule bacteria and the formation process of root nodule has been obtained through the 3D imaging of GFP reporting fluorescence.

  12. Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP).

    Science.gov (United States)

    Huber, Reinhard C; Remuge, Liliana; Carlisle, Ailsa; Lillico, Simon; Sandøe, Peter; Sørensen, Dorte B; Whitelaw, C Bruce A; Olsson, I Anna S

    2012-08-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology--animal welfare--has not been approached through systematic assessment and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals along various stages of post natal development. The protocol used covered reproductory performance and behaviour in GFP and wildtype sows and general health and development, social behaviour, exploratory behaviour and emotionality in GFP and wildtype littermates from birth until an age of roughly 4 months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs expressing GFP as healthy. Although the results are not surprising in the light of previous experience, they give a more solid fundament to the evaluation of GFP expression as being relatively non-invasive in pigs. The present study may furthermore serve as starting point for researchers aiming at a systematic characterization of welfare relevant effects in the line of transgenic pigs they are working with.

  13. H-NS silences gfp, the green fluorescent protein gene: gfpTCD is a genetically Remastered gfp gene with reduced susceptibility to H-NS-mediated transcription silencing and with enhanced translation.

    Science.gov (United States)

    Corcoran, Colin P; Cameron, Andrew D S; Dorman, Charles J

    2010-09-01

    The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein.

  14. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

  15. A two-photon fluorescent turn-on probe for imaging of SO{sub 2} derivatives in living cells and tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Xiaoyan; Zhu, Longming; Liu, Hong-wen; Hu, Xiaoxiao; Peng, Rui-zi; Zhang, Jing; Zhang, Xiao-Bing, E-mail: xbzhang@hnu.edu.cn; Tan, Weihong, E-mail: tan@chem.ufl.edu

    2016-09-21

    SO{sub 2} and its derivatives (bisulfite/sulfite) play crucial roles in several physiological processes. Therefore, development of reliable analytical methods for monitoring SO{sub 2} and its derivatives in biological systems is very significant. In this paper, a FRET-based two-photon fluorescent turn-on probe, A-HCy, was proposed for specific detection of SO{sub 2} derivatives through the bisulfite/sulfite-promoted Michael addition reaction. In this FRET system, an acedan (2-acetyl-6-dialkylaminonaphthalene) moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite. A-HCy exhibited excellent selectivity and rapid response to HSO{sub 3}{sup −} with a detection limit of 0.24 μM. More importantly, probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO{sub 2} derivatives in living cells and tissues, suggesting its great potential for practical application in biological systems. - Highlights: • A FRET-based two-photon fluorescent probe for detecting SO{sub 2} derivatives was proposed. • The probe shows high sensitivity, excellent selectivity, rapid response as well as low cytotoxicity. • The probe was capable of detecting SO{sub 2} derivatives up to 170 μm depth in tissues.

  16. Metallization and investigation of electrical properties of in vitro recrystallized mSbsC-eGFP assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Korkmaz, Nuriye; Roedel, Gerhard [Institut fuer Genetik, Technische Universitaet Dresden, 01217 Dresden (Germany); Boerrnert, Felix; Mendes, Rafael G; Bachmatiuk, Alicja; Ruemmeli, Mark H; Buechner, Bernd [Leibniz-Institut fuer Festkoerper- und Werkstoffforschung Dresden, 01069 Dresden (Germany); Koehler, Denny; Eng, Lukas M, E-mail: korkmaz@yahoo.com, E-mail: f.boerrnert@ifw-dresden.de, E-mail: denny.koehler@iapp.de, E-mail: r.g.mendes@ifw-dresden.de, E-mail: a.bachmatiuk@ifw-dresden.de, E-mail: m.ruemmeli@ifw-dresden.de, E-mail: b.buechner@ifw-dresden.de, E-mail: lukas.eng@iapp.de, E-mail: gerhard.roedel@tu-dresden.de [Institut fuer Angewandte Photophysik, Technische Universitaet Dresden, 01062 Dresden (Germany)

    2011-09-16

    Surface layer (SL) proteins are self-assembling nanosized arrays which can be recrystallized in solution or on surfaces. In this paper, we investigate the metallization, contact potential difference and conductivity of in vitro recrystallized mSbsC-eGFP tube-like assemblies for possible applications in nanobiotechnology. Treatment of mSbsC-eGFP tube-like structures with 150 mM Pt salt solution resulted in the formation of metallized SL assemblies decorated with Pt nanoparticles (> 3 nm) which were closely packed and aggregated into metal clusters. Kelvin probe force microscopy (KPFM) measurements revealed that metallized and unmetallized SL templates showed different surface potential behaviours, demonstrating that the metal coating changes the electrostatic surface characteristics of SL assemblies. In situ conductivity measurements showed that unmetallized SL assemblies were not conductive. Metallized samples showed linear I-V dependence between - 1 and + 1 V with a conductivity of {approx} 10{sup 3} S m{sup -1}.

  17. Metallization and investigation of electrical properties of in vitro recrystallized mSbsC-eGFP assemblies

    International Nuclear Information System (INIS)

    Korkmaz, Nuriye; Roedel, Gerhard; Boerrnert, Felix; Mendes, Rafael G; Bachmatiuk, Alicja; Ruemmeli, Mark H; Buechner, Bernd; Koehler, Denny; Eng, Lukas M

    2011-01-01

    Surface layer (SL) proteins are self-assembling nanosized arrays which can be recrystallized in solution or on surfaces. In this paper, we investigate the metallization, contact potential difference and conductivity of in vitro recrystallized mSbsC-eGFP tube-like assemblies for possible applications in nanobiotechnology. Treatment of mSbsC-eGFP tube-like structures with 150 mM Pt salt solution resulted in the formation of metallized SL assemblies decorated with Pt nanoparticles ( > 3 nm) which were closely packed and aggregated into metal clusters. Kelvin probe force microscopy (KPFM) measurements revealed that metallized and unmetallized SL templates showed different surface potential behaviours, demonstrating that the metal coating changes the electrostatic surface characteristics of SL assemblies. In situ conductivity measurements showed that unmetallized SL assemblies were not conductive. Metallized samples showed linear I-V dependence between - 1 and + 1 V with a conductivity of ∼ 10 3 S m -1 .

  18. A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors

    Directory of Open Access Journals (Sweden)

    Godement Pierre

    2007-11-01

    Full Text Available Abstract Background Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing. Results We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type. Conclusion The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.

  19. 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...

  20. 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...

  1. Are "g" and the General Factor of Personality (GFP) Correlated?

    Science.gov (United States)

    Irwing, Paul; Booth, Tom; Nyborg, Helmuth; Rushton, J. Philippe

    2012-01-01

    We examined whether the General Factor of Personality (GFP) is related to the "g" factor of cognitive ability using data from the Vietnam Experience Study which randomly sampled 4462 Vietnam War veterans from a total sample of about five million Vietnam era army veterans. Exclusionary criteria included passing a fitness test, achieving a…

  2. Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

    DEFF Research Database (Denmark)

    Huber, Reinhard C.; Remuge, Liliana; Carlisle, Ailsa

    2012-01-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology—animal welfare—has not been approached through systematic assessment...... and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals...... months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs...

  3. hNIS-IRES-eGFP Dual Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2005-04-01

    Full Text Available The human and rodent sodium iodide symporters (NIS have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES-linked human NIS (hNIS-enhanced green fluorescent protein (eGFP hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.

  4. Inoculating plants with the endophytic bacterium Pseudomonas sp. Ph6-gfp to reduce phenanthrene contamination.

    Science.gov (United States)

    Sun, Kai; Liu, Juan; Gao, Yanzheng; Sheng, Yuehui; Kang, Fuxing; Waigi, Michael Gatheru

    2015-12-01

    Plant organic contamination poses a serious threat to the safety of agricultural products and human health worldwide, and the association of endophytic bacteria with host plants may decrease organic pollutants in planta. In this study, we firstly determined the growth response and biofilm formation of endophytic Pseudomonas sp. Ph6-gfp, and then systematically evaluated the performance of different plant colonization methods (seed soaking (SS), root soaking (RS), leaf painting (LP)) for circumventing the risk of plant phenanthrene (PHE) contamination. After inoculation for 48 h, strain Ph6-gfp grew efficiently with PHE, oxalic acid, or malic acid as the sole sources of carbon and energy. Moreover, strain Ph6-gfp could form robust biofilms in LB medium. In greenhouse hydroponic experiments, strain Ph6-gfp could actively colonize inoculated plants internally, and plants colonized with Ph6-gfp showed a higher capacity for PHE removal. Compared with the Ph6-gfp-free treatment, the accumulations of PHE in Ph6-gfp-colonized plants via SS, RS, and LP were 20.1, 33.1, and 7.1 %, respectively, lower. Our results indicate that inoculating plants with Ph6-gfp could lower the risk of plant PHE contamination. RS was most efficient for improving PHE removal in whole plant bodies by increasing the cell numbers of Ph6-gfp in plant roots. The findings in this study provide an optimized method to strain Ph6-gfp reduce plant PAH residues, which may be applied to agricultural production in PAH-contaminated soil.

  5. Confinement effect of organic nanotubes toward green fluorescent protein (GFP) depending on the inner diameter size.

    Science.gov (United States)

    Kameta, Naohiro; Minamikawa, Hiroyuki; Someya, Yuu; Yui, Hiroharu; Masuda, Mitsutoshi; Shimizu, Toshimi

    2010-04-12

    Transportation, release behavior, and stability of a green fluorescent protein (GFP, 3x4 nm) in self-assembled organic nanotubes with three different inner diameters (10, 20, and 80 nm) have been studied in terms of novel nanocontainers. Selective immobilization of a fluorescent acceptor dye on the inner surface enabled us to not only visualize the transportation of GFP in the nanochannels but to also detect release of the encapsulated GFP to the bulk solution in real time, based on fluorescence resonance energy transfer (FRET). Obtained diffusion constants and release rates of GFP markedly decreased as the inner diameter of the nanotubes was decreased. An endo-sensing procedure also clarified the dependence of the thermal and chemical stabilities of the GFP on the inner diameters. The GFP encapsulated in the 10 nm nanochannel showed strong resistance to heat and to a denaturant. On the other hand, the 20 nm nanochannel accelerated the denaturation of the encapsulated GFP compared with the rate of denaturation of the free GFP in bulk and the encapsulated GFP in the 80 nm nanochannels. The confinement effect based on rational fitting of the inner diameter to the size of GFP allowed us to store it stably and without denaturation under high temperatures and high denaturant concentrations.

  6. Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

    International Nuclear Information System (INIS)

    Wang Hongmin; Monteiro, Mervyn J.

    2007-01-01

    Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases

  7. Observation of cytoskeleton dynamics in Arabidopsis-GFP-MAP4, -GFP-ABD2 plants under clinorotation

    Science.gov (United States)

    Shevchenko, Galina

    With the aim to investigate the impact of simulated microgravity on the cytoskeleton arrangement and plant cell growth we observed Arabidopsis thaliana transfected with GFP- MAP4 and GFP-ABD2 under clinorotation (2 rpm). This allowed us to visualize the tubulin microtubules and actin microfilaments in live cells and helped to follow the dynamics of cytoskeleton rearrangements in simulated microgravity. Ability to investigate the changes in live plant cells opens the wide perspectives for onboard research. We applied the pharmacological approach and investigated the behavior of microtubules when the microfilaments were disrupted and vice versa. Measurements of cell parameters in the distal elongation zone of plant root showed the slight discoordination of growth in cells with damaged actin microfilament. It is suggested that mutual structural interrelations of the cytoskeleton elements is essential for growth coordination in plant root. Clinorotation experiments revealed that root cell growth is less dependent upon the mutual co-alignment between the cytoskeleton elements. In this connection, we assume the involvement of cytoskeleton in the mechanism of growth restriction which is hidden under constant g-vector and is unmasked in simulated microgravity.

  8. Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-Aequorin reporter

    Directory of Open Access Journals (Sweden)

    Tou Cheu eXiong

    2014-02-01

    Full Text Available Calcium (Ca2+ is a second messenger involved in many plant signalling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A, has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues.Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

  9. Patterning protein complexes on DNA nanostructures using a GFP nanobody.

    Science.gov (United States)

    Sommese, R F; Hariadi, R F; Kim, K; Liu, M; Tyska, M J; Sivaramakrishnan, S

    2016-11-01

    DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies. © 2016 The Protein Society.

  10. Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study.

    Directory of Open Access Journals (Sweden)

    Bing Yang

    Full Text Available Insect prophenoloxidase (PPO is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+ was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.

  11. Function and structure of GFP-like proteins in the protein data bank.

    Science.gov (United States)

    Ong, Wayne J-H; Alvarez, Samuel; Leroux, Ivan E; Shahid, Ramza S; Samma, Alex A; Peshkepija, Paola; Morgan, Alicia L; Mulcahy, Shawn; Zimmer, Marc

    2011-04-01

    The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.

  12. The dielectric response to photoexcitation of GFP: A molecular dynamics study

    Science.gov (United States)

    Xu, Yao; Gnanasekaran, Ramachandran; Leitner, David M.

    2013-03-01

    The dielectric response to photoexcitation of the Green Fluorescent Protein (GFP) chromophore and contributions to it from water and GFP are computed by molecular simulations using a force field for the chromophore parametrized by ab initio calculations of ground and excited states. The chromophore is embedded in the β-barrel where it is surrounded by about 10 waters, which are found to play a significant role in slow dielectric relaxation. Dynamics of hydrogen bonds between water and GFP is examined and found to be slower and more heterogeneous for hydrogen bonds inside the β-barrel than in the hydration layer around GFP.

  13. Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase

    Science.gov (United States)

    Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

    2016-01-01

    Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins. PMID:26258682

  14. Cellular Activation of the Self-Quenched Fluorescent Reporter Probe in Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Alexei A. Bogdanov, Jr.

    2002-01-01

    Full Text Available The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF self-quenched macromolecular probe (PGC-Cy5.5 has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 μm showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total and GFP-negative (19% total populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFPnegative cells (2.5-3.0% had seven times higher NIRF intensity than the majority of GFP-positive or GFPnegative cells (372 and 55 AU, respectively. Highly NIRF-positive, FP-negative cells were CD45-and MAC3-positive. Our results indicate that: 1 intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2 tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

  15. The General Factor of Personality (GFP) and parental support: testing a prediction from Life History Theory

    NARCIS (Netherlands)

    Linden, D. van der; Figueredo, A.J.; Leeuw, R.N.H. de; Scholte, R.H.J.; Engels, R.C.M.E.

    2012-01-01

    In the present study, we tested whether the General Factor of Personality (GFP) is related to the level of parental support. The GFP is assumed to occupy the apex of the hierarchy of human personality structure and is believed to reflect a socially and sexually selected aggregate of behavioral

  16. The expression of GFP under the control of fibroin promotor in ...

    Indian Academy of Sciences (India)

    ... mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector (pGFP-N2/Fib) was constructed by use of replacing the CMV promoter with the fibroin promoter. The results of visual screening under a fluorescent inverted microscope and Western blot analysis indicated that the GFP gene was expressed in ...

  17. 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....

  18. Green to red photoconversion of GFP for protein tracking in vivo.

    Science.gov (United States)

    Sattarzadeh, Amirali; Saberianfar, Reza; Zipfel, Warren R; Menassa, Rima; Hanson, Maureen R

    2015-07-07

    A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

  19. Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum.

    Science.gov (United States)

    Riedel, Marko; Calmin, Gautier; Belbahri, Lassaad; Lefort, Francois; Götz, Monika; Wagner, Stefan; Werres, Sabine

    2009-01-01

    Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl₂-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron, one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non-labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum. Limitations of the technology are discussed.

  20. Sequential melting of two hydrophobic clusters within the green fluorescent protein GFP-cycle3.

    Science.gov (United States)

    Melnik, Tatiana N; Povarnitsyna, Tatiana V; Glukhov, Anatoly S; Uversky, Vladimir N; Melnik, Bogdan S

    2011-09-13

    The analysis of the three-dimensional structure of green fluorescent protein (GFP-cycle3) revealed the presence of two well-defined hydrophobic clusters located on the opposite sides of the GFP β-can that might contribute to the formation of partially folded intermediate(s) during GFP unfolding. The microcalorimetric analysis of the nonequilibrium melting of GFP-cycle3 and its two mutants, I14A and I161A, revealed that due to the sequential melting of the mentioned hydrophobic clusters, the temperature-induced denaturation of this protein most likely occurs in three stages. The first and second stages involve melting of a smaller hydrophobic cluster formed around the residue I161, whereas a larger hydrophobic cluster (formed around the residues I14) is melted only at the last GFP-cycle3 denaturation step or remains rather structured even in the denatured state.

  1. Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

    Science.gov (United States)

    Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

    2013-01-04

    RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  2. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

    Science.gov (United States)

    Slubowski, Christian J; Funk, Alyssa D; Roesner, Joseph M; Paulissen, Scott M; Huang, Linda S

    2015-04-01

    Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance. Copyright © 2015 John Wiley & Sons, Ltd.

  3. Synthesis and properties of the para-trimethylammonium analogues of green fluorescence protein (GFP) chromophore: The mimic of protonated GFP chromophore.

    Science.gov (United States)

    Fanjiang, Ming-Wei; Li, Ming-Ju; Sung, Robert; Sung, Kuangsen

    2018-04-01

    At low pH, protons from the external, bulk solution can protonate the phenoxide group of the p-HBDI chromophore in wild-type green fluorescent protein (wtGFP) and its mutants, and likely continue to tentatively protonate the phenol hydroxyl group of the same chromophores. Because the protonated GFP chromophore is a transient, we prepare the stable p-trimethylammonium analogues (2a and 2b) of the GFP chromophore to mimic it and explore their properties. What we found is that the p-trimethylammonium analogues of the GFP chromophore have the highly electrophilic amidine carbon, blue-shifted electronic absorption, smaller molar absorptivity, smaller fluorescent quantum yield, and faster E-Z thermoisomerization rate. The amidine carbon of the p-trimethylammonium analogue (2b) of the GFP chromophore is the only site that is attacked by very weak nucleophile of water, resulting in ring-opening of the imidazolinone moiety. The half-life of its decay rate in D 2 O is around 33 days. Actually, acid-catalyzed hydrolysis of p-HBDI also results in ring-opening of the imidazolinone moiety. The ratio of the acid-catalyzed hydrolysis rate constants [k obs (p-HBDI)/k obs (1b)] between p-HBDI and 1b (p-dimethylammonium analogue of the GFP chromophore) is dramatically increased from 0.30 at pH = 2 to 0.63 at pH = 0. This is the evidence that more and more phenol hydroxyl groups of p-HBDI are tentatively protonated in a low-pH aqueous solution and that accelerates hydrolysis of p-HBDI in the way similar to the quaternary ammonium derivatives 2a and 2b in water. With this view point, 2a and 2b still can partially mimic the cationic p-HBDI with the protonated phenol hydroxyl group. Implication of the experiment is that the amidine carbon of the chromophore in wtGFP and its mutants at very low pH should be highly electrophilic. Whether ring-opening of the imidazolinone moiety of the GFP chromophore would occur or not depends on if water molecules can reach the amidine carbon of

  4. Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP.

    Science.gov (United States)

    Solovyov, K V; Polyakov, D S; Grudinina, N A; Egorov, V V; Morozova, I V; Aleynikova, T D; Shavlovsky, M M

    2011-01-01

    The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.

  5. Mobile probes

    DEFF Research Database (Denmark)

    Ørngreen, Rikke; Jørgensen, Anna Neustrup; Noesgaard, Signe Schack

    2016-01-01

    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.......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...

  6. Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells

    Directory of Open Access Journals (Sweden)

    Douglas Ganini

    2017-08-01

    Full Text Available Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2 per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2•– and H2O2 in the presence of NADH. Generation of the free radical O2•– and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.

  7. 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.)

  8. Dynamics of protein and its hydration water: neutron scattering studies on fully deuterated GFP.

    Science.gov (United States)

    Nickels, Jonathan D; O'Neill, Hugh; Hong, Liang; Tyagi, Madhusudan; Ehlers, Georg; Weiss, Kevin L; Zhang, Qiu; Yi, Zheng; Mamontov, Eugene; Smith, Jeremy C; Sokolov, Alexei P

    2012-10-03

    We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Fluorescent sperm in a transparent worm: validation of a GFP marker to study sexual selection.

    Science.gov (United States)

    Marie-Orleach, Lucas; Janicke, Tim; Vizoso, Dita B; Eichmann, Micha; Schärer, Lukas

    2014-06-30

    Sexual selection has initially been thought to occur exclusively at the precopulatory stage in terms of contests among males and female mate choice, but research over the last four decades revealed that it often continues after copulation through sperm competition and cryptic female choice. However, studying these postcopulatory processes remains challenging because they occur internally and therefore are often difficult to observe. In the transparent free-living flatworm Macrostomum lignano, a recently established transgenic line that expresses green fluorescent protein (GFP) in all cell types, including sperm, offers a unique opportunity to non-invasively visualise and quantify the sperm of a GFP-expressing donor inside the reproductive tract of wild-type recipients in vivo. We here test several aspects of the reproductive performance of the transgenic individuals and the accuracy of the techniques involved in assessing the GFP-expressing worms and their sperm. We then show the usefulness of these methods in a study on sperm displacement. GFP-expressing worms do not differ from wild-type worms in terms of morphology, mating rate and reproductive success. In addition, we show that the GFP signal is reliably and unequivocally expressed by all GFP-expressing individuals observed under epifluorescence illumination. However, the intensity of the GFP signal emitted by sperm of GFP expressing donors can vary (which we show to be at least in part due to sperm ageing) and the GFP marker is inherited according to Mendel's laws in most, but not all, of the individuals. Nevertheless, we argue these two issues can be addressed with an appropriate experimental design. Finally, we demonstrate the value of the GFP-techniques by comparing the number of GFP-expressing sperm in a wild-type recipient before and after mating with a competing sperm donor, providing clear experimental evidence for sperm displacement in M. lignano. This result suggests that sperm donors can displace

  10. Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

    Science.gov (United States)

    Plaunt, Adam J; Harmatys, Kara M; Wolter, William R; Suckow, Mark A; Smith, Bradley D

    2014-04-16

    Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

  11. 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

  12. Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ

    Science.gov (United States)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2009-02-01

    Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

  13. Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

    Science.gov (United States)

    Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

    2016-01-01

    Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.

  14. 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.

  15. Detection of targeted GFP-Hox gene fusions during mouse embryogenesis.

    Science.gov (United States)

    Godwin, A R; Stadler, H S; Nakamura, K; Capecchi, M R

    1998-10-27

    The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13(GFPneo) embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13(lacZneo) embryos. Similarly, Hoxa1(GFPneo) expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1(GFPneo) embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

  16. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

    Science.gov (United States)

    Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

    2017-07-03

    We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins.

    Science.gov (United States)

    Okuno, Takashi; Yamabayashi, Hiroko; Kogure, Kentaro

    2010-03-01

    Nubp1 (also known as Nbp35) and Nubp2 (also known as Cfd1) proteins are known to be responsible for regulating centrosome duplication in mouse and ribosome biogenesis in yeast. Nubp proteins contribute to diverse physiological functions. It is thought that Nubp1 and Nubp2 proteins interact with each other and regulate their functions. However, little is known about the intracellular localization of Nubp proteins. In this study, we compared the intracellular localization of human Nubp1 and Nubp2 by fusing these proteins with green fluorescent protein (GFP) in HeLa cells. The nuclear transfer of Nubp1-GFP, where GFP was fused to the C-terminus, was not observed. However, GFP-Nubp1, where GFP was fused to the N-terminus, did accumulate in the nucleus. In addition, GFP-modification at the N-terminal of Nubp2 induced nuclear transformation. Our data suggest that the C-terminal region of Nubp1 is important for nuclear transfer and the N-terminal of Nubp2 contributes to the morphology of the nucleus.

  18. Functional characterisation of filamentous actin probe expression in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Shrujna Patel

    Full Text Available Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

  19. Pollution Probe.

    Science.gov (United States)

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  20. 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

  1. In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy.

    Science.gov (United States)

    Kokona, Despina; Jovanovic, Joël; Ebneter, Andreas; Zinkernagel, Martin S

    2017-11-11

    Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1 gfp/gfp ) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases 1 , 2 , 3 , 4 , 5 , 6 . This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1 gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 µm and 6 µm, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies 7 , which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate 8 .

  2. HSP70-Inducible hNIS-IRES-eGFP Reporter Imaging: Response to Heat Shock

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2007-11-01

    Full Text Available A retroviral vector pQHSP70/hNIS-IRES-eGFP (pQHNIG70 was constructed containing the hNIS-IRES-eGFP dual-reporter genes under the control of an inducible human heat shock protein (HSP70 promoter and RG2-pQHSP70/hNIS-IRES-eGFP (RG2-pQHNIG70 transduced cells were generated. Heat-induced expression of both reporter genes in RG2-pQHNIG70 cells was validated by enhanced green fluorescent protein (eGFP fluorescence-activated cell sorter, in vitro radiotracer assays, and immunoblot and immunocytochemistry. A 2.2- to 6.1-fold (131I−, a 6.1- to 14.4-fold (99mTcO4−, and a 5.1- to 39-fold (fluorescence increase above baseline was observed in response to graded hyperthermia (39–43°C. Increases in eGFP fluorescence and radiotracer uptake were first noted at 6 hours, reached a maximum at 24 hours, and fell toward baseline at 72 hours. A stable ratio of radiotracer uptake to eGFP fluorescence and to heat shock protein (HSP70 protein was demonstrated over a wide range of expression levels, induced by different levels of heating. We also demonstrate that the local application of heat on RG2-pQHNIG70 xenografts can effectively induce hNIS and eGFP gene expression in vivo and that this expression can be efficiently visualized by fluorescence, scintigraphic, and micro–positron emission tomography imaging. Endogenous HSP70 protein and reporter expression was confirmed by postmortem tissue evaluations (immunoblot and immunohistochemistry. The pQHNIG70 reporter system can be used to study stress and drug responses in transduced cells and tissues.

  3. GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

    Science.gov (United States)

    Weber, Eva; Guth, Christina; Weiss, Ingrid M

    2012-01-01

    Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

  4. GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

    Directory of Open Access Journals (Sweden)

    Eva Weber

    Full Text Available Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3 (- as the first ionic interaction partner, but not necessarily for Ca(2+. The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

  5. Colonization study of gfp-tagged Achromobacter marplatensis strain in sugar beet.

    Science.gov (United States)

    Shi, YingWu; Li, Chun; Yang, HongMei; Zhang, Tao; Gao, Yan; Chu, Min; Zeng, Jun; Lin, Qing; OuTiKuEr; Li, YuGuo; Huo, Xiangdong; Lou, Kai

    2017-04-01

    This study details the introduction of a gfp marker into an endophytic bacterial strain (Achromobacter marplatensis strain 17, isolated from sugar beet) to monitor its colonization of sugar beet (Beta. vulgaris L.). Stability of the plasmid encoding the gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under nonselective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated sugar beet plants that grew for 10 or 20 days. gfp-Expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the Achromobacter marplatensis 17:gfp strain in plants 20 days after inoculation, even in the absence of selective pressure, suggests that it is good colonizer. These results also suggest that this strain could be a useful tool for the delivery of enzymes or other proteins into plants. In addition, the study highlights that sugar beet plants can be used effectively for detailed in vitro studies on the interactions between A. marplatensis strain 17 and its host, particularly if a gfp-tagged strain of the pathogen is used.

  6. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    Science.gov (United States)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  7. Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker.

    Science.gov (United States)

    Torres, Adalgisa Ribeiro; Araújo, Welington Luiz; Cursino, Luciana; de Barros Rossetto, Priscilla; Mondin, Mateus; Hungria, Mariangela; Azevedo, João Lúcio

    2013-07-01

    This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.

  8. Tracing GFP-labeled WJMSCs in vivo using a chronic salpingitis model: an animal experiment.

    Science.gov (United States)

    Li, Zhe; Zhang, Zhao; Ming, Wai-Kit; Chen, Xin; Xiao, Xiao-Min

    2017-12-01

    The present study was conducted to evaluate the distribution of Wharton's jelly-derived mesenchymal stem cells (WJMSCs) and their repairing function on the oviduct. WJMSCs were transfected with the LV3-GFP-PURO lentivirus. Female New Zealand rabbits (n = 24) were divided randomly into control A and B groups and experimental C and D groups to establish inflammation models. Sterile saline solution or WJMSCs were injected into rabbits via ear veins and/or genital tract perfusion once weekly for 3 weeks. All rabbits were humanely sacrificed 1 week after the last perfusion to collect the oviduct, uterus, liver, and bladder for examination. Green fluorescent protein (GFP) and cytokeratin 7 (CK7) were imaged using a Leica Qwin Plus V3 fluorescence confocal microscope and analyzed as mean optical densities in an Image-Pro Plus analysis system. We found that lentivirus expressing the GFP gene produced an efficient transfection. The mean optical density values of GFP and CK7 in the oviducts were higher in the experimental D group than those in the control A and experimental C groups. No GFP fluorescence deposits occurred in the bladder of the control A group or experimental C group. Colocalization of CK7 and WJMSCs was observed in the oviducts in all groups. WJMSCs exhibited homing characteristics and migrated to the injured oviduct to promote epithelial cell growth. Additionally, local treatment resulted in higher efficiency.

  9. Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

    International Nuclear Information System (INIS)

    Fujimura, Juri; Ogawa, Rei; Mizuno, Hiroshi; Fukunaga, Yoshitaka; Suzuki, Hidenori

    2005-01-01

    Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders

  10. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    Science.gov (United States)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  11. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Science.gov (United States)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  12. Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

    Directory of Open Access Journals (Sweden)

    Walsh Rama

    2003-12-01

    Full Text Available Abstract Background Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE, despite being transcribed in companion cells (CC. Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants. Results In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs. Conclusion A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

  13. [Analysis of bacterial colonization associated with Gigaspora margarita spores by green fluorescence protein (GFP) marked technology].

    Science.gov (United States)

    Long, Liangkun; Yao, Qing; Ai, Yuncan; Zhu, Honghui

    2009-05-01

    We analyzed bacterial colonization associated with spores of arbuscular mycorrhizal fungi (AMF) Gigaspora margarita, to indicate their ecological niche, and to provide information for further researches on their populations or functions. Six bacteria strains (Peanibacillus sp. M060106-1, Peanibacillus sp. M061122-2, Peanibacillus sp. M061122-6, Bacillus sp. M061122-4, Bacillus sp. M061122-10 and Brevibacillus sp. M061122-12) isolated from G. margarita spores were tagged with green fluorescence protein (GFP) using the carrier plasmid pNF8 (gfp-mut1). We analyzed the ecological niche and population dynamics of tagged strains on G. margarita under different conditions by using fluorescent microscope and/or plate counts. Four strains (M060106-1, M061122-6, M061122-10 and M061122-12) were tagged with GFP, showing high plasmid stability. These tagged strains possessed the basic characteristics identical to their original strains and, hence, were fit for short-term study of environmental colonization. All four GFP-tagged strains colonized the spore wall of G. margarita, and M061122-6 and M061122-12 further colonized the fungal hyphae. Under different pH conditions,the population dynamic of each GFP-tagged strain on the spores showed the same trend, i.e. first increased and then decreased, and the effects on the population size varied with different pH value. GFP-tagged strains colonized the spores of low viability more easily than those of high viability, and the population dynamic on the spores of high viability was different for each tagged strain. The isolated bacteria associated with G. margarita spores can re-colonize the fungal spores, whereas their colonizing ability depends on their characteristics and environmental factors. These data contributes to the further understanding of populations and functions of AMF-associated bacteria.

  14. The hTH-GFP reporter rat model for the study of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Lorraine Iacovitti

    Full Text Available Parkinson disease (PD is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH-expressing dopaminergic (DA neurons of the substantia nigra pars compacta (SNpc. These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus. As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson's disease research.

  15. Laser microbeam abalation of GFP-labeled nuclear organelles in a living cell.

    Science.gov (United States)

    LaMorte, Vickie J.; Krasieva, Tatiana B.; Evans, Ronald M.; Berns, Michael W.; Tromberg, Bruce J.

    1997-05-01

    Cancer, development, cellular growth and differentiation are governed by gene expression. Recent molecular and cellular advances to visualize and perturb the pathways of transcriptional regulation, nascent RNA processing, and protein trafficking at the single cell level have been developed. More recently, applications utilizing the green fluorescent marker (GFP) from Aequorea victoria have facilitated visualization of these molecular events in a living cell. Specifically, we will describe a novel approach to perturb cellular processes by labeling discrete cellular components of interest with GFP and subsequently altering/ablating them with a laser microbeam.

  16. Structure of the green fluorescent protein NowGFP with an anionic tryptophan-based chromophore.

    Science.gov (United States)

    Pletnev, Vladimir Z; Pletneva, Nadya V; Sarkisyan, Karen S; Mishin, Alexander S; Lukyanov, Konstantin A; Goryacheva, Ekaterina A; Ziganshin, Rustam H; Dauter, Zbigniew; Pletnev, Sergei

    2015-08-01

    A green-emitting fluorescent variant, NowGFP, with a tryptophan-based chromophore (Thr65-Trp66-Gly67) was recently developed from the cyan mCerulean by introducing 18 point mutations. NowGFP is characterized by bright green fluorescence at physiological and higher pH and by weak cyan fluorescence at low pH. Illumination with blue light induces irreversible photoconversion of NowGFP from a green-emitting to a cyan-emitting form. Here, the X-ray structures of intact NowGFP at pH 9.0 and pH 4.8 and of its photoconverted variant, NowGFP_conv, are reported at 1.35, 1.18 and 2.5 Å resolution, respectively. The structure of NowGFP at pH 9.0 suggests the anionic state of Trp66 of the chromophore to be the primary cause of its green fluorescence. At both examined pH values Trp66 predominantly adopted a cis conformation; only ∼ 20% of the trans conformation was observed at pH 4.8. It was shown that Lys61, which adopts two distinct pH-dependent conformations, is a key residue playing a central role in chromophore ionization. At high pH the side chain of Lys61 forms two hydrogen bonds, one to the indole N atom of Trp66 and the other to the carboxyl group of the catalytic Glu222, enabling an indirect noncovalent connection between them that in turn promotes Trp66 deprotonation. At low pH, the side chain of Lys61 is directed away from Trp66 and forms a hydrogen bond to Gln207. It has been shown that photoconversion of NowGFP is accompanied by decomposition of Lys61, with a predominant cleavage of its side chain at the C(γ)-C(δ) bond. Lys61, Glu222, Thr203 and Ser205 form a local hydrogen-bond network connected to the indole ring of the chromophore Trp66; mutation of any of these residues dramatically affects the spectral properties of NowGFP. On the other hand, an Ala150Val replacement in the vicinity of the chromophore indole ring resulted in a new advanced variant with a 2.5-fold improved photostability.

  17. FRET ratiometric probes reveal the chiral-sensitive cysteine-dependent H2S production and regulation in living cells

    Science.gov (United States)

    Wei, Lv; Yi, Long; Song, Fanbo; Wei, Chao; Wang, Bai-Fan; Xi, Zhen

    2014-04-01

    Hydrogen sulfide (H2S) is an endogenously produced gaseous signalling molecule with multiple biological functions. In order to visualize and quantify the endogenous in situ production of H2S in living cells, here we developed two new sulphide ratiometric probes (SR400 and SR550) based on fluorescence resonance energy transfer (FRET) strategy for live capture of H2S. The FRET-based probes show excellent selectivity toward H2S in a high thiol background under physiological buffer. The probe can be used to in situ visualize cysteine-dependent H2S production in a chiral-sensitive manner in living cells. The ratiometric imaging studies indicated that D-Cys induces more H2S production than that of L-Cys in mitochondria of human embryonic kidney 293 cells (HEK293). The cysteine mimics propargylglycine (PPG) has also been found to inhibit the cysteine-dependent endogenous H2S production in a chiral-sensitive manner in living cells. D-PPG inhibited D-Cys-dependent H2S production more efficiently than L-PPG, while, L-PPG inhibited L-Cys-dependent H2S production more efficiently than D-PPG. Our bioimaging studies support Kimura's discovery of H2S production from D-cysteine in mammalian cells and further highlight the potential of D-cysteine and its derivatives as an alternative strategy for classical H2S-releasing drugs.

  18. Identification of Cells at Early and Late Stages of Polarization During Odontoblast Differentiation Using pOBCol3.6GFP and pOBCol2.3GFP Transgenic Mice

    Science.gov (United States)

    Balic, Anamaria; Aguila, H. Leonardo; Mina, Mina

    2010-01-01

    Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stage of differentiation along a lineage. In the present study we used primary cell cultures derived from the dental pulp from pOBCol3.6GFP and pOBCol2.3GFP transgenic mice as a model to develop markers for early stages of odontoblast differentiation from progenitor cells. We analyzed the temporal and spatial expression of 2.3-GFP and 3.6-GFP during in vitro mineralization. Using FACS to separate cells based on GFP expression, we obtained relatively homogenous sub-populations of cells and analyzed their dentinogenic potentials and their progression into odontoblasts. Our observations showed that these transgenes were activated before the onset of matrix deposition and in cells at different stages of polarization. The 3.6-GFP transgene was activated in cells in early stages of polarization whereas the 2.3-GFP transgene was activated at a later stage of polarization just before or at the time of formation of secretory odontoblast. PMID:20728593

  19. Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

    Science.gov (United States)

    Ewe, Daniela; Río Bártulos, Carolina; Kroth, Peter G.; Gruber, Ansgar

    2016-01-01

    An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns. PMID:27635322

  20. Co-transfer of gfp , CHS and hptII genes into Oncidium Sharry Baby ...

    African Journals Online (AJOL)

    One month after bombardment, the PLB were subjected to 5 mg/ml hygromycin selection for 8 months. A total of 137 regenerated putative transformants were randomly selected and verified by polymerase chain reaction (PCR) analysis. The results indicated the presence of the transgenes gfp, hptII and antisense CHS in 28, ...

  1. Green Fluorescent Protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum

    Science.gov (United States)

    Marko Riedel; Gautier Calmin; Lassaad Belbahri; Francois Lefort; Monika Gotz; Stefan Wagner; Sabine. Werres

    2009-01-01

    Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen...

  2. Analysis of computational models for an accurate study of electronic excitations in GFP

    DEFF Research Database (Denmark)

    Schwabe, Tobias; Beerepoot, Maarten; Olsen, Jógvan Magnus Haugaard

    2015-01-01

    Using the chromophore of the green fluorescent protein (GFP), the performance of a hybrid RI-CC2 / polarizable embedding (PE) model is tested against a quantum chemical cluster pproach. Moreover, the effect of the rest of the protein environment is studied by systematically increasing the size...

  3. Visualization of coral host-pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

    Science.gov (United States)

    Pollock, F. Joseph; Krediet, Cory J.; Garren, Melissa; Stocker, Roman; Winn, Karina; Wilson, Bryan; Huete-Stauffer, Carla; Willis, Bette L.; Bourne, David G.

    2015-06-01

    The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host-pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain's virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen's interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.

  4. A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Ishag, H Z A; Liu, M J; Yang, R S; Xiong, Q Y; Feng, Z X; Shao, G Q

    2016-04-28

    Mycoplasma hyopneumoniae (M. hyopneumoniae) causes porcine enzootic pneumonia (PEP) that significantly affects the pig industry worldwide. Despite the availability of the whole genome sequence, studies on the pathogenesis of this organism have been limited due to the lack of a genetic manipulation system. Therefore, the aim of the current study was to generate a general GFP reporter vector based on a replicating plasmid. Here, we describe the feasibility of GFP reporter expression in M. hyopneumoniae (strain 168L) controlled by the p97 gene promoter of this mycoplasma. An expression plasmid (pMD18-TOgfp) containing the p97 gene promoter, and origin of replication (oriC) of M. hyopneumoniae, tetracycline resistant marker (tetM), and GFP was constructed and used to transform competent M. hyopneumoniae cells. We observed green fluorescence in M. hyopneumoniae transformants under fluorescence microscopy, which indicates that there was expression of the GFP reporter that was driven by the p97 gene promoter. Additionally, an electroporation method for M. hyopneumoniae with an efficiency of approximately 1 x 10(-6) transformants/μg plasmid DNA was optimized and is described herein. In conclusion, our data demonstrate the susceptibility of M. hyopneumoniae to genetic manipulation whereby foreign genes are expressed. This work may encourage the development of genetic tools to manipulate the genome of M. hyopneumoniae for functional genomic analyses.

  5. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

    Directory of Open Access Journals (Sweden)

    Arthur M Talman

    2010-02-01

    Full Text Available The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

  6. Co-transfer of gfp, CHS and hptII genes into Oncidium Sharry Baby ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... crop plant for cut-flower in Malaysia. The fragrant types provide additional commercial values. However, some of these fragrant orchids possess dull ..... culture. Determining the most suitable observation time for gfp expression after bombardment is also essential to opti- mize the transformation system.

  7. Use of targetable gfp-tagged neuropeptide for visualizing neuropeptide release following execution of a behavior.

    Science.gov (United States)

    Husain, Qasim M; Ewer, John

    2004-05-01

    Previous work has shown that a transgene consisting of a fusion between the rat atrial natriuretic peptide and a green fluorescent protein reporter (ANF-gfp) is processed, localized, and released, as would be an endogenous neuropeptide when it is expressed in the nervous system of Drosophila melanogaster using the GAL4/UAS expression system. Here we have tested the utility of this targetable transgene for detecting neuropeptide release following the execution of a peptide-controlled behavior. For the behavior we used ecdysis, the behavior expressed by insects to shed their old cuticle at the end of the molt. We found that larval ecdysis was accompanied by a readily detectable reduction in gfp fluorescence from relevant secretory cells in the periphery and peptidergic neurons in the CNS. We also found that expression of the ANF-gfp products did not have detrimental effects on larval ecdysis or adult circadian rhythmicity, when the transgene was expressed in peptidergic cells that are known to control these behaviors. Finally, we used a broadly expressed GAL4 driver to show that the UAS-ANF-gfp transgene could be used to identify axons that show a reduction in gfp fluorescence following the expression of ecdysis behavior. These findings, coupled with the availability of an increasing number of strains bearing different GAL4 drivers, suggest that this transgene will be a useful tool for identifying peptidergic neurons and secretory cells (and, eventually, their secretory product) that release their peptide content during the occurrence, in the intact animal, of a developmental, physiological or behavioral process of interest. Copyright 2004 Wiley Periodicals, Inc. J Neurobiol 59: 181-191, 2004

  8. Comparative characterization of mesenchymal stem cells from eGFP transgenic and non-transgenic mice

    Directory of Open Access Journals (Sweden)

    Bunnell Bruce A

    2009-01-01

    Full Text Available Abstract Background Adipose derived- and bone marrow-derived murine mesenchymal stem cells (mMSCs may be used to study stem cell properties in an in vivo setting for the purposes of evaluating therapeutic strategies that may have clinical applications in the future. If these cells are to be used for transplantation, the question arises of how to track the administered cells. One solution to this problem is to transplant cells with an easily identifiable genetic marker such as enhanced green fluorescent protein (eGFP. This protein is fluorescent and therefore does not require a chemical substrate for identification and can be visualized in living cells. This study seeks to characterize and compare adipose derived- and bone marrow-derived stem cells from C57Bl/6 mice and eGFP transgenic C57Bl/6 mice. Results The expression of eGFP does not appear to affect the ability to differentiate along adipogenic or osteogenic lineages; however it appears that the tissue of origin can influence differentiation capabilities. The presence of eGFP had no effect on cell surface marker expression, and mMSCs derived from both bone marrow and adipose tissue had similar surface marker profiles. There were no significant differences between transgenic and non-transgenic mMSCs. Conclusion Murine adipose derived and bone marrow derived mesenchymal stem cells from non-transgenic and eGFP transgenic C57Bl/6 mice have very similar characterization profiles. The availability of mesenchymal stem cells stably expressing a genetic reporter has important applications for the advancement of stem cell research.

  9. Distinct fluorescent pattern of KAT1::GFP in the plasma membrane of Vicia faba guard cells.

    Science.gov (United States)

    Homann, Ulrike; Meckel, Tobias; Hewing, Jennifer; Hütt, Marc-Thorsten; Hurst, Annette C

    2007-08-01

    The organisation of membrane proteins into certain domains of the plasma membrane (PM) has been proposed to be important for signalling in yeast and animal cells. Here we describe the formation of a very distinct pattern of the K(+) channel KAT1 fused to the green fluorescent protein (KAT1::GFP) when transiently expressed in guard cells of Vicia faba. Using confocal laser scanning microscopy we observed a radially striped pattern of KAT1::GFP fluorescence in the PM in about 70% of all transfected guard cells. This characteristic pattern was found to be cell type and protein specific and independent of the stomatal aperture and the cytoskeleton. Staining of the cell wall of guard cells with Calcofluor White revealed a great similarity between the arrangement of cellulose microfibrils and the KAT1::GFP pattern. Furthermore, the radial pattern of KAT1::GFP immediately disappeared when turgor pressure was strongly decreased by changing from hypotonic to hypertonic conditions. The pattern reappeared within 15 min upon reestablishment of high turgor pressure in hypotonic solution. Evaluation of the staining pattern by a mathematical algorithm further confirmed this reversible abolishment of the radial pattern during hypertonic treatment. We therefore conclude that the radial organisation of KAT1::GFP depends on the close contact between the PM and cell wall in turgid guard cells. These results offer the first indication for a role of the cell wall in the localisation of ion channels. We propose a model in which KAT1 is located in the cellulose fibrils intermediate areas of the PM and discuss the physiological role of this phenomenon.

  10. Knowing what to do in social situations: The general factor of personality (GFP) and the performance on situational judgment tests

    NARCIS (Netherlands)

    Van der Linden, D.; Oostrom, J.K.; Born, M.P.; Van der Molen, H.T.; Serlie, A.W.

    2014-01-01

    Two studies examined the general factor of personality (GFP) in relationship to performance on video-based situational tests measuring social knowledge and skills. Study 1 (N = 180 candidates of an assessment center) showed that high-GFP individuals were better able to indicate the appropriate

  11. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation

    DEFF Research Database (Denmark)

    Gram, G J; Nielsen, S D; Hansen, J E

    1998-01-01

    We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo). This was done to determine whether hGFP could function as a marker gene...

  12. Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

    Science.gov (United States)

    Wierer, S.; Elgass, K.; Bieker, S.; Zentgraf, U.; Meixner, A. J.; Schleifenbaum, F.

    2011-02-01

    The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplast-located Grx1-roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.

  13. Studies of patulin diffusion in apple using a gfp transformant of Penicillium expansum

    DEFF Research Database (Denmark)

    Jensen, Birgit; Nielsen, Per Væggemose; Jensen, Dan Funck

    To study whether patulin diffuses in front of the pathogen or vice versa, we have inserted a constitutively expressed gfp-construct into Penicillium expansum. Spores and hyphal growth of the transformant IBT21525gfp4 in apple tissue was visualized directly by fluorescence mocroscopy. Using HPLC...

  14. Rosa26-GFP direct repeat (RaDR-GFP mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

    Directory of Open Access Journals (Sweden)

    Michelle R Sukup-Jackson

    2014-06-01

    Full Text Available Homologous recombination (HR is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

  15. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation

    DEFF Research Database (Denmark)

    Gram, G J; Nielsen, S D; Hansen, J E

    1998-01-01

    packaging cells returned to untreated control levels within 2 weeks. Using flow cytometric analysis, hGFP expression was detected in up to 15% of transduced MT4 cells (a CD4+ lymphocytic cell line) after coculturing with packaging cells for 4 days. A 3-day postcoculture treatment with 5-azacytidine......We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo). This was done to determine whether hGFP could function as a marker gene...... in a retroviral vector and to investigate the expression of genes in a retroviral vector. Surprisingly, clonal vector packaging cell lines showed variable levels of hGFP expression, and expression was detected in as few as 49% of the cells in a clonally derived culture. This indicated that hGFP expression...

  16. Isolation of mesenchymal stem cells (MSCs) from green fluorescent protein positive (GFP+) transgenic rodents: the grass is not always green(er).

    Science.gov (United States)

    Harting, Matthew T; Jimenez, Fernando; Cox, Charles S

    2009-01-01

    Cellular therapy is under intense basic science and clinical investigation as a therapeutic intervention. One of the challenges lies in tracking these cells in vivo. While there are many ways to label and track cells--each with strengths and weaknesses--the green fluorescent protein (GFP) is a reporter gene commonly employed. We report a significant and consistent reduction in the expression of GFP with the culture of mesenchymal stem cells (MSCs) isolated from the bone marrow of GFP(+) transgenic rodents. After MSC isolation and immunophenotype characterization, along with co-localization with GFP, MSCs were evaluated for GFP expression through flow cytometry and fluorescent microscopy, revealing that only 50% of the cells expressed GFP. Differentiation of the cells to adipocytes did not alter the GFP expression significantly. Incubation with an anti-GFP antibody increased the fluorescent intensity of the GFP-expressing and some of the GFP nonexpressing cells. Incubation of MSCs with a histone deacetylase inhibitor, trichostatin A, did not significantly alter GFP expression, while incubation with a DNA demethylation reagent, 5-azacytidine, increased GFP expression, suggesting that epigenetic modification by DNA methylation may play a role in GFP expression among MSCs.

  17. Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

    Science.gov (United States)

    Mess, Andrea; Perecin, Felipe; Bressan, Fabiana Fernandes; Mesquita, Ligia Garcia; Miglino, Maria Angelica; Pimentel, José RodrigoValim; Neto, Paulo Fantinato; Meirelles, Flávio Vieira

    2013-01-01

    Background To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously

  18. 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...

  19. Behavioral Evaluation of hMSC-GFP+ Transplantation in an Hemiparkinson Experimental Model in Wistar Rat

    Directory of Open Access Journals (Sweden)

    Jéssica Paola Alcázar Arzuza

    2017-05-01

    Full Text Available The effect of hMSCs-GFP+ transplantation was evaluated in an experimental model of Parkinson's disease (PD in 27 Wistar rats, or in three experimental groups: control (CON  n=7, injured (LES n=10 and transplanted (LES+T n=10. In order to evaluate the influence of the transplantation on the motor behavior, one month after the injury, rotation behavior induced by apomorphine, neurological test, transversal bar and SNpc cells positive to TH were developed. Using the Anova test, there was a decrease in the number of turns in transplanted animals (p=0.005 as well as in the neurological test (p=0.0004 and in the transverse bar that lead to this group in an intermediate position regarding LES and CON groups. There is a possible recovery of the transplantation-mediated nigroestriatal pathway of hMSC-GFP +.

  20. Isolation of progenitor cells from GFP-transgenic pigs and transplantation to the retina of allorecipients

    DEFF Research Database (Denmark)

    Klassen, Henry; Warfvinge, Karin; Schwartz, Philip H

    2008-01-01

    Work in rodents has demonstrated that progenitor transplantation can achieve limited photoreceptor replacement in the mammalian retina; however, replication of these findings on a clinically relevant scale requires a large animal model. To evaluate the ability of porcine retinal progenitor cells...... to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower...... expression in subretinal grafts was high in cells expressing vimentin and lower in cells expressing photoreceptor markers, again consistent with possible downregulation during differentiation. Cells survived transplantation to the injured retina of allorecipients at all time points examined (up to 10 weeks...

  1. Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection

    Energy Technology Data Exchange (ETDEWEB)

    Roberto, F.F.; Barnes, J.M.; Bruhn, D.F.

    2002-03-28

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as B-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  2. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    CERN Document Server

    Awazu, K; Tamiya, E

    2002-01-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 mu m. The maximum transfer efficiency was about 0.5%.

  3. Aptamer-Based Dual-Functional Probe for Rapid and Specific Counting and Imaging of MCF-7 Cells.

    Science.gov (United States)

    Yang, Bin; Chen, Beibei; He, Man; Yin, Xiao; Xu, Chi; Hu, Bin

    2018-02-06

    Development of multimodal detection technologies for accurate diagnosis of cancer at early stages is in great demand. In this work, we report a novel approach using an aptamer-based dual-functional probe for rapid, sensitive, and specific counting and visualization of MCF-7 cells by inductively coupled plasma-mass spectrometry (ICP-MS) and fluorescence imaging. The probe consists of a recognition unit of aptamer to catch cancer cells specifically, a fluorescent dye (FAM) moiety for fluorescence resonance energy transfer (FRET)-based "off-on" fluorescence imaging as well as gold nanoparticles (Au NPs) tag for both ICP-MS quantification and fluorescence quenching. Due to the signal amplification effect and low spectral interference of Au NPs in ICP-MS, an excellent linearity and sensitivity were achieved. Accordingly, a limit of detection of 81 MCF-7 cells and a relative standard deviation of 5.6% (800 cells, n = 7) were obtained. The dynamic linear range was 2 × 10 2 to 1.2 × 10 4 cells, and the recoveries in human whole blood were in the range of 98-110%. Overall, the established method provides quantitative and visualized information on MCF-7 cells with a simple and rapid process and paves the way for a promising strategy for biomedical research and clinical diagnostics.

  4. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein.

    Science.gov (United States)

    Nakanishi, T; Ikawa, M; Yamada, S; Parvinen, M; Baba, T; Nishimune, Y; Okabe, M

    1999-04-23

    We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome, a membrane limited organelle overlying the nucleus. The sperm showed normal fertilizing ability and the integrity of their acrosome was easily examined in a non-invasive manner by tracing the GFP in individual 'live' sperm with fluorescent microscopy. The time required for the dispersal of acrosomal contents was demonstrated to be approximately 3 s after the onset of acrosome reaction.

  5. PA-GFP: a window into the subcellular adventures of the individual mitochondrion.

    Science.gov (United States)

    Haigh, Sarah E; Twig, Gilad; Molina, Anthony A J; Wikstrom, Jakob D; Deutsch, Motti; Shirihai, Orian S

    2007-01-01

    Mitochondrial connectivity is characterized by matrix lumen continuity and by dynamic rewiring through fusion and fission events. While these mechanisms homogenize the mitochondrial population, a number of studies looking at mitochondrial membrane potential have demonstrated that mitochondria exist as a heterogeneous population within individual cells. To address the relationship between mitochondrial dynamics and heterogeneity, we tagged and tracked individual mitochondria over time while monitoring their mitochondrial membrane potential (deltapsi(m)). By utilizing photoactivatible-GFP (PA-GFP), targeted to the mitochondrial matrix, we determined the boundaries of the individual mitochondrion. A single mitochondrion is defined by the continuity of its matrix lumen. The boundaries set by luminal continuity matched those set by electrical coupling, indicating that the individual mitochondrion is equipotential throughout the entire organelle. Similar results were obtained with PA-GFP targeted to the inner membrane indicating that matrix continuity parallels inner membrane continuity. Sequential photoconversion of matrix PA-GFP in multiple locations within the mitochondrial web reveals that each ramified mitochondrial structure is composed of juxtaposed but discontinuous units. Moreover, as many as half of the events in which mitochondria come into contact, do not result in fusion. While all fission events generated two electrically uncoupled discontinuous matrices, the two daughter mitochondria frequently remained juxtaposed, keeping the tubular appearance unchanged. These morphologically invisible fission events illustrate the difference between mitochondrial fission and fragmentation; the latter representing the movement and separation of disconnected units. Simultaneous monitoring of deltapsi(m) of up to four individual mitochondria within the same cell revealed that subcellular heterogeneity in deltapsi(m) does not represent multiple unstable mitochondria that

  6. Signal/noise analysis of FRET-based sensors.

    Science.gov (United States)

    Woehler, Andrew; Wlodarczyk, Jakub; Neher, Erwin

    2010-10-06

    Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

    Science.gov (United States)

    Faull, Sarah V; Brown, Anwen E; Haq, Imran; Irving, James A

    2017-01-01

    Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

  8. Expression of Green Fluorescent Protein (GFP using In Vitro translation cell free system

    Directory of Open Access Journals (Sweden)

    M Mohamadipoor

    2009-03-01

    Full Text Available ABSTRACT Background and the purpose of the study: One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. Methods: pIVEX2.3-GFP plasmid was cloned to E. coli   and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Results: Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Major conclusion: Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification.

  9. Nestin-GFP transgene reveals neural precursor cells in adult skeletal muscle.

    Science.gov (United States)

    Birbrair, Alexander; Wang, Zhong-Min; Messi, Maria Laura; Enikolopov, Grigori N; Delbono, Osvaldo

    2011-02-03

    Therapy for neural lesions or degenerative diseases relies mainly on finding transplantable active precursor cells. Identifying them in peripheral tissues accessible for biopsy, outside the central nervous system, would circumvent the serious immunological and ethical concerns impeding cell therapy. In this study, we isolated neural progenitor cells in cultured adult skeletal muscle from transgenic mice in which nestin regulatory elements control GFP expression. These cells also expressed the early neural marker Tuj1 and light and heavy neurofilament but not S100β, indicating that they express typical neural but not Schwann cell markers. GFP+/Tuj1+ cells were also negative for the endothelial and pericyte markers CD31 and α-smooth muscle actin, respectively. We established their a) functional response to glutamate in patch-clamp recordings; b) interstitial mesenchymal origin; c) replicative capacity; and d) the environment necessary for their survival after fluorescence-activated cell sorting. We propose that the decline in nestin-GFP expression in muscle progenitor cells and its persistence in neural precursor cells in muscle cultures provide an invaluable tool for isolating a population of predifferentiated neural cells with therapeutic potential.

  10. Nestin-GFP transgene reveals neural precursor cells in adult skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Alexander Birbrair

    2011-02-01

    Full Text Available Therapy for neural lesions or degenerative diseases relies mainly on finding transplantable active precursor cells. Identifying them in peripheral tissues accessible for biopsy, outside the central nervous system, would circumvent the serious immunological and ethical concerns impeding cell therapy.In this study, we isolated neural progenitor cells in cultured adult skeletal muscle from transgenic mice in which nestin regulatory elements control GFP expression. These cells also expressed the early neural marker Tuj1 and light and heavy neurofilament but not S100β, indicating that they express typical neural but not Schwann cell markers. GFP+/Tuj1+ cells were also negative for the endothelial and pericyte markers CD31 and α-smooth muscle actin, respectively. We established their a functional response to glutamate in patch-clamp recordings; b interstitial mesenchymal origin; c replicative capacity; and d the environment necessary for their survival after fluorescence-activated cell sorting.We propose that the decline in nestin-GFP expression in muscle progenitor cells and its persistence in neural precursor cells in muscle cultures provide an invaluable tool for isolating a population of predifferentiated neural cells with therapeutic potential.

  11. Generation and characterization of neurogenin1-GFP transgenic medaka with potential for rapid developmental neurotoxicity screening

    International Nuclear Information System (INIS)

    Fan Chunyang; Simmons, Steven O.; Law, Sheran H.W.; Jensen, Karl; Cowden, John; Hinton, David; Padilla, Stephanie; Ramabhadran, Ram

    2011-01-01

    Fish models such as zebrafish and medaka are increasingly used as alternatives to rodents in developmental and toxicological studies. These developmental and toxicological studies can be facilitated by the use of transgenic reporters that permit the real-time, noninvasive observation of the fish. Here we report the construction and characterization of transgenic medaka lines expressing green fluorescent protein (GFP) under the control of the zebrafish neurogenin 1 (ngn1) gene promoter. Neurogenin (ngn1) is a helix-loop-helix transcription factor expressed in proliferating neuronal progenitor cells early in neuronal differentiation and plays a crucial role in directing neurogenesis. GFP expression was detected from 24 h post-fertilization until hatching, in a spatial pattern consistent with the previously reported zebrafish ngn1 expression. Temporal expression of the transgene parallels the expression profile of the endogenous medaka ngn1 transcript. Further, we demonstrate that embryos from the transgenic line permit the non-destructive, real-time screening of ngn1 promoter-directed GFP expression in a 96-well format, enabling higher throughput studies of developmental neurotoxicants. This strain has been deposited with and maintained by the National BioResource Project and is available on request ( (http://www.shigen.nig.ac.jp/medaka/strainDetailAction.do?quickSearch=true and strainId=5660)).

  12. Interaction of PLP with GFP-MAL2 in the human oligodendroglial cell line HOG.

    Directory of Open Access Journals (Sweden)

    Raquel Bello-Morales

    Full Text Available The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP, the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested.

  13. GFP sheds light on the infection process of avocado roots by Rosellinia necatrix.

    Science.gov (United States)

    Pliego, C; Kanematsu, S; Ruano-Rosa, D; de Vicente, A; López-Herrera, C; Cazorla, F M; Ramos, C

    2009-02-01

    In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron microscopy (SEM) images allowed detailed visualisation of the hyphal network generated by invasion of R. necatrix through the epidermal, cortical and vascular cells, including hyphal anastomosis and branching points. To our knowledge, this is the first report describing the construction of GFP-tagged strains belonging to the genus Rosellinia for monitoring white root rot using CLSM and SEM.

  14. Introduction of green fluorescent protein (GFP) into hippocampal neurons through viral infection.

    Science.gov (United States)

    Malinow, Roberto; Hayashi, Yasunori; Maletic-Savatic, Mirjana; Zaman, Shahid H; Poncer, Jean-Christophe; Shi, Song-Hai; Esteban, José A; Osten, Pavel; Seidenman, Ken

    2010-04-01

    Expression of green fluorescent protein (GFP), its more fluorescent mutant forms (e.g., EGFP [enhanced GFP]), or their fusion protein derivatives, affords a number of informative possibilities in cellular neuroscience. EGFP is a soluble protein and appears to be homogeneously distributed within the cytosol of neurons when expressed. Thus, it reveals the structure of the neuron, including the cell body, and axonal and dendritic arbors. It is also sufficiently bright to reveal detailed structures such as axonal boutons and dendritic spines. When expressed as a fusion protein, EGFP can provide information about the distribution characteristics of the proteins within neurons. Furthermore, during single-cell electrophysiological studies, such expression can direct the investigator to record from a cell carrying a foreign gene. In this protocol, we describe the use of the Sindbis pseudovirus expression system to deliver GFP to neurons. Sindbis is a member of the alphaviruses, which are plus-stranded RNA viruses. This protocol uses the DH(26S) strain, which preferentially infects neurons over glia (50:1). Two infection methods are given: one for dissociated hippocampal cultured neurons and one for organotypic hippocampal slices.

  15. Efficient transformation and expression of gfp gene in Valsa mali var. mali.

    Science.gov (United States)

    Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

    2015-01-01

    Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

  16. A novel binary T-vector with the GFP reporter gene for promoter characterization.

    Directory of Open Access Journals (Sweden)

    Shu-Ye Jiang

    Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

  17. 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.

  18. Temperature influences on the expression of GFP promoted by the upstream sequence of cpcB from Arthrospira platensis

    Science.gov (United States)

    Lu, Yongzhong; Zhang, Xuecheng

    2007-07-01

    In order to investigate the regulation mechanism of the phycocyanin gene, a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene (gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence, suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation initiation region (TIR) revealed that RNA thermosensor might account for the temperature regulation.

  19. Synthesis and Properties of the p-Sulfonamide Analogue of the Green Fluorescent Protein (GFP) Chromophore: The Mimic of GFP Chromophore with Very Strong N-H Photoacid Strength.

    Science.gov (United States)

    Chen, Yi-Hui; Sung, Robert; Sung, Kuangsen

    2018-03-12

    The para-sulfonamide analogue ( p-TsABDI) of a green fluorescent protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its S 1 excited-state p K a * value in dimethylsulfoxide (DMSO) is -1.5, which is strong enough to partially protonate dipolar aprotic solvents and causes excited-state proton transfer (ESPT), so it can partially mimic the GFP chromophore to further study the ESPT-related photophysics and the blinking phenomenon of GFP. In comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (p K a = 7.4, p K a * = 1.3 in water), p-TsABDI (p K a = 6.7, p K a * = -1.5 in DMSO) is a better photoacid for pH-jump studies.

  20. Elaboration and quality control of the inoculum of the experimental vaccine Brucella S19-tn7-GFP for use in white animals and associated serological test for the detection of anti-GFP antibodies

    International Nuclear Information System (INIS)

    Salas Alfaro, Dariana

    2014-01-01

    The preparation of the inoculum of the experimental vaccine Brucella S19-Tn7-GFP is optimized for application in white animals. An associated serological test has allowed differentiating infected animals from those vaccinated with the experimental strain. The same bacteriological and biological properties of the B. abortus S19-Tn7-GFP strain have maintained in the parental vaccine strain S19 and is stable over time. A protocol for the inoculums of strain S19-Tn7-GFP is established for its preparation and use in white animals and quality control. The inoculum stability is evaluated through the simulation of conditions that can be presented in the transportation and application process in the field. An enzyme immunoassay ELISA is optimized for the detection of anti-GFP antibodies in cattle [es

  1. An intein with genetically selectable markers provides a new approach to internally label proteins with GFP

    Directory of Open Access Journals (Sweden)

    Davis Trisha N

    2011-06-01

    Full Text Available Abstract Background Inteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein. Results The Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1 aminoglycoside phosphotransferase; 2 imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3 hygromycin B phosphotransferase; and 4 the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene CMD1. The DNA was transformed into yeast and integrants obtained by direct selection for the intein's marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker. Conclusions Inteins continue to show their flexibility as tools in molecular biology. The Pch PRP8 intein can successfully

  2. Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei.

    Science.gov (United States)

    Pérez-Arellano, Isabel; Pérez-Martínez, Gaspar

    2003-05-16

    An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFP(UV)) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFP(UV) expression, followed by aeration and a temperature of 30 degrees C. Environmental factors favoring GFP(UV) accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 microg of GFP per mg of total protein, from which 55% was fluorescent.

  3. Establishing the substantive interpretation of the GFP by considering evidence from research on personality disorders and Animal Personality

    NARCIS (Netherlands)

    M.P. Hengartner (Michael P.); D. van der Linden (Dimitri); C.S. Dunkel (Curtis)

    2017-01-01

    textabstractIn research on individual differences, various structural models aim at providing a comprehensive description of personality. These models assume multiple, mostly independent personality dimensions. More recently, the so-called General Factor of Personality (GFP) has become a

  4. Development of GFP fusions for examination of the effects of the space environment on gene expression in Escherichia coli

    Science.gov (United States)

    Mancinelli, R.; Fahlen, T.

    The goal of the In situ Space Gene Expression on Nano-satillites (ISGEN) program is to be ready to fly technology that can support a fully automated experiment to quantify changes in model organisms in situ in low earth orbit in a free flyer platform in less than two years. A straightforward gene expression assay that meets the ISGEN flight objective for testing flight hardware as well as return data regarding the effects of microgravity on gene expression has been developed. Escherichia coli K-12, a bacterium that exhibits changes in its growth pattern when flown in micro-gravity on the Space Shuttle, was used. The scientific objective of this work is to determine if there is a discernable change in metabolic and stress pathway gene expression due to growth in the space environment. To that end, we have linked the green fluorescent protein (GFP) reporter gfp to phoP, a gene that responds to extracellular Mg2+ levels, and pykF, a gene involved in the glycolytic pathway that responds to changes in intracellular pyruvate. These genes respond to the metabolic needs of the cell and may be altered in the micro-gravity environment. E. coli cells containing a plasmid encoding the phoP-gfp-mut3 reporter construct were grown with or without MgSO_4. The effect of the added MgSO_4 is the repression of the expression of GFP. This is the expected result if GFP expression were under the control of a magnesium-regulated promoter such as phoP. Consistent with the negative feedback loop, we observe repression of GFP production in cells containing our pykF-gfp plasmid construct, when grown in the presence of excess glucose. Thus, the pykF-gfp fusion functions as a glucose sensor.

  5. Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect.

    Science.gov (United States)

    Delvigne, Frank; Brognaux, Alison; Francis, Frédéric; Twizere, Jean-Claude; Gorret, Nathalie; Sorensen, Soren J; Thonart, Philippe

    2011-08-01

    Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great extent in the case of a culture operated in well-mixed fed-batch mode, by comparison with those operated in SDR. GFP leakage seems to be correlated to a higher membrane permeability, confirming previous studies highlighting a better cell viability in cultures operated in a fluctuating environment. Our results suggest that GFP leakage could be used in parallel to the normal GFP biosensor function in order to assess microbial viability in process conditions. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. THE ORNL ATOM PROBE

    OpenAIRE

    Miller, M.

    1986-01-01

    The ORNL Atom Probe is a microanalytical tool for studies in materials science. The instrument is a combination of a customized version of the vacuum system of the VG FIM-100 atom probe, an ORNL-designed microcomputer-controlled digital timing system, and a double curved CEMA Imaging Atom Probe detector. The atom probe combines four instruments into one - namely a field ion microscope, an energy compensated time-of-flight mass spectrometer, an imaging atom probe, and a pulsed laser atom probe.

  7. 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....

  8. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

    Directory of Open Access Journals (Sweden)

    Margaret Rohrbaugh

    Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

  9. Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP.

    Science.gov (United States)

    Xu, Jing; Zhu, Wenhua; Guo, Yuanxu; Jiang, Congshan; Hussain, Nazim; Meng, Liesu; Lu, Shemin

    2017-02-01

    The pLKO.1-TRC plasmid has been a popular and widely used vector due to its simple handling and stability. The huge RNAi database, a TRC library, has been established based on this vector. However, this plasmid only has a puromycin-resisted gene for selecting, which limits its application in microscopy and fluorescence-activated cell sorting (FACS). In the present work, PCR, restriction endonuclease digestion and molecular cloning techniques were used to insert the gene decoding green fluorescent protein (GFP) without changing the structure of original plasmid to extend its application. To demonstrate the function of new plasmid, we constructed shNC and shGAPDH plasmids based on newly constructed pLKO-TurboGFP-TRC (pLKOG) and original pLKO plasmids, and then packaged lentivirus particles by 293T cells. The supernatant containing lentiviral particles was collected and then incubated with RAW264.7 cells for infection. After selection for 7 days by using puromycin, the cells were harvested. RT-qPCR and Western blotting were used to detect the target gene expression. FACS was used to detect the green fluorescent of cells. Our results showed that the newly constructed pLKOG plasmid, as a lentiviral vector carrying shRNA, could knock down the target gene expression efficiently and express TurboGFP protein efficiently in the host cells. We conclude that the new plasmid is a convenient vector for selecting positive cells with shRNA by using fluorescent microscope and FACS.

  10. Construction of new GFP-tagged fusants for Trichoderma harzianum with enhanced biocontrol activity

    Directory of Open Access Journals (Sweden)

    Kowsari Mojegan

    2014-07-01

    Full Text Available Trichoderma is one of the most exploited biocontrol agents for the management of plant diseases. In biocontrol ecology, the critical factors are detection, and the monitoring and recovery of specific biocontrol agents either naturally present or deliberately released into the environment. Protoplast fusion is an appropriate tool for the improvement of biocontrol Trichoderma strains. Protoplast isolation from Trichoderma harzianum was achieved using 24 h culture age, 6.6 mg/ml Novazym L 1412 at 30°C which resulted the maximum protoplast yield of 5 × 108/ml. The self-fused protoplasts were regenerated and 12 fusants were selected based on their growth rate on 2% colloidal chitin medium. Next, a comparison was done for chitinase and antagonistic activity. Transcriptomic analysis based on quantitative real-time RT-PCR, demonstrated that T8-05 fusant expressed 1.5 fold of chit42 transcript as compared with the parental line. This fusant with 7.02±0.15U chitinase activity showed a higher growth inhibition rate (100% than the parent strain, against Rhizoctonia solani. To obtain a genetically marked fusant that can be used as a biomonitor, the fusant was cotransformed with the gfp and amdS genes. The morphology and viability of selected cotransformant (FT8-7MK-05-2 was similar to the parent. Green fluorescing conidia were observed within the first 2 days of incubation in the soil, and this was followed by the formation of chlamydopores after 60 days. The colonisation of the gfp-tagged fusant was also monitored visually on R. solani sclerotia by scanning electron microscopy. Production of gfp-tagged fusant of Trichoderma spp. provides a potentially useful tool for monitoring hyphal growth patterns and the population of biocontrol agent isolates introduced into environmental systems.

  11. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    International Nuclear Information System (INIS)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

    2007-01-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

  12. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    Energy Technology Data Exchange (ETDEWEB)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application.

  13. Stylophora pistillata in the Red Sea demonstrate higher GFP fluorescence under ocean acidification conditions

    Science.gov (United States)

    Grinblat, Mila; Fine, Maoz; Tikochinski, Yaron; Loya, Yossi

    2018-03-01

    Ocean acidification is thought to exert a major impact on calcifying organisms, including corals. While previous studies have reported changes in the physiological response of corals to environmental change, none have described changes in expression of the ubiquitous host pigments—fluorescent proteins (FPs)—to ocean acidification. The function of FPs in corals is controversial, with the most common consideration being that these primarily regulate the light environment in the coral tissue and protect the host from harmful UV radiation. Here, we provide for the first time experimental evidence that increased fluorescence of colonies of the coral Stylophora pistillata is independent of stress and can be regulated by a non-stressful decrease in pH. Stylophora pistillata is the most abundant and among the most resilient coral species in the northern Gulf of Eilat/Aqaba (GoE/A). Fragmented "sub-colonies" ( n = 72) incubated for 33 days under three pH treatments (ambient, 7.9, and 7.6), under ambient light, and running seawater showed no stress or adverse physiological performance, but did display significantly higher fluorescence, with lower pH. Neither the average number of planulae shed from the experimental sub-colonies nor planulae green fluorescent protein (GFP) expression changed significantly among pH treatments. Sub-colonies incubated under the lower-than-ambient pH conditions showed an increase in both total protein and GFP expression. Since extensive protein synthesis requires a high level of transcription, we suggest that GFP constitutes a UV protection mechanism against potential RNA as well as against DNA damage caused by UV exposure. Manipulating the regulation of FPs in adult corals and planulae, under controlled and combined effects of pH, light, and temperature, is crucial if we are to obtain a better understanding of the role played by this group of proteins in cnidarians.

  14. Actin Microfilament Organization in the Transition Zone of Arabidopsis-ABD2-GFP Roots Under Clinorotation

    Science.gov (United States)

    Shevchenko, Galina

    2012-12-01

    Seedlings of Arabidopsis thaliana-ABD2-GFP were grown under slow clinorotation (2 rpm) and treated with actin and tubulin disrupting drugs in order to characterize the role of actin microfilaments in cell growth and gravisensing. Changes in microfilament organization and cell parameters have shown that the transition root zone (TZ) is rather sensitive to microfilament disruption in control plants. It is assumed that under clinorotation, organization of actin cytoskeleton in the TZ is coordinated in a different way than in the control. Organization of microfilaments depends upon organization of microtubules and clinorotation does not influence this interrelation significantly.

  15. Facilitation of yeast-lethal membrane protein production by detoxifying with GFP tagging.

    Science.gov (United States)

    Oshikane, Hiroyuki; Watabe, Masahiko; Nakaki, Toshio

    2018-03-27

    Recombinant techniques for target protein production have been rapidly established and widely utilised in today's biological research. Nevertheless, methods for membrane protein production have yet to be developed, since membrane proteins generally tend to be expressed at low levels, easily aggregated, and/or even toxic to their host cells. Here we report that a GFP-tagging technique can be applied for the stable production of membrane proteins that are toxic to their host cells when overexpressed, paving the way for future advances in membrane protein biochemistry and drug development. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Regional Differences in Striatal Neuronal Ensemble Excitability Following Cocaine and Extinction Memory Retrieval in Fos-GFP Mice.

    Science.gov (United States)

    Ziminski, Joseph J; Sieburg, Meike C; Margetts-Smith, Gabriella; Crombag, Hans S; Koya, Eisuke

    2018-03-01

    Learned associations between drugs of abuse and the drug administration environment have an important role in addiction. In rodents, exposure to a drug-associated environment elicits conditioned psychomotor activation, which may be weakened following extinction (EXT) learning. Although widespread drug-induced changes in neuronal excitability have been observed, little is known about specific changes within neuronal ensembles activated during the recall of drug-environment associations. Using a cocaine-conditioned locomotion (CL) procedure, the present study assessed the excitability of neuronal ensembles in the nucleus accumbens core and shell (NAc core and NAc shell ), and dorsal striatum (DS) following cocaine conditioning and EXT in Fos-GFP mice that express green fluorescent protein (GFP) in activated neurons (GFP+). During conditioning, mice received repeated cocaine injections (20 mg/kg) paired with a locomotor activity chamber (Paired) or home cage (Unpaired). Seven to 13 days later, both groups were re-exposed to the activity chamber under drug-free conditions and Paired, but not Unpaired, mice exhibited CL. In a separate group of mice, CL was extinguished by repeatedly exposing mice to the activity chamber under drug-free conditions. Following the expression and EXT of CL, GFP+ neurons in the NAc core (but not NAc shell and DS) displayed greater firing capacity compared to surrounding GFP- neurons. This difference in excitability was due to a generalized decrease in GFP- excitability following CL and a selective increase in GFP+ excitability following its EXT. These results suggest a role for both widespread and ensemble-specific changes in neuronal excitability following recall of drug-environment associations.

  17. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    Science.gov (United States)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  18. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  19. Application of FRET probes in the analysis of neuronal plasticity

    Directory of Open Access Journals (Sweden)

    Yoshibumi eUeda

    2013-10-01

    Full Text Available Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since GFP was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET, which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo.

  20. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP

    Directory of Open Access Journals (Sweden)

    Griesbeck Oliver

    2003-05-01

    Full Text Available Abstract Background The green fluorescent protein (GFP has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET. However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins. Results By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells. Conclusion "T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.

  1. A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis.

    Science.gov (United States)

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Yudina, O S; Omelyanchuk, L V

    2009-05-01

    The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with gamma-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.

  2. Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials.

    Science.gov (United States)

    Wu, Jing-Jing; Liu, Yu-Wen; Sun, Meng-Xiang

    2011-07-01

    Green fluorescent proteins (GFPs) are widely used in tracing transgene expression and have been known as convenient and efficient markers for plant transformation. However, sometimes researchers are still puzzled by the weak fluorescence since it makes the observation of GFP signals and confirmation of transgenic plants difficult. In this investigation, we explored the possibility of enhancing the weak signals by changing the pH environment of detection and took microplate reader as a more effective instrument compared to traditional fluorescent microscope to detect the weak signals. It was found that the fluorescence intensity of enhanced GFP (EGFP) in transgenic plants can be increased 2-6 folds by altering the environmental pH, and the concentration of EGFP at a large scale (ranged from 20 ng/ml to 20 μg/ml) can be detected and quantified. It can exclude the influence of degradation fragment and hence facilitate later analysis; these advantages were further verified by comparing with western blotting and confocal microscopy. It was reliable and effective for the qualitative and quantitative analysis of transgenic plants and was more suitable for the detection of very weak fluorescent signals.

  3. In Vivo GFP Knockdown by Cationic Nanogel-siRNA Polyplexes

    Directory of Open Access Journals (Sweden)

    Arun R. Shrivats

    2015-07-01

    Full Text Available RNA interference (RNAi is a powerful tool to treat diseases and elucidate target gene function. Prior to clinical implementation, however, challenges including the safe, efficient and targeted delivery of siRNA must be addressed. Here, we report cationic nanogel nanostructured polymers (NSPs prepared by atom transfer radical polymerization (ATRP for in vitro and in vivo siRNA delivery in mammalian models. Outcomes from siRNA protection studies suggested that nanogel NSPs reduce enzymatic degradation of siRNA within polyplexes. Further, the methylation of siRNA may enhance nuclease resistance without compromising gene knockdown potency. NSP-mediated RNAi treatments against Gapdh significantly reduced GAPDH enzyme activity in mammalian cell culture models supplemented with 10% serum. Moreover, nanogel NSP-mediated siRNA delivery significantly inhibited in vivo GFP expression in a mouse model. GFP knockdown was siRNA sequence-dependent and facilitated by nanogel NSP carriers. Continued testing of NSP/siRNA compositions in disease models may produce important new therapeutic options for patient care.

  4. The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.

    Science.gov (United States)

    Chen, Zhi-jie; Tian, Ziqi; Kallio, Karen; Oleson, April L; Ji, Ao; Borchardt, Dan; Jiang, De-en; Remington, S James; Ai, Hui-wang

    2016-04-13

    Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

  5. Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

    Science.gov (United States)

    Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

    2018-02-20

    Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel

  6. Rapid-response flood mapping during Hurricanes Harvey, Irma and Maria by the Global Flood Partnership (GFP)

    Science.gov (United States)

    Cohen, S.; Alfieri, L.; Brakenridge, G. R.; Coughlan, E.; Galantowicz, J. F.; Hong, Y.; Kettner, A.; Nghiem, S. V.; Prados, A. I.; Rudari, R.; Salamon, P.; Trigg, M.; Weerts, A.

    2017-12-01

    The Global Flood Partnership (GFP; https://gfp.jrc.ec.europa.eu) is a multi-disciplinary group of scientists, operational agencies and flood risk managers focused on developing efficient and effective global flood management tools. Launched in 2014, its aim is to establish a partnership for global flood forecasting, monitoring and impact assessment to strengthen preparedness and response and to reduce global disaster losses. International organizations, the private sector, national authorities, universities and research agencies contribute to the GFP on a voluntary basis and benefit from a global network focused on flood risk reduction. At the onset of Hurricane Harvey, GFP was `activated' using email requests via its mailing service. Soon after, flood inundation maps, based on remote sensing analysis and modeling, were shared by different agencies, institutions, and individuals. These products were disseminated, to varying degrees of effectiveness, to federal, state and local agencies via emails and data-sharing services. This generated a broad data-sharing network which was utilized at the early stages of Hurricane Irma's impact, just two weeks after Harvey. In this presentation, we will describe the extent and chronology of the GFP response to both Hurricanes Harvey, Irma and Maria. We will assess the potential usefulness of this effort for event managers in various types of organizations and discuss future improvements to be implemented.

  7. Electrical resistivity probes

    Science.gov (United States)

    Lee, Ki Ha; Becker, Alex; Faybishenko, Boris A.; Solbau, Ray D.

    2003-10-21

    A miniaturized electrical resistivity (ER) probe based on a known current-voltage (I-V) electrode structure, the Wenner array, is designed for local (point) measurement. A pair of voltage measuring electrodes are positioned between a pair of current carrying electrodes. The electrodes are typically about 1 cm long, separated by 1 cm, so the probe is only about 1 inch long. The electrodes are mounted to a rigid tube with electrical wires in the tube and a sand bag may be placed around the electrodes to protect the electrodes. The probes can be positioned in a borehole or on the surface. The electrodes make contact with the surrounding medium. In a dual mode system, individual probes of a plurality of spaced probes can be used to measure local resistance, i.e. point measurements, but the system can select different probes to make interval measurements between probes and between boreholes.

  8. Improvement of AdCMV-GFP gene transfection efficiency induced by heavy-ion beam irradiation on murine melanoma cells

    International Nuclear Information System (INIS)

    Duan Xin; Min Fengling; Liu Bing; Zhou Qingming; Li Xiaoda; Wang Yanling; Chinese Academy of Sciences, Beijing; Zhang Hong; Qiu Rong; Hao Jifang; Zhou Guangming; Gao Qingxiang

    2007-01-01

    The effect of 12 C 6+ beam irradiation on AdCMV-GFP (a replication deficient recombinant adenoviral vector containing CMV promoter and green fluorescent protein) gene transfection efficiency for murine melanoma cell B16 has been investigated. B16 cells infected with AdCMV-GFP were irradiated by different doses of 12 C 6+ beam. The transfection efficiency was assessed by flow cytometry (FCM). Results show that 12 C 6+ beam irradiation can improve transfection efficiency of AdCMV-GFP on murine melanoma cell B16 in a dose-dependent manner. In addition, the transfection efficiency in pre-tranfection plus irradiation group is higher than that in pre-irradiation plus transfection group at the same dose irradiation dose. (authors)

  9. Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

    DEFF Research Database (Denmark)

    Delvigne, Frank; Brognaux, Alison; Francis, Frédéric

    2011-01-01

    Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down......-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose...... results suggest that GFP leakage could be used in parallel to the normal GFP biosensor function in order to assess microbial viability in process conditions...

  10. Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): Snipping the proton wire

    Science.gov (United States)

    Stoner-Ma, Deborah; Jaye, Andrew A.; Ronayne, Kate L.; Nappa, Jérôme; Tonge, Peter J.; Meech, Stephen R.

    2008-06-01

    Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild-type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A∗ excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state.

  11. Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

    Science.gov (United States)

    Chen, Mei-Shu; Lin, Hua-Kuo; Chiu, Hsun; Lee, Don-Ching; Chung, Yu-Fen; Chiu, Ing-Ming

    2015-03-01

    FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (-540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP(+) cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP(+) cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP(+) cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP(+) cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP(+) -neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP(+) /TH(+) cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.

  12. Rare distribution of green fluorescent protein (GFP) in hydroids from Porto Cesareo, Lecce, Italy, with reference to biological meaning of this rarity

    OpenAIRE

    Kubota, Shin; Gravili, Cinzia

    2011-01-01

    A reliable taxonomic character, distributional pattern of green fluorescent protein (GFP), that is contributable to species demarcation by observing living materials under the fluorescent microscope, has not been much done in hydroids in contrast to hydromedusae. We carried out such a fundamental study in diverse hydroids collected from Porto Cesareo, Lecce, Italy. In contrast to diversified distribution of GFP in the hydromedusae, GFP distribution in hydroids are unexpectedly very rare, and ...

  13. Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death

    Directory of Open Access Journals (Sweden)

    Somerville Chris R

    2005-03-01

    Full Text Available Abstract Background A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and / or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations. Results While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30 – 60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death. Conclusion Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and

  14. Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death

    Science.gov (United States)

    Cutler, Sean R; Somerville, Chris R

    2005-01-01

    Background A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and / or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations. Results While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30 – 60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death. Conclusion Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and biochemical analysis. PMID

  15. Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality

    Directory of Open Access Journals (Sweden)

    Michael Bugiel

    2015-11-01

    Full Text Available Microspheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein.

  16. Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot

    Directory of Open Access Journals (Sweden)

    Cecilia Armesto

    2012-09-01

    Full Text Available Blister spot (Colletotrichum gloeosporioides is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.

  17. Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis.

    Science.gov (United States)

    Danish, Azeem; Lee, Sang-Yong; Müller, Christa E

    2017-10-07

    A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A 2A receptor (A 2A AR), and connexin 43 (Cx43). Quantification of SV2A and A 2A AR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

  18. Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

    Science.gov (United States)

    Perez-Cunningham, Jessica; Boyer, Scott W; Landon, Mark; Forsberg, E Camilla

    2016-08-01

    Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  19. A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

    DEFF Research Database (Denmark)

    Wendel, Sofie; Christian Fischer, Emil; Martinez, Virginia

    2016-01-01

    for evaluating and developing surface display systems is missing.Results: Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid...... nanobody: GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies...

  20. Evaluation of (fli:GFP) Casper Zebrafish Embryos as a Model for Human Conjunctival Melanoma.

    Science.gov (United States)

    Pontes, Kelly Cristine de Sousa; Groenewoud, Arwin; Cao, Jinfeng; Ataide, Livia Maria Silva; Snaar-Jagalska, Ewa; Jager, Martine J

    2017-12-01

    Conjunctival melanoma (CM) is a rare malignant disease that can lead to recurrences and metastases. There is a lack of effective treatments for the metastases, and we set out to develop a new animal model to test potential therapies. Zebrafish are being used as a model for many diseases, and our goal was to test whether this animal could be used to study CM. Three human CM cell lines (CRMM-1 and CM2005.1, which both harbor a B-RAF mutation, and CRMM-2, which has an N-RAS mutation) were injected into the yolk sac, around the eye, and into the duct of Cuvier of transgenic (fli:GFP) Casper zebrafish embryos. Fluorescent and confocal images were taken to assess the phenotype and the behavior of engrafted cells and to test the effect of Vemurafenib as a treatment against CM. While the cells that had been injected inside the yolk sac died and those injected around the eye sporadically went into the circulation, the cells that had been injected into the duct of Cuvier colonized the zebrafish: cells from all three cell lines proliferated and disseminated to the eyes, where they formed clusters, and to the tail, where we noticed extravasation and micrometastases. Vemurafenib, a potent agent for treatment of B-RAF V600E-positive melanoma, inhibited outgrowth of CRMM-1 and CM2005.1 cells in a mutation-dependent way. The (fli:GFP) Casper zebrafish embryo can be used as an efficient animal model to study metastatic behavior of human CM cells and warrants further testing of drug efficacy to aid care of CM patients.

  1. High temperature probe

    Science.gov (United States)

    Swan, Raymond A.

    1994-01-01

    A high temperature probe for sampling, for example, smokestack fumes, and is able to withstand temperatures of 3000.degree. F. The probe is constructed so as to prevent leakage via the seal by placing the seal inside the water jacket whereby the seal is not exposed to high temperature, which destroys the seal. The sample inlet of the probe is also provided with cooling fins about the area of the seal to provide additional cooling to prevent the seal from being destroyed. Also, a heated jacket is provided for maintaining the temperature of the gas being tested as it passes through the probe. The probe includes pressure sensing means for determining the flow velocity of an efficient being sampled. In addition, thermocouples are located in various places on the probe to monitor the temperature of the gas passing there through.

  2. Microneurosurgical water probe.

    Science.gov (United States)

    Pogády, P; Wurm, G

    2005-04-01

    When constructing the micro-neurosurgical water ball probe, the authors have simply combined the properties of a ball probe with an irrigational function and the supportive role of water current to form a new irrigating ball dissector. The micro-instrument has an outlet mechanism with which the surgeon can regulate the flow of physiological solution into the operational field. Its point has the properties of a ball probe, and the overall bayonet shape facilitates surgical interventions in deep tissues under microscopic control. The water probe therefore enables the surgeon to perform precise mechanical preparation supported by a regulated current of water and a targeted irrigation in the operational field. The physiological solution in the pressure infusion cuff is under minimal pressure and directly connected to the probe. Due to the fact that one device can be used for various purposes the water ball probe represents an advantageous alternative to conventional micro-neurosurgical preparation.

  3. Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150+ long-term repopulating murine hematopoietic stem cells.

    Science.gov (United States)

    Tornack, Julia; Kawano, Yohei; Garbi, Natalio; Hämmerling, Günter J; Melchers, Fritz; Tsuneto, Motokazu

    2017-09-01

    The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long-term repopulating HSCs (LT-HSC) are routinely enriched as Lin - Sca1 + c-Kit + CD34 - Flt3 - CD150 + CD48 - cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L-eGFP reporter mice, we show that endogenous Flt3L-eGFP-reporter RNA expression correlates with eGFP-protein expression. This Flt3L-eGFP-reporter expression distinguishes two LT-HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L-eGFP-reporter low cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporter high cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporter low cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporter high HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporter low cells develop to Flt3L-eGFP-reporter high cells in vitro, although Flt3L-eGFP-reporter high cells remain Flt3L-eGFP-reporter high . CD150 + Flt3L-eGFP-reporter low cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporter high cells do express EPCR but not CD41. Thus, FACS-enrichment of Flt3/ Flt3L-eGFP-reporter negative, Lin - CD150 + CD48 - EPCR + CD41 + HSCs allows a further 5-fold enrichment of functional LT-HSCs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Generation of the Fluorescent HMGB1-GFP Fusion Protein in Insect Cells and Evaluation of its Immunogenicity in Two Mice Models.

    Science.gov (United States)

    Anvar, Ali; Vahabpour, Rouhollah; Salahshourifar, Iman; Bolhassani, Azam

    2017-01-01

    High mobility group box 1 (HMGB1) is a highly conserved protein present in the nuclei and cytoplasm of cells which has an important role as a mediator of inflammation in the extracellular environment. HMGB1 was identified as an innate adjuvant that induces immune responses against soluble antigens in vivo. Our goal is the generation of recombinant HMGB1-GFP fusion protein in insect cells for evaluation of immune responses in mouse model. In the current study, we used a baculovirus expression system for insect cells that was based on expression of HMGB1 with target gene (GFP), and purified the recombinant HMGB1- GFP fusion protein. We then demonstrated whether immunogenicity of GFP changes in the presence or absence of recombinant HMGB1 acting as an adjuvant in C57BL/6 and BALB/c mice. Our data showed that HMGB1 had a major influence on antibody immune responses induced by GFP in both animal models. The groups receiving HMGB1-GFP fusion protein showed total IgG and IgG2a responses significantly higher than IgG1 in BALB/c mice. Indeed, a mixed IgG1/IgG2a response was observed with high intensity toward IgG2a. In contrast, C57BL/6 mice immunized by HMGB1-GFP protein elicited the same levels of IgG1 and IgG2a. However, the levels of IgG2a and total IgG against the recombinant GFP (rGFP) in C57BL/6 mice were lower than those in BALB/c mice. We concluded that fusion of HMGB1 with GFP was immunologically more effective than GFP alone. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Medial prefrontal cortex neuronal activation and synaptic alterations after stress-induced reinstatement of palatable food seeking: a study using c-fos-GFP transgenic female rats.

    Science.gov (United States)

    Cifani, Carlo; Koya, Eisuke; Navarre, Brittany M; Calu, Donna J; Baumann, Michael H; Marchant, Nathan J; Liu, Qing-Rong; Khuc, Thi; Pickel, James; Lupica, Carl R; Shaham, Yavin; Hope, Bruce T

    2012-06-20

    Relapse to maladaptive eating habits during dieting is often provoked by stress and there is evidence for a role of ovarian hormones in stress responses and feeding. We studied the role of these hormones in stress-induced reinstatement of food seeking and medial prefrontal cortex (mPFC) neuronal activation in c-fos-GFP transgenic female rats, which express GFP in strongly activated neurons. Food-restricted ovariectomized or sham-operated c-fos-GFP rats were trained to lever-press for palatable food pellets. Subsequently, lever-pressing was extinguished and reinstatement of food seeking and mPFC neuronal activation was assessed after injections of the pharmacological stressor yohimbine (0.5-2 mg/kg) or pellet priming (1-4 noncontingent pellets). Estrous cycle effects on reinstatement were also assessed in wild-type rats. Yohimbine- and pellet-priming-induced reinstatement was associated with Fos and GFP induction in mPFC; both reinstatement and neuronal activation were minimally affected by ovarian hormones in both c-fos-GFP and wild-type rats. c-fos-GFP transgenic rats were then used to assess glutamatergic synaptic alterations within activated GFP-positive and nonactivated GFP-negative mPFC neurons following yohimbine-induced reinstatement of food seeking. This reinstatement was associated with reduced AMPA receptor/NMDA receptor current ratios and increased paired-pulse facilitation in activated GFP-positive but not GFP-negative neurons. While ovarian hormones do not appear to play a role in stress-induced relapse of food seeking in our rat model, this reinstatement was associated with unique synaptic alterations in strongly activated mPFC neurons. Our paper introduces the c-fos-GFP transgenic rat as a new tool to study unique synaptic changes in activated neurons during behavior.

  6. The impact of plasmid on regeneration and expression efficiencies of gfp gene in tobacco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Niko PAVLIN

    2015-12-01

    Full Text Available Tobacco leaf explants were transformed by bacteria Agrobacterium tumefaciens (A. t. and plasmid pBIN mgfp5-ER, which has a single copy of the green fluorescent gfp gene and A. t.-pART27 2mgfp5-ER, which has two copies of the gfp gene. Both plasmids have a built-in selection nptII gene for resistance to the antibiotic kanamycin. The presence of the green fluorescent mGFP-ER protein was detected with the epifluorescent microscope in the individual cells 3 days after transformation with A. t.-pART27 2mgfp5-ER and after 6 days in cells transformed with A.t.-pBIN mgfp5-ER. After infection by A. t.-pART27 2mgfp5-ER, in most cases the regeneration was direct, without intermediate stages of callus and faster, as the first globular structures were formed 10–12 days after transformation and a 204 % regeneration was achieved, while the first globular structure, after infection with A. t.-pBIN mgfp5-ER, occurred after 18 days and formed more callus and the regeneration was only 78.4 %. The duplex PCR analysis, performed on all 149 resulting regenerants, confirmed the presence of fragments of length 650 bp specific to the selection nptII gene and length of 422 bp specific for gfp marker gene.

  7. Factors affecting the excretion of GFP Salmonella enterica serovar typhimurium by adult house flies (Diptera: Muscidae; Musca domestica L.)

    Science.gov (United States)

    House flies harbor and disseminate food-borne pathogens. Salmonella Typhimurium (S. Typhimurium) is a zoonotic pathogen shed by livestock that causes gastroenteritis in humans. We previously demonstrated that GFP-S. Typhimurium fed to house flies persist in the digestive for 24h. The excretion dynam...

  8. Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium

    NARCIS (Netherlands)

    Franz, Eelco; Visser, Anna A; Van Diepeningen, Anne D; Klerks, Michel M; Termorshuizen, Aad J; van Bruggen, Ariena H C

    The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca

  9. Deployment of a Prototype Plant GFP Imager at the Arthur Clarke Mars Greenhouse of the Haughton Mars Project

    Directory of Open Access Journals (Sweden)

    Robert J. Ferl

    2008-04-01

    Full Text Available The use of engineered plants as biosensors has made elegant strides in the past decades, providing keen insights into the health of plants in general and particularly in the nature and cellular location of stress responses. However, most of the analytical procedures involve laboratory examination of the biosensor plants. With the advent of the green fluorescence protein (GFP as a biosensor molecule, it became at least theoretically possible for analyses of gene expression to occur telemetrically, with the gene expression information of the plant delivered to the investigator over large distances simply as properly processed fluorescence images. Spaceflight and other extraterrestrial environments provide unique challenges to plant life, challenges that often require changes at the gene expression level to accommodate adaptation and survival. Having previously deployed transgenic plant biosensors to evaluate responses to orbital spaceflight, we wished to develop the plants and especially the imaging devices required to conduct such experiments robotically, without operator intervention, within extraterrestrial environments. This requires the development of an autonomous and remotely operated plant GFP imaging system and concomitant development of the communications infrastructure to manage dataflow from the imaging device. Here we report the results of deploying a prototype GFP imaging system within the Arthur Clarke Mars Greenhouse (ACMG an autonomously operated greenhouse located within the Haughton Mars Project in the Canadian High Arctic. Results both demonstrate the applicability of the fundamental GFP biosensor technology and highlight the difficulties in collecting and managing telemetric data from challenging deployment environments.

  10. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    Science.gov (United States)

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  11. Gene Amplification by PCR and Subcloning into a GFP-Fusion Plasmid Expression Vector as a Molecular Biology Laboratory Course

    Science.gov (United States)

    Bornhorst, Joshua A.; Deibel, Michael A.; Mulnix, Amy B.

    2004-01-01

    A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the…

  12. Monitoring the active conformation of green fluorescent protein (GFP) and β-glucosidase adsorbed on soil particles.

    Science.gov (United States)

    Morales-Belpaire, Isabel; Gerin, Patrick A

    2012-01-01

    In order to determine the effect of various soil components on the activity of proteins, we monitored the fluorescence and the enzymatic activity of, respectively, green fluorescent protein (GFP) and β-glucosidase adsorbed on fine soil particles. We also monitored the activity of these proteins in the presence of components that are representative of soil colloids: a montmorillonite clay, goethite and organic matter extracted from soil. Upon adsorption on clay and goethite, GFP lost its fluorescence properties while β-glucosidase suffered only a partial loss of its catalytic activity. Extractable organic matter had an inactivating role on GFP while it did not cause inactivation of β-glucosidase. When GFP and β-glucosidase adsorbed on particles from natural soil samples, their behaviour was consistent with the behaviour observed for these proteins in the presence of the separate components, suggesting that the macroscopic activity of proteins adsorbed on soil particles corresponds to an average of the activities of proteins adsorbed on a mixture of surfaces. The monitoring of the proteins on soil particles with different organic matter contents has also shown that organic matter can have different effects (protecting or inactivating) on different proteins.

  13. Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium

    NARCIS (Netherlands)

    Franz, E.; Visser, A.A.; Diepeningen, van A.D.; Klerks, M.M.; Termorshuizen, A.J.; Bruggen, van A.H.C.

    2007-01-01

    The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca

  14. Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells.

    Science.gov (United States)

    Staudacher, Verena; Trujillo, Madia; Diederichs, Tim; Dick, Tobias P; Radi, Rafael; Morgan, Bruce; Deponte, Marcel

    2018-04-01

    Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Probe tests microweld strength

    Science.gov (United States)

    1965-01-01

    Probe is developed to test strength of soldered, brazed or microwelded joints. It consists of a spring which may be adjusted to the desired test pressure by means of a threaded probe head, and an indicator lamp. Device may be used for electronic equipment testing.

  16. Variable episomal silencing of a recombinant herpesvirus renders its encoded GFP an unreliable marker of infection in primary cells.

    Directory of Open Access Journals (Sweden)

    Thomas J Ellison

    Full Text Available The availability of reliable recombinant reporter virus systems has been a great boon to the study of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8. Unexpectedly, we found that expression of the ostensibly constitutive green fluorescent protein (GFP marker was progressively lost during unselected passage in primary rat mesenchymal precursor cells (MM, despite efficient maintenance of latent viral gene expression and episomal partitioning. This repression of EF1-α promoter-driven GFP expression appeared to be passage-dependent, however, since functionally immortalized MM cells derived from long serial passage retained stable expression of GFP following rKSHV.219 infection. Chromatin analysis of cultures that we had infected in parallel demonstrated an increase in repressive H3K27 tri-methylation across the viral episome with the exception of the LANA control region in MM cells infected at early rather than late passage post-isolation. The silencing of GFP expression in the MM cells was reversible in a dose-dependent fashion by the histone deacetylase inhibitor valproic acid, further implicating cellular silencing on incoming viral genomes, and underscoring potential differences in viral gene regulation between primary and functionally immortalized cells. Furthermore, using multispectral imaging flow cytometry, we also determined that the extent of GFP expression per cell among those that were positive did not correlate with the number of LANA dots per nucleus nor the extent of overall LANA expression per cell. This suggests a more complex mode of local gene regulation, rather than one that simply reflects the relative intracellular viral copy number. In sum, we have demonstrated the significant potential for false-negative data when using a constitutive marker gene as a sole means of evaluating herpesviral infection, especially in primary cells.

  17. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

    Directory of Open Access Journals (Sweden)

    Macmaster Suzanne

    2002-06-01

    Full Text Available Abstract Background Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP in embryonic stem (ES cells and mice 2. Results In this study we have used embryonic stem (ES cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP and enhanced yellow fluorescent protein (EYFP, two mutant and spectrally distinct color variants of wild type (wt GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid diploid and polarized (tetraploid diploid chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. Conclusions GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or

  18. Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

    Science.gov (United States)

    Mares, Rosa E; Meléndez-López, Samuel G; Ramos, Marco A

    2011-01-01

    Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI

  19. Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo.

    Directory of Open Access Journals (Sweden)

    Claudia Laperchia

    Full Text Available Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype.With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity of dendritic cells (DCs, and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.

  20. Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution

    Science.gov (United States)

    Novikov, Eugene G.; Skakun, Victor V.; Borst, Jan Willem; Visser, Antonie J. W. G.

    2018-01-01

    The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

  1. Gas Replacements for GFP to Track Microbial Dynamics in Soils and Sediments

    Science.gov (United States)

    Cheng, Hsiao-Ying; Silberg, Jonathan; Masiello, Caroline

    2016-04-01

    Metagenomic analyses offer unprecedented views of soil microbial communities, and additionally provide a host of testable hypotheses about the biological mechanisms driving global biogeochemical fluxes. Outside the biogeosciences, hypotheses generated by metagenomics are often tested using biosensors, microbes programmed to respond in a detectable way to either changes in their metabolism or changes in the environment. A very large number of microbial behaviors can be monitored using biosensors, but these sensors typically report in ways that are undetectable in soils, e.g. by releasing green fluorescent protein (GFP). We are building a new class of biosensors that report by releasing easily-detected gases. We will provide an overview of the potential uses of gas-reporting biosensors in geobiology, and will report the current development these sensors. One goal in the development of these sensors is to make tractable the testing of gene expression hypotheses derived from metagenomics data. Examples of processes that could be tracked non-invasively with gas sensors include coordination of biofilm formation, nitrification, rhizobial infection of plant roots, and at least some forms of methanogenesis, all of which are managed by the easily-engineered acyl homoserine lactone cell-cell communication system. Another relatively simple process to track with gas sensors is horizontal gene transfer. We will report on the progress of these proof-of-concept examples.

  2. Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization

    DEFF Research Database (Denmark)

    Xu, Wen-Ming; Zhang, Si-Zhong; Qiu, Wei-Min

    2004-01-01

    To use green fluorescent protein as a marker to study the localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification, and then cloned into pGEM-Teasy vector. After the double enzyme...... cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1. Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP). Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed...... that green fluorescence protein expressed over the whole cell when transfected with vector pEGFP-N1. While after the transfection with pEGFP-ZNF230, the fluorescence located mainly on the nuclei of the cells. We demonstrated that the transfected Cos cell line can express human ZNF230 and mouse znf230...

  3. Determination of Histone 2B-Green Fluorescent Protein (GFP) Retention in Intestinal Stem Cells.

    Science.gov (United States)

    Hughes, Kevin R; Mahida, Yashwant R

    2018-01-01

    The epithelium of the gastrointestinal tract represents the interface between the luminal contents of the gut and that of the host tissues and plays a central role not only in regulating absorption of dietary nutrients but also in providing a barrier to prevent the entry of bacteria and other pathogens. Repair and replacement of damaged aging cells within the epithelium is modulated by stem cells, which are located in the intestinal crypts of the small intestine.Two distinct populations of intestinal stem cells have been described in the literature, one population at the very base of the crypt and a second population of long-lived stem cells located just above the Paneth cell zone. Herein, we describe a method to label this population of long-lived GFP label retaining cells. This method is free from confounding factors of previous methodologies based on radioactive tracers and also enables functional studies not previously possible using the radioactive tracer techniques described in the literature.

  4. Neutrons as a probe

    International Nuclear Information System (INIS)

    Iizumi, Masashi

    1993-01-01

    As an introduction to the symposium a brief overview will be given about the features of neutrons as a probe. First it will be pointed out that the utilization of neutrons as a probe for investigating the structural and dynamical properties of condensed matters is a benign gift eventuated from the release of atomic energy initiated by Enrico Fermi exactly half century ago. Features of neutrons as a probe are discussed in accordance with the four basic physical properties of neutrons as an elementary particle; (1) no electric charge (the interaction with matter is nuclear), (2) the mass of neutron is 1 amu, (3) spin is 1/2 and (4) neutrons have magnetic dipole moment. Overview will be given on the uniqueness of neutrons as a probe and on the variety in the way they are used in the wide research area from the pure science to the industrial applications. (author)

  5. Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli.

    Science.gov (United States)

    Delvigne, Frank; Boxus, Mathieu; Ingels, Sophie; Thonart, Philippe

    2009-02-25

    Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude. The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP). It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population. The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low

  6. Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli

    Directory of Open Access Journals (Sweden)

    Boxus Mathieu

    2009-02-01

    Full Text Available Abstract Background Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude. Results The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP. It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population. Conclusion The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial

  7. Noninvasive ultrasonic probes

    International Nuclear Information System (INIS)

    Barnes, S.R.; Galer, D.R.; Leard, R.S.

    1987-01-01

    An ultrasonic probe is described for insonifying the ascending aorta of a supine or reclining human patient from a location within the suprasternal notch of the patient. The probe comprises: a transducer head and an elongated handle; housing propagates ultrasonic energy and for intercept-frequency-shifted, reflected radiant energy; the handle has a proximate portion and a distal portion and a non-circular cross-sectional configuration with at least one longitudinal edge which furnishes a gripping surface; this facilitates tactile positioning of the probe; the transducer head is integral with the handle of the probe at the exposed end of the proximate portion; the transducer head has a generally arcuate cross-sectional configuration and a generally trapezoidal profile; the transducer head is oriented at right angles to the proximate portion of the handle and has an exposed, patient contacting end in which the transducer means are located; this facilitates the orientation of the transducer means housed in the head relative to the ascending aorta of the patient; and the distal end portion of the elongated probe handle is integral with and immovably oriented at a severe angle relative to the proximate end of that handle, and lies in the same plane as the proximate end of the handle; the transducer head of the probe is placed with facility within the suprasternal notch of the patient by an operator positioned behind the head of the patient

  8. Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3' untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos.

    Science.gov (United States)

    Kawakami, Y; Ishihara, M; Saito, T; Fujimoto, T; Adachi, S; Arai, K; Yamaha, E

    2012-12-01

    Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.

  9. Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration

    DEFF Research Database (Denmark)

    Hansen, M.C.; Palmer, R.J.; Udsen, C.

    2001-01-01

    Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH. There are conflicting data on these limitations, however, and systematic...... studies to assess the importance of these factors for growing bacterial cultures are lacking. In the present study, homogeneous expression of the gfpmut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of Streptococcus gordonii DL1....... A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 0.1 p.p.m. dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0.025 p.p.m. dissolved oxygen...

  10. A Light-Induced Reaction with Oxygen Leads to Chromophore Decomposition and Irreversible Photobleaching in GFP-Type Proteins.

    Science.gov (United States)

    Grigorenko, Bella L; Nemukhin, Alexander V; Polyakov, Igor V; Khrenova, Maria G; Krylov, Anna I

    2015-04-30

    Photobleaching and photostability of proteins of the green fluorescent protein (GFP) family are crucially important for practical applications of these widely used biomarkers. On the basis of simulations, we propose a mechanism for irreversible bleaching in GFP-type proteins under intense light illumination. The key feature of the mechanism is a photoinduced reaction of the chromophore with molecular oxygen (O2) inside the protein barrel leading to the chromophore's decomposition. Using quantum mechanics/molecular mechanics (QM/MM) modeling we show that a model system comprising the protein-bound Chro(-) and O2 can be excited to an electronic state of the intermolecular charge-transfer (CT) character (Chro(•)···O2(-•)). Once in the CT state, the system undergoes a series of chemical reactions with low activation barriers resulting in the cleavage of the bridging bond between the phenolic and imidazolinone rings and disintegration of the chromophore.

  11. [Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells].

    Science.gov (United States)

    Liu, Jun-Pin; Li, Hong-Tao; Li, Wei; Liu, Hong; Zhang, Ling; Min, Jie; Zhou, Ting; Zhou, Lei; Zhang, Zhi-Bing

    2016-07-01

    To construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells. Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy. The construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells. Conclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.

  12. Fluorescent labeling of newborn dentate granule cells in GAD67-GFP transgenic mice: a genetic tool for the study of adult neurogenesis.

    Directory of Open Access Journals (Sweden)

    Shengli Zhao

    Full Text Available Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP(+ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP(+ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.

  13. Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

    Science.gov (United States)

    Nienhaus, Karin; Nienhaus, G. Ulrich

    2016-11-01

    Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

  14. Looking at the Green Fluorescent Protein (GFP) chromophore from a different perspective: A computational insight

    Science.gov (United States)

    Paul, Bijan Kumar; Guchhait, Nikhil

    2013-02-01

    In the present contribution Density Functional Theory (DFT) has been applied to explore molecular dipole moment, frontier molecular orbital (FMO) features, chemical hardness, and the molecular electrostatic potential surface (MEPS) characteristics for optimized molecular geometry of the Green Fluorescent Protein (GFP) chromophore p-hydroxybenzylideneimidazolinone (HBDI) both in its protonated (neutral) and deprotonated (anion) forms. The distribution of atomic charges over the entire molecular framework as obtained from Natural Bond Orbital (NBO) analysis is found to faithfully replicate the predictions from the MEP map in respect of reactivity map of HBDI (neutral and anion) and possible sites for hydrogen bonding interactions etc. The three dimensional MEP map encompassing the entire molecule yields a reliable reactivity map of HBDI molecule also displaying the most probable regions for non-covalent interactions. The differential distribution of the electrostatic potential over the neutral and anionic species of HBDI is authentically reflected on MEP map and NBO charge distribution analysis. Thermodynamic properties such as heat capacity, thermal energy, enthalpy, entropy have been calculated and the correlation of the various thermodynamic functions with temperature has been established for neutral molecule. More importantly, however, the computational approach has been employed to unveil the nonlinear optical (NLO) properties of protonated (neutral) and deprotonated (anion) HBDI. Also in an endeavor to achieve a fuller understanding on this aspect the effect of basis set on the NLO properties of the title molecule has been investigated. Our computations delineate the discernible differences in NLO properties between the neutral and anionic species of HBDI whereby indicating the possibility of development of photoswitchable NLO device.

  15. Differential equation methods for simulation of GFP kinetics in non-steady state experiments.

    Science.gov (United States)

    Phair, Robert D

    2018-03-15

    Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Current methods, although useful and powerful, depend on the assumption that the cellular system being studied is in a steady state, that is, the assumption that all the molecular concentrations and fluxes are constant for the duration of the experiment. Here, we derive new tracer kinetic analytical methods for non-steady state biological systems by constructing mechanistic nonlinear differential equation models of the underlying cell biological processes and linking them to a separate set of differential equations governing the kinetics of the fluorescent tracer. Linking the two sets of equations is based on a new application of the fundamental tracer principle of indistinguishability and, unlike current methods, supports correct dependence of tracer kinetics on cellular dynamics. This approach thus provides a general mathematical framework for applications of GFP fluorescence microscopy (including photobleaching [FRAP, FLIP] and photoactivation to frequently encountered experimental protocols involving physiological or pharmacological perturbations (e.g., growth factors, neurotransmitters, acute knockouts, inhibitors, hormones, cytokines, and metabolites) that initiate mechanistically informative intracellular transients. When a new steady state is achieved, these methods automatically reduce to classical steady state tracer kinetic analysis. © 2018 Phair. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  16. Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

    International Nuclear Information System (INIS)

    Nienhaus, Karin; Nienhaus, G Ulrich

    2016-01-01

    Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments. (topical review)

  17. Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

    Directory of Open Access Journals (Sweden)

    Meiling Yu

    2017-09-01

    Full Text Available Porcine transmissible gastroenteritis virus (TGEV and porcine epidemic diarrhea virus (PEDV are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. To develop an effective bivalent oral vaccine against TGEV and PEDV infection, the D antigenic site of the TGEV spike (S protein and the major antigen site (core neutralizing epitope—COE of the PEDV S protein were used as immunogens, and the enhanced green fluorescent protein (eGFP gene was used as a reporter to construct genetically engineered Lactobacillus casei rLpPGF-T7g10-eGFP-6D-COE. The expression of proteins of interest by the recombinant L. casei was confirmed by confocal laser scanning microscopy and a Western blot assay, and the immunogenicity of rLpPGF-T7g10-eGFP-6D-COE in orally immunized mice was evaluated. The results showed that levels of anti-PEDV and anti-TGEV serum immunoglobulin G (IgG and mucosal secreted immunoglobulin A (sIgA antibodies obtained from the mice immunized with rLpPGF-T7g10-eGFP-6D-COE, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (PBS or rLpPG-T7g10. Moreover, the serum IgG antibodies showed neutralizing effects against PEDV and TGEV. Our data suggest that the antibiotic resistance-free genetically engineered L. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against PEDV and TGEV.

  18. Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus.

    Science.gov (United States)

    Yu, Meiling; Wang, Li; Ma, Sunting; Wang, Xiaona; Wang, Yusai; Xiao, Ya; Jiang, Yanping; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

    2017-09-25

    Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. To develop an effective bivalent oral vaccine against TGEV and PEDV infection, the D antigenic site of the TGEV spike (S) protein and the major antigen site (core neutralizing epitope-COE) of the PEDV S protein were used as immunogens, and the enhanced green fluorescent protein (eGFP) gene was used as a reporter to construct genetically engineered Lactobacillus casei rLpPG F -T7g10-eGFP-6D-COE. The expression of proteins of interest by the recombinant L. casei was confirmed by confocal laser scanning microscopy and a Western blot assay, and the immunogenicity of rLpPG F -T7g10-eGFP-6D-COE in orally immunized mice was evaluated. The results showed that levels of anti-PEDV and anti-TGEV serum immunoglobulin G (IgG) and mucosal secreted immunoglobulin A (sIgA) antibodies obtained from the mice immunized with rLpPG F -T7g10-eGFP-6D-COE, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (PBS) or rLpPG-T7g10. Moreover, the serum IgG antibodies showed neutralizing effects against PEDV and TGEV. Our data suggest that the antibiotic resistance-free genetically engineered L. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against PEDV and TGEV.

  19. Green fluorescent protein (GFP) color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1) transfected endothelial modification.

    Science.gov (United States)

    Wurster, Thomas; Pölzelbauer, Catharina; Schönberger, Tanja; Paul, Angela; Seizer, Peter; Stellos, Konstantinos; Schuster, Andreas; Botnar, Rene M; Gawaz, Meinrad; Bigalke, Boris

    2012-01-01

    Human Parvovirus B19 (PVB19) has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP) color reporter gene in the non-structural segment 1 (NS1) of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304). The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber). NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; PGFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; PGFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (PGFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

  20. GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard

    OpenAIRE

    Piotrowska-Seget, Zofia; Beściak, Grażyna; Bernaś, Tytus; Kozdrój, Jacek

    2011-01-01

    The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bear...

  1. Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay.

    Science.gov (United States)

    Brunkard, Jacob O; Burch-Smith, Tessa M; Runkel, Anne M; Zambryski, Patricia

    2015-01-01

    Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

  2. Gene amplification by PCR and subcloning into a GFP-fusion plasmid expression vector as a molecular biology laboratory course*.

    Science.gov (United States)

    Bornhorst, Joshua A; Deibel, Michael A; Mulnix, Amy B

    2004-05-01

    A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the course of a single semester in a weekly 3-h laboratory period. Students designed PCR primers for amplification of the selected gene using computational DNA sequence analysis tools. During the experimental portion of the course, students amplified and ligated the target DNA into a commercially available GFP expression vector. Following transformation of the ligation product, plasmids were harvested from the resulting bacterial colonies and were analyzed by restriction digestion to confirm the creation of the chimeric GFP-DNA. This course gave students valuable experience with commonly used molecular techniques in an authentic research project. In addition, students gained experience with experimental design and execution. The techniques presented here are flexible and can be generalized for use with almost any DNA sequence and expression vector. This series also serves as an example of how faculty can adapt their ongoing research projects to the undergraduate laboratory. Copyright © 2004 International Union of Biochemistry and Molecular Biology, Inc.

  3. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH fusion to gfp (green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Bat-Erdene Jugder

    2016-07-01

    Full Text Available Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2. Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

  4. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein).

    Science.gov (United States)

    Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady; Marquis, Christopher P

    2016-01-01

    Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

  5. Diversity of retinal ganglion cells identified by transient GFP transfection in organotypic tissue culture of adult marmoset monkey retina.

    Directory of Open Access Journals (Sweden)

    Satoru Moritoh

    Full Text Available The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus monkey as a "super-model" of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells, and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]. By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina.

  6. Deciphering the Niches of Colonisation of Vitis vinifera L. by the Esca-Associated Fungus Phaeoacremonium aleophilum Using a gfp Marked Strain and Cutting Systems.

    Directory of Open Access Journals (Sweden)

    Romain Pierron

    Full Text Available Esca disease has become a major threat for viticulture. Phaeoacremonium aleophilum is considered a pioneer of the esca complex pathosystem, but its colonisation behaviour inside plants remains poorly investigated.In this study, P. aleophilum::gfp7 colonisation was assessed six and twelve weeks post-inoculation in two different types of tissues: in the node and the internode of one year-old rooted cuttings of Cabernet Sauvignon. These processes of colonisation were compared with the colonisation by the wild-type strain using a non-specific lectin probe Alexa Fluor 488-WGA.Data showed that six weeks post-inoculation of the internode, the fungus had colonised the inoculation point, the bark and xylem fibres. Bark, pith and xylem fibres were strongly colonised by the fungus twelve weeks post-inoculation and it can progress up to 8 mm from the point of inoculation using pith, bark and fibres. P. aleophilum was additionally detected in the lumen of xylem vessels in which tyloses blocked its progression. Different plant responses in specific tissues were additionally visualised. Inoculation of nodes led to restricted colonisation of P. aleophilum and this colonisation was associated with a plant response six weeks post-inoculation. The fungus was however detected in xylem vessels, bark and inside the pith twelve weeks post-inoculation.These results demonstrate that P. aleophilum colonisation can vary according to the type of tissues and the type of spread using pith, bark and fibres. Woody tissues can respond to the injury and to the presence of this fungus, and xylem fibres play a key role in the early colonisation of the internode by P. aleophilum before the fungus can colonise xylem vessels.

  7. Model for resonant plasma probe.

    Energy Technology Data Exchange (ETDEWEB)

    Warne, Larry Kevin; Johnson, William Arthur; Hebner, Gregory Albert; Jorgenson, Roy E.; Coats, Rebecca Sue

    2007-04-01

    This report constructs simple circuit models for a hairpin shaped resonant plasma probe. Effects of the plasma sheath region surrounding the wires making up the probe are determined. Electromagnetic simulations of the probe are compared to the circuit model results. The perturbing effects of the disc cavity in which the probe operates are also found.

  8. Convective heat flow probe

    Science.gov (United States)

    Dunn, James C.; Hardee, Harry C.; Striker, Richard P.

    1985-01-01

    A convective heat flow probe device is provided which measures heat flow and fluid flow magnitude in the formation surrounding a borehole. The probe comprises an elongate housing adapted to be lowered down into the borehole; a plurality of heaters extending along the probe for heating the formation surrounding the borehole; a plurality of temperature sensors arranged around the periphery of the probe for measuring the temperature of the surrounding formation after heating thereof by the heater elements. The temperature sensors and heater elements are mounted in a plurality of separate heater pads which are supported by the housing and which are adapted to be radially expanded into firm engagement with the walls of the borehole. The heat supplied by the heater elements and the temperatures measured by the temperature sensors are monitored and used in providing the desired measurements. The outer peripheral surfaces of the heater pads are configured as segments of a cylinder and form a full cylinder when taken together. A plurality of temperature sensors are located on each pad so as to extend along the length and across the width thereof, with a heating element being located in each pad beneath the temperature sensors. An expansion mechanism driven by a clamping motor provides expansion and retraction of the heater pads and expandable packer-type seals are provided along the probe above and below the heater pads.

  9. Following the infection process of vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species.

    Science.gov (United States)

    Dubert, Javier; Nelson, David R; Spinard, Edward J; Kessner, Linda; Gomez-Chiarri, Marta; da Costa, Fiz; Prado, Susana; Barja, Juan L

    2016-01-01

    Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was

  10. Multispectral imaging probe

    Science.gov (United States)

    Sandison, David R.; Platzbecker, Mark R.; Descour, Michael R.; Armour, David L.; Craig, Marcus J.; Richards-Kortum, Rebecca

    1999-01-01

    A multispectral imaging probe delivers a range of wavelengths of excitation light to a target and collects a range of expressed light wavelengths. The multispectral imaging probe is adapted for mobile use and use in confined spaces, and is sealed against the effects of hostile environments. The multispectral imaging probe comprises a housing that defines a sealed volume that is substantially sealed from the surrounding environment. A beam splitting device mounts within the sealed volume. Excitation light is directed to the beam splitting device, which directs the excitation light to a target. Expressed light from the target reaches the beam splitting device along a path coaxial with the path traveled by the excitation light from the beam splitting device to the target. The beam splitting device directs expressed light to a collection subsystem for delivery to a detector.

  11. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    Science.gov (United States)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Förster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI. PMID:20599730

  12. Genetic transformation of Drosophila willistoni using piggyBac transposon and GFP

    Directory of Open Access Journals (Sweden)

    Manuela Finokiet

    2007-01-01

    Full Text Available Studies were carried out on the use of piggyBac transposable element as vector and the green fluorescent protein (EGFP from the jellyfish, Aquorea victoria, as a genetic marker for the transformation of Drosophila willistoni. Preblastoderm embryos of D. willistoni white mutant were microinjected with a plasmid containing the EGFP marker and the piggyBac ITRs, together with a helper plasmid containing the piggyBac transposase placed under the control of the D. melanogaster hsp70 promoter. G0 adults transformants were recovered at a frequency of approximately 67%. Expression of EGFP in larvae, pupae and adults was observed up to the third generation, suggesting that this transposon was not stable in D. willistoni. Transformed individuals displayed high levels of EGFP expression during larvae and adult stages in the eye, abdomen, thorax and legs, suggesting a wide expression pattern in this species than reported to other species of Drosophilidae.Descrevemos neste trabalho a transformação genética de Drosophila willistoni empregando o elemento transponível piggyBac como vetor e o gene EGFP (green fluorescent protein retirado da água-viva Aquorea victoria, como marcador de transformação. Embriões de D. willistoni em estágio pré-blastoderme, mutantes para o gene white, foram microinjetados com plasmídio contendo o marcador EGFP e as regiões ITRs do transposon piggyBac concomitantemente com um plasmídio auxiliar possuindo o gene da transposase de piggyBac sobre o controle do promotor do gene hsp70 de Drosophila melanogaster. Adultos transformantes Go foram gerados em uma taxa de 67%. A expressão de GFP em larvas, pupas e adultos foi observada somente até a terceira geração, sugerindo que este transposon não é estável em D. willistoni. Os indivíduos transformados exigem um alto nível de expressão de EGFP durante os estágios de larva e, também em adultos o gene marcador é expresso nos olhos, abdome, tórax e patas, mostrando um

  13. A General Factor of Personality (GFP) in the personality disorders: three studies of the Dimensional Assessment of Personality Pathology - Basic Questionnaire (DAPP-BQ).

    Science.gov (United States)

    Rushton, J Philippe; Irwing, Paul; Booth, Tom

    2010-08-01

    We used structural equation modeling to test the hypothesis that a General Factor of Personality (GFP) occupies the apex of the hierarchy of personality disorders in three validation samples of the Dimensional Assessment of Personality Pathology - Basic Questionnaire (DAPP-BQ). In a general population sample (N = 942), we found a GFP explained 34% of the variance in four first-order factors and 33% of the variance in all 18 scales. In a twin sample (N = 1,346), a GFP explained 35% of the variance in four first-order factors and 34% of the variance in all 18 scales. In a clinical sample (N = 656), a GFP explained 34% of the variance in four first-order factors and 30% of the variance in all 18 scales.

  14. GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies

    DEFF Research Database (Denmark)

    Lübeck, M.; Knudsen, I.M.B.; Jensen, B.

    2002-01-01

    Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The beta-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated...... into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro....... In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley...

  15. Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies.

    Science.gov (United States)

    Sper, Renan B; Koh, Sehwon; Zhang, Xia; Simpson, Sean; Collins, Bruce; Sommer, Jeff; Petters, Robert M; Caballero, Ignacio; Platt, Jeff L; Piedrahita, Jorge A

    2017-01-01

    Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3-5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model.

  16. GAD67-GFP+ neurons in the Nucleus of Roller: a possible source of inhibitory input to hypoglossal motoneurons. I. Morphology and firing properties.

    Science.gov (United States)

    van Brederode, J F M; Yanagawa, Y; Berger, A J

    2011-01-01

    In this study we examined the electrophysiological and morphological properties of inhibitory neurons located just ventrolateral to the hypoglossal motor (XII) nucleus in the Nucleus of Roller (NR). In vitro experiments were performed on medullary slices derived from postnatal day 5 (P5) to P15 GAD67-GFP knock-in mouse pups. on cell recordings from GFP+ cells in NR in rhythmic slices revealed that these neurons are spontaneously active, although their spiking activity does not exhibit inspiratory phase modulation. Morphologically, GFP+ cells were bi- or multipolar cells with small- to medium-sized cell bodies and small dendritic trees that were often oriented parallel to the border of the XII nucleus. GFP+ cells were classified as either tonic or phasic based on their firing responses to depolarizing step current stimulation in whole cell current clamp. Tonic GFP+ cells fired a regular train of action potentials (APs) throughout the duration of the pulse and often showed rebound spikes after a hyperpolarizing step. In contrast, phasic GFP+ neurons did not fire throughout the depolarizing current step but instead fired fewer than four APs at the onset of the pulse or fired multiple APs, but only after a marked delay. Phasic cells had a significantly smaller input resistance and shorter membrane time constant than tonic GFP+ cells. In addition, phasic GFP+ cells differed from tonic cells in the shape and time course of their spike afterpotentials, the minimum firing frequency at threshold current amplitude, and the slope of their current-frequency relationship. These results suggest that GABAergic neurons in the NR are morphologically and electrophysiologically heterogeneous cells that could provide tonic inhibitory synaptic input to HMs.

  17. Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP).

    Science.gov (United States)

    Young, Katie R; Arthus-Cartier, Guillaume; Yam, Karen K; Lavoie, Pierre-Olivier; Landry, Nathalie; D'Aoust, Marc-André; Vézina, Louis-Philippe; Couture, Manon M-J; Ward, Brian J

    2015-09-01

    Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines. © FASEB.

  18. Probing the Solar Interior

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 3; Issue 3. Probing the Solar Interior Hearing the Heartbeats of the Sun. Ashok Ambastha. General ... Author Affiliations. Ashok Ambastha1. Joint In-Charge Udaipur Solar Observatory Physical Research laboratory P.O. Box No. 198 Udaipur 313 001, India ...

  19. Probing the Solar Interior

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 3; Issue 3. Probing the Solar Interior Hearing the Heartbeats of the Sun. Ashok Ambastha. General Article Volume 3 Issue 3 March 1998 pp 18-31. Fulltext. Click here to view fulltext PDF. Permanent link:

  20. Terahertz scanning probe microscope

    NARCIS (Netherlands)

    Klapwijk, T.M.

    2014-01-01

    The invention provides aterahertz scanning probe microscope setup comprising (i) a terahertz radiation source configured to generate terahertz radiation; (ii) a terahertz lens configured to receive at least part of the terahertz radiation from the terahertz radiation source; (iii) a cantilever unit

  1. One-Probe Search

    DEFF Research Database (Denmark)

    Östlin, Anna; Pagh, Rasmus

    2002-01-01

    We consider dictionaries that perform lookups by probing a single word of memory, knowing only the size of the data structure. We describe a randomized dictionary where a lookup returns the correct answer with probability 1 - e, and otherwise returns don't know. The lookup procedure uses an expan...

  2. Probing the Solar System

    Science.gov (United States)

    Wilkinson, John

    2013-01-01

    Humans have always had the vision to one day live on other planets. This vision existed even before the first person was put into orbit. Since the early space missions of putting humans into orbit around Earth, many advances have been made in space technology. We have now sent many space probes deep into the Solar system to explore the planets and…

  3. Thermal conductivity probe

    Science.gov (United States)

    Navickas, J.

    1969-01-01

    Low-mass probe accurately measures the thermal conductivity of polyurethane foam /and other thermal insulating materials/ while exposed to either hydrogen of helium permeation in temperature ranges from ambient to cryogenic. The thermal conductivity of a specimen is determined from an experimentally determined increase in temperature.

  4. The Impact of GFP Reporter Gene Transduction and Expression on Metabolomics of Placental Mesenchymal Stem Cells Determined by UHPLC-Q/TOF-MS

    Directory of Open Access Journals (Sweden)

    Jinfeng Yang

    2017-01-01

    Full Text Available Introduction. Green fluorescent protein (GFP is widely used as a reporter gene in regenerative medicine research to label and track stem cells. Here, we examined whether expressing GFP gene may impact the metabolism of human placental mesenchymal stem cells (hPMSCs. Methods. The GFP gene was transduced into hPMSCs using lentiviral-based infection to establish GFP+hPMSCs. A sensitive 13C/12C-dansyl labeling LC-MS method targeting the amine/phenol submetabolome was used for in-depth cell metabolome profiling. Results. A total of 1151 peak pairs or metabolites were detected from 12 LC-MS runs. Principal component analysis and partial least squares discriminant analysis showed poor separation, and the volcano plots demonstrated that most of the metabolites were not significantly changed when hPMSCs were tagged with GFP. Overall, 739 metabolites were positively or putatively identified. Only 11 metabolites showed significant changes. Metabolic pathway analyses indicated that three of the identified metabolites were involved in nine pathways. However, these metabolites are unlikely to have a large impact on the metabolic pathways due to their nonessential roles and limited hits in pathway analysis. Conclusion. This study indicated that the expression of ectopic GFP reporter gene did not significantly alter the metabolomics pathways covered by the amine/phenol submetabolome.

  5. Contrasting colonization and plant growth promoting capacity between wild type and gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

    Energy Technology Data Exchange (ETDEWEB)

    Weyens N.; van der Lelie D.; Boulet, J.; Adriaensen, D.; Timmermans, J.-P.; Prinsen, E.; Van Oevelen, S.; D" Haen, J.; Smeets, K.; Taghavi, S.; Vangronsveld, J.

    2011-06-09

    This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.

  6. Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein.

    Science.gov (United States)

    Greussing, Ruth; Unterluggauer, Hermann; Koziel, Rafal; Maier, Andrea B; Jansen-Dürr, Pidder

    2012-11-10

    Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins (1, 2). Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response (3), cell cycle regulation and cellular differentiation (4) or in immune system response (5). The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases (4, 6). Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging (7, 8, 9, 10). Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable (11)) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

  7. Calibration Fixture For Anemometer Probes

    Science.gov (United States)

    Lewis, Charles R.; Nagel, Robert T.

    1993-01-01

    Fixture facilitates calibration of three-dimensional sideflow thermal anemometer probes. With fixture, probe oriented at number of angles throughout its design range. Readings calibrated as function of orientation in airflow. Calibration repeatable and verifiable.

  8. Performance of popular XC-functionals for the description of excitation energies in GFP-like chromophore models

    DEFF Research Database (Denmark)

    List, Nanna Holmegaard; Olsen, Jógvan Magnus; Rocha-Rinza, Tomás

    2012-01-01

    this task. We present an evaluation of the performance of commonly used XC-functionals for the prediction of excitation energies of GFP-like chromophores. In particular, we have considered the TD-DFT vertical excitation energies of chromophores displaying different charge states. We compare the quality...... are found to give the overall best performance. The Coulomb-attenuated CAM-B3LYP functional systematically overestimates the excitation energies of the charged states; however, its error has the attractive feature of being size-independent and almost identical for the considered anionic and cationic systems...

  9. Heavy ion beam probing

    Energy Technology Data Exchange (ETDEWEB)

    Hickok, R L

    1980-07-01

    This report consists of the notes distributed to the participants at the IEEE Mini-Course on Modern Plasma Diagnostics that was held in Madison, Wisconsin in May 1980. It presents an overview of Heavy Ion Beam Probing that briefly describes the principles and discuss the types of measurements that can be made. The problems associated with implementing beam probes are noted, possible variations are described, estimated costs of present day systems, and the scaling requirements for large plasma devices are presented. The final chapter illustrates typical results that have been obtained on a variety of plasma devices. No detailed calculations are included in the report, but a list of references that will provide more detailed information is included.

  10. Heavy ion beam probing

    International Nuclear Information System (INIS)

    Hickok, R.L.

    1980-07-01

    This report consists of the notes distributed to the participants at the IEEE Mini-Course on Modern Plasma Diagnostics that was held in Madison, Wisconsin in May 1980. It presents an overview of Heavy Ion Beam Probing that briefly describes the principles and discuss the types of measurements that can be made. The problems associated with implementing beam probes are noted, possible variations are described, estimated costs of present day systems, and the scaling requirements for large plasma devices are presented. The final chapter illustrates typical results that have been obtained on a variety of plasma devices. No detailed calculations are included in the report, but a list of references that will provide more detailed information is included

  11. Gravity Probe B Inspection

    Science.gov (United States)

    2000-01-01

    The space vehicle Gravity Probe B (GP-B) is the relativity experiment developed at Stanford University to test two extraordinary predictions of Albert Einstein's general theory of relativity. The experiment will measure, very precisely, the expected tiny changes in the direction of the spin axes of four gyroscopes contained in an Earth-orbiting satellite at a 400-mile altitude. So free are the gyroscopes from disturbance that they will provide an almost perfect space-time reference system. They will measure how space and time are very slightly warped by the presence of the Earth, and, more profoundly, how the Earth's rotation very slightly drags space-time around with it. These effects, though small for the Earth, have far-reaching implications for the nature of matter and the structure of the Universe. GP-B is among the most thoroughly researched programs ever undertaken by NASA. This is the story of a scientific quest in which physicists and engineers have collaborated closely over many years. Inspired by their quest, they have invented a whole range of technologies that are already enlivening other branches of science and engineering. In this photograph, engineer Gary Reynolds is inspecting the inside of the probe neck during probe thermal repairs. GP-B is scheduled for launch in April 2004 and managed for NASA by the Marshall Space Flight Center. Development of the GP-B is the responsibility of Stanford University along with major subcontractor Lockheed Martin Corporation. (Image credit to Russ Leese, Gravity Probe B, Stanford University)

  12. Novel Eddycurrent Probe Development.

    Science.gov (United States)

    1981-12-01

    oVCut MVc AVu inc F C -- -(1)(-jlOOO AZC (23)0 --2--M F1 (23) If the Thevenin source voltages, V0, are adjusted so that Vinc is the same for both the...as small as 1.2 cm. The differential probe assembly was spring loaded about a pivot post (see Figure 12) so it could scan noncircular or eccentric

  13. Induced current heating probe

    International Nuclear Information System (INIS)

    Thatcher, G.; Ferguson, B.G.; Winstanley, J.P.

    1984-01-01

    An induced current heating probe is of thimble form and has an outer conducting sheath and a water flooded flux-generating unit formed from a stack of ferrite rings coaxially disposed in the sheath. The energising coil is made of solid wire which connects at one end with a coaxial water current tube and at the other end with the sheath. The stack of ferrite rings may include non-magnetic insulating rings which help to shape the flux. (author)

  14. Atom probe crystallography

    OpenAIRE

    Gault, Baptiste; Moody, Michael P.; Cairney, Julie M.; Ringer, Simon P.

    2012-01-01

    This review addresses new developments in the emerging area of “atom probe crystallography”, a materials characterization tool with the unique capacity to reveal both composition and crystallographic structure at the atomic scale. This information is crucial for the manipulation of microstructure for the design of both structural and functional materials with optimized mechanical, electric, optoelectronic, magnetic, or superconducting properties that will find application in, for example, nan...

  15. Probing gravitation with pulsars

    Science.gov (United States)

    Kramer, Michael

    2013-03-01

    Radio pulsars are fascinating and extremely useful objects. Despite our on-going difficulties in understanding the details of their emission physics, they can be used as precise cosmic clocks in a wide-range of experiments - in particular for probing gravitational physics. While the reader should consult the contributions to these proceedings to learn more about this exciting field of discovering, exploiting and understanding pulsars, we will concentrate here on on the usage of pulsars as gravity labs.

  16. A vector carrying the GFP gene (Green fluorescent protein as a yeast marker for fermentation processes Um vetor com o gene da GFP (Green fluorescent protein para a marcação de leveduras em processos fermentativos

    Directory of Open Access Journals (Sweden)

    Luiz Humberto Gomes

    2000-12-01

    Full Text Available Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3. Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.Leveduras contaminantes podem causar grandes perdas em processos fermentativos quando infectam culturas puras e degradam a qualidade do produto final. Estas leveduras podem ser detectadas por diversos métodos mas nenhum deles oferece resultados com a exatidão e precisão necessárias, quando os contaminantes estão em baixa freqüência. Culturas puras contendo um gene marcador podem ser utilizadas para a direta identificação de células e colônias contaminantes. Detecção rápida e fácil é desejada e marcadores morfológicos podem auxiliar na visualização da cultura marcada. O gene da GFP (green fluorescent protein extraído da Aequorea victoria

  17. Nanoscale thermal probing

    Directory of Open Access Journals (Sweden)

    Yanan Yue

    2012-03-01

    Full Text Available Nanoscale novel devices have raised the demand for nanoscale thermal characterization that is critical for evaluating the device performance and durability. Achieving nanoscale spatial resolution and high accuracy in temperature measurement is very challenging due to the limitation of measurement pathways. In this review, we discuss four methodologies currently developed in nanoscale surface imaging and temperature measurement. To overcome the restriction of the conventional methods, the scanning thermal microscopy technique is widely used. From the perspective of measuring target, the optical feature size method can be applied by using either Raman or fluorescence thermometry. The near-field optical method that measures nanoscale temperature by focusing the optical field to a nano-sized region provides a non-contact and non-destructive way for nanoscale thermal probing. Although the resistance thermometry based on nano-sized thermal sensors is possible for nanoscale thermal probing, significant effort is still needed to reduce the size of the current sensors by using advanced fabrication techniques. At the same time, the development of nanoscale imaging techniques, such as fluorescence imaging, provides a great potential solution to resolve the nanoscale thermal probing problem.

  18. Einstein Inflationary Probe (EIP)

    Science.gov (United States)

    Hinshaw, Gary

    2004-01-01

    I will discuss plans to develop a concept for the Einstein Inflation Probe: a mission to detect gravity waves from inflation via the unique signature they impart to the cosmic microwave background (CMB) polarization. A sensitive CMB polarization satellite may be the only way to probe physics at the grand-unified theory (GUT) scale, exceeding by 12 orders of magnitude the energies studied at the Large Hadron Collider. A detection of gravity waves would represent a remarkable confirmation of the inflationary paradigm and set the energy scale at which inflation occurred when the universe was a fraction of a second old. Even a strong upper limit to the gravity wave amplitude would be significant, ruling out many common models of inflation, and pointing to inflation occurring at much lower energy, if at all. Measuring gravity waves via the CMB polarization will be challenging. We will undertake a comprehensive study to identify the critical scientific requirements for the mission and their derived instrumental performance requirements. At the core of the study will be an assessment of what is scientifically and experimentally optimal within the scope and purpose of the Einstein Inflation Probe.

  19. GLP-1 receptor antagonist as a potential probe for pancreatic β-cell imaging

    International Nuclear Information System (INIS)

    Mukai, Eri; Toyoda, Kentaro; Kimura, Hiroyuki; Kawashima, Hidekazu; Fujimoto, Hiroyuki; Ueda, Masashi; Temma, Takashi; Hirao, Konomu; Nagakawa, Kenji; Saji, Hideo; Inagaki, Nobuya

    2009-01-01

    We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic β-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [ 125 I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [ 125 I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120 min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [ 125 I]BH-exendin(9-39) injection into transgenic mice with pancreatic β-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic β-cell imaging.

  20. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  1. Quadruplex-Flanking Stem Structures Modulate the Stability and Metal Ion Preferences of RNA Mimics of GFP.

    Science.gov (United States)

    Ageely, Eman A; Kartje, Zachary J; Rohilla, Kushal J; Barkau, Christopher L; Gagnon, Keith T

    2016-09-16

    The spinach family of RNA aptamers are RNA mimics of green fluorescent protein (GFP) that have previously been designed to address the challenges of imaging RNA inside living cells. However, relatively low levels of free intracellular magnesium limited the practical use of these aptamers. Recent cell-based selections identified the broccoli RNA aptamer, which requires less magnesium for fluorescence, but the basis for magnesium preference remained unclear. Here, we find that the broccoli RNA structure is very similar to that of baby spinach, a truncated version of the spinach aptamer. Differences in stability and metal ion preferences between these two aptamers, and among broccoli mutants, are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Mutation of purine-purine pairs in broccoli at the terminal stem-quadruplex transition caused reversion of broccoli to a higher magnesium dependence. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs likely explain their sensitivity to magnesium for stability and fluorescence. Thus, optimizations designed to improve aptamers should take into consideration the role of metal ions in stabilizing the transitions and interactions between independently folding RNA structural motifs.

  2. Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

    Science.gov (United States)

    Sommer, Jeffrey R; Alderson, Jon; Laible, Goetz; Petters, Robert M

    2006-06-01

    We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.

  3. Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

    Science.gov (United States)

    Smith, E. G.; D'Angelo, C.; Salih, A.; Wiedenmann, J.

    2013-06-01

    Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.

  4. Uncovering the role of the flexible C-terminal tail: A model study with Strep-tagged GFP

    Directory of Open Access Journals (Sweden)

    Michael W. Lassalle

    2016-06-01

    Full Text Available Recently, it has been recognized that, much like an electric current in an electric circuit, dynamic disruptions from flexible, unstructured regions distal to the active region are transferred through the contact network to the active site and influence protein stability and/or function. As transmembrane proteins frequently possess the β-barrel structure, studies of proteins with this topology are required. The unstructured lid segments of the β-barrel GFP protein are conserved and could play a role in the backbone stabilization required for chromophore function. A study of the disordered C-terminus and the function within the lid is necessary. In this study, we entirely truncated the flexible C-terminal tail and investigated the N-terminal Strep-tagged GFP by fluorescence spectroscopy, and the temperature- and GdnHCl-induced unfolding by circular dichroism. The introduction of the unstructured Strep-tag itself changed the unfolding pathway. Truncating the entire flexible tail did not decrease the fluorescence intensity to a large extent; however, the protein stability changed dramatically. The temperature for half-denaturation T1/2 changed significantly from 79 °C for the wild-type to 72.8 °C for the mutant. Unfolding kinetics at different temperatures have been induced by 4 M GdnHCl, and the apparent Arrhenius activation energy decreased by 40% as compared to the wild-type.

  5. A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.

    Science.gov (United States)

    Wendel, Sofie; Fischer, Emil C; Martínez, Virginia; Seppälä, Susanna; Nørholm, Morten H H

    2016-05-03

    Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing. Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency. We have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech.

  6. Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation.

    Science.gov (United States)

    Bi, Ruiye; Fan, Yi; Luo, En; Yuan, Quan; Mannstadt, Michael

    2017-03-14

    Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre + mice with Rosa-mTmG fl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

  7. [Construction and identification of eukaryotic plasmid pGC-silencer-U6/Neo/GFP/ABCG2].

    Science.gov (United States)

    Yu, Yanping; Zhang, Song; Kong, Weijia

    2010-09-01

    To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, and to detect the RNAi effect of shRNA. Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The recombinant plasmids were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and Western blot. The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed. The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells, at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3 (P < 0.05), with an inhibition ratio of 75% at the mRNA level and 68% at the protein level. pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.

  8. The membrane skeleton in Paramecium: Molecular characterization of a novel epiplasmin family and preliminary GFP expression results.

    Science.gov (United States)

    Pomel, Sébastien; Diogon, Marie; Bouchard, Philippe; Pradel, Lydie; Ravet, Viviane; Coffe, Gérard; Viguès, Bernard

    2006-02-01

    Previous attempts to identify the membrane skeleton of Paramecium cells have revealed a protein pattern that is both complex and specific. The most prominent structural elements, epiplasmic scales, are centered around ciliary units and are closely apposed to the cytoplasmic side of the inner alveolar membrane. We sought to characterize epiplasmic scale proteins (epiplasmins) at the molecular level. PCR approaches enabled the cloning and sequencing of two closely related genes by amplifications of sequences from a macronuclear genomic library. Using these two genes (EPI-1 and EPI-2), we have contributed to the annotation of the Paramecium tetraurelia macronuclear genome and identified 39 additional (paralogous) sequences. Two orthologous sequences were found in the Tetrahymena thermophila genome. Structural analysis of the 43 sequences indicates that the hallmark of this new multigenic family is a 79 aa domain flanked by two Q-, P- and V-rich stretches of sequence that are much more variable in amino-acid composition. Such features clearly distinguish members of the multigenic family from epiplasmic proteins previously sequenced in other ciliates. The expression of Green Fluorescent Protein (GFP)-tagged epiplasmin showed significant labeling of epiplasmic scales as well as oral structures. We expect that the GFP construct described herein will prove to be a useful tool for comparative subcellular localization of different putative epiplasmins in Paramecium.

  9. Development of Mackintosh Probe Extractor

    Science.gov (United States)

    Rahman, Noor Khazanah A.; Kaamin, Masiri; Suwandi, Amir Khan; Sahat, Suhaila; Jahaya Kesot, Mohd

    2016-11-01

    Dynamic probing is a continuous soil investigation technique, which is one of the simplest soil penetration test. It basically consist of repeatedly driving a metal tipped probe into the ground using a drop weight of fixed mass and travel. Testing was carried out continuously from ground level to the final penetration depth. Once the soil investigation work done, it is difficult to pull out the probe rod from the ground, due to strong soil structure grip against probe cone and prevent the probe rod out from the ground. Thus, in this case, a tool named Extracting Probe was created to assist in the process of retracting the probe rod from the ground. In addition, Extracting Probe also can reduce the time to extract the probe rod from the ground compare with the conventional method. At the same time, it also can reduce manpower cost because only one worker involve to handle this tool compare with conventional method used two or more workers. From experiment that have been done we found that the time difference between conventional tools and extracting probe is significant, average time difference is 155 minutes. In addition the extracting probe can reduce manpower usage, and also labour cost for operating the tool. With all these advantages makes this tool has the potential to be marketed.

  10. eGFP expression under the Uchl1 promoter labels corticospinal motor neurons and a subpopulation of degeneration resistant spinal motor neurons in ALS mouse models

    Science.gov (United States)

    Yasvoina, Marina V.

    Current understanding of basic cellular and molecular mechanisms for motor neuron vulnerability during motor neuron disease initiation and progression is incomplete. The complex cytoarchitecture and cellular heterogeneity of the cortex and spinal cord greatly impedes our ability to visualize, isolate, and study specific neuron populations in both healthy and diseased states. We generated a novel reporter line, the Uchl1-eGFP mouse, in which cortical and spinal components of motor neuron circuitry are genetically labeled with eGFP under the Uchl1 promoter. A series of cellular and anatomical analyses combined with retrograde labeling, molecular marker expression, and electrophysiology were employed to determine identity of eGFP expressing cells in the motor cortex and the spinal cord of novel Uchl1-eGFP reporter mice. We conclude that eGFP is expressed in corticospinal motor neurons (CSMN) in the motor cortex and a subset of S-type alpha and gamma spinal motor neurons (SMN) in the spinal cord. hSOD1G93A and Alsin-/- mice, mouse models for amyotrophic lateral sclerosis (ALS), were bred to Uchl1-eGFP reporter mouse line to investigate the pathophysiology and underlying mechanisms of CSMN degeneration in vivo. Evidence suggests early and progressive degeneration of CSMN and SMN in the hSOD1G93A transgenic mice. We show an early increase of autophagosome formation in the apical dendrites of vulnerable CSMN in hSOD1G93A-UeGFP mice, which is localized to the apical dendrites. In addition, labeling S-type alpha and gamma SMN in the hSOD1G93A-UeGFP mice provide a unique opportunity to study basis of their resistance to degeneration. Mice lacking alsin show moderate clinical phenotype and mild CSMN axon degeneration in the spinal cord, which suggests vulnerability of CSMN. Therefore, we investigated the CSMN cellular and axon defects in aged Alsin-/- mice bred to Uchl1-eGFP reporter mouse line. We show that while CSMN are preserved and lack signs of degeneration, CSMN axons

  11. Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus.

    Science.gov (United States)

    Moreau, Morgane J J; Schaeffer, Patrick M

    2013-12-01

    The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.

  12. AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells.

    Science.gov (United States)

    Fassina, L; Magenes, G; Inzaghi, A; Palumbo, S; Allavena, G; Miracco, C; Pirtoli, L; Biggiogera, M; Comincini, S

    2012-10-11

    An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

  13. Development of a robust, higher throughput green fluorescent protein (GFP)-based Epstein-Barr Virus (EBV) micro-neutralization assay.

    Science.gov (United States)

    Lin, Rui; Heeke, Darren; Liu, Hui; Rao, Eileen; Marshall, Jason D; Chio, Vera; Cataniag, Floro; Yu, Li; Zuo, Fengrong; McCarthy, Michael P

    2017-09-01

    The goal of most prophylactic vaccines is to elicit robust and effective neutralizing antibodies against the human pathogen target. The titer of neutralizing antibodies to Epstein-Barr Virus (EBV) is a useful biomarker for evaluating EBV vaccines. Here, the development and optimization of a 96-well micro-neutralization fluorescent imaging assay (FIA) using an EBV virus-encoding green fluorescent protein (GFP) to infect adherent EBV recipient cells is reported. The conditions were optimized for generating reproducible EBV-GFP virus, for maintaining viral infectivity for months, and for efficient viral infection of recipient cell culture. The utility of the EBV-GFP FIA neutralization assay was demonstrated in a mouse study of an investigational adjuvanted EBV gp350 subunit vaccine. This assay confirmed the generation of high titers of anti-EBV-neutralizing antibodies which correlated well with the established Raji cell-based flow cytometry-based EBV neutralization assay, as well as with anti-gp350 IgG titers. In naturally infected EBV+ human serum samples, a good correlation between anti-gp350 IgG ELISA titer and EBV-GFP FIA neutralization antibody titer was also observed. Taken together, these results demonstrate the establishment of a scalable high throughput EBV-GFP FIA micro-neutralization assay suitable to measure humoral EBV vaccine response in a large-scale human trial. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Mobile Probing Kit

    DEFF Research Database (Denmark)

    Larsen, Jakob Eg; Sørensen, Lene Tolstrup; Sørensen, J.K.

    2007-01-01

    Mobile Probing Kit is a low tech and low cost methodology for obtaining inspiration and insights into user needs, requirements and ideas in the early phases of a system's development process. The methodology is developed to identify user needs, requirements and ideas among knowledge workers...... characterized as being highly nomadic and thus potential users of mobile and ubiquitous technologies. The methodology has been applied in the 1ST MAGNET Beyond project in order to obtain user needs and requirements in the process of developing pilot services. We report on the initial findings from applying...... this methodology in the early phases of this large scale research and development process....

  15. High spatial resolution Kelvin probe force microscopy with coaxial probes

    International Nuclear Information System (INIS)

    Brown, Keith A; Westervelt, Robert M; Satzinger, Kevin J

    2012-01-01

    Kelvin probe force microscopy (KPFM) is a widely used technique to measure the local contact potential difference (CPD) between an AFM probe and the sample surface via the electrostatic force. The spatial resolution of KPFM is intrinsically limited by the long range of the electrostatic interaction, which includes contributions from the macroscopic cantilever and the conical tip. Here, we present coaxial AFM probes in which the cantilever and cone are shielded by a conducting shell, confining the tip–sample electrostatic interaction to a small region near the end of the tip. We have developed a technique to measure the true CPD despite the presence of the shell electrode. We find that the behavior of these probes agrees with an electrostatic model of the force, and we observe a factor of five improvement in spatial resolution relative to unshielded probes. Our discussion centers on KPFM, but the field confinement offered by these probes may improve any variant of electrostatic force microscopy. (paper)

  16. A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence.

    Science.gov (United States)

    Davis, I; Girdham, C H; O'Farrell, P H

    1995-08-01

    The central role of gene expression in regulating development has largely been studied by in situ hybridization and antibody staining techniques in fixed material. However, rapid temporal and spatial changes in gene expression are often difficult to correlate with complex morphogenetic movements. A green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, can be used as a real-time reporter for gene expression and could aid analysis of dynamic events during embryogenesis. Here, we describe a transgenic Drosophila line ubiquitously expressing a nuclear GFP fusion protein that highlights morphogenesis, cell movement, and mitosis in living embryos. The fusion protein is highly fluorescent when maternally supplied, but there is a long delay between its zygotic expression and the appearance of fluorescence. GFP is thus an excellent marker for the expression of stable gene products, but a poor reporter for dynamic zygotic gene expression in early Drosophila embryos.

  17. A Model to Study the Phenotypic Changes of Insect Cell Transfection by Copepod Super Green Fluorescent Protein (cop-GFP) in Baculovirus Expression System.

    Science.gov (United States)

    Shokrollahi, Narjes; Shahbazzadeh, Delavar; Pooshang-Bagheri, Kamran; Habibi-Anbouhi, Mahdi; Jahanian-Najafabadi, Ali; Behdani, Mahdi

    2016-07-01

    Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells. The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein (copGFP). In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells. Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis. The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection.

  18. Transduction of an immortalized olfactory ensheathing glia cell line with the green fluorescent protein (GFP) gene: Evaluation of its neuroregenerative capacity as a proof of concept.

    Science.gov (United States)

    Plaza, N; Simón, D; Sierra, J; Moreno-Flores, M T

    2016-01-26

    Olfactory ensheathing glia (OEG) cells are known to foster axonal regeneration of central nervous system (CNS) neurons. Several lines of reversibly immortalized human OEG (ihOEG) have been previously established that enabled to develop models for their validation in vitro and in vivo. In this work, a constitutively GFP-expressing ihOEG cell line was obtained, and named Ts14-GFP. Ts14-GFP neuroregenerative ability was similar to that found for the parental line Ts14 and it can be assayed using in vivo transplantation experimental paradigms, after spinal cord or optic nerve damage. Additionally, we have engineered a low-regenerative ihOEG line, hTL2, using lentiviral transduction of the large T antigen from SV40 virus, denominated from now on Ts12. Ts12 can be used as a low regeneration control in these experiments. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Combined use of different Gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa

    DEFF Research Database (Denmark)

    Boldt, T.S.; Sørensen, J.; Karlsson, U.

    2004-01-01

    Single-cell localization and activity of Pseudomonas,fluorescens F113, colonizing alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1 ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of different stability. The monitoring systems permitted non...... of chlorinated biphenyl was constructed, using another gfp fusion with the meta-pathway Pin promoter from Pseudomonas putida (TOL plasmid). Expression of this promoter, which is strongly induced by the PCB-2 degradation product, 3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on alfalfa...... roots using the P. fluorescens F113rifpcb reporter. A small but distinct fraction of the introduced bacteria activated the Pm promoter and thus appeared to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading cells, which by design were identical to the sensing cells, were located...

  20. Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum

    Directory of Open Access Journals (Sweden)

    Vahid NASIRI

    2016-10-01

    Full Text Available Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development. 

  1. An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin, tyrosine hydroxylase, and RFamide-related peptide neurons in mice.

    Science.gov (United States)

    Smith, Jeremy T; Reichenbach, Alex; Lemus, Moyra; Mani, Bharath K; Zigman, Jeffrey M; Andrews, Zane B

    2013-09-01

    Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Jim Silver

    Full Text Available Replication-competent adenovirus type 5 (Ad5 vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells.

  3. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

    Science.gov (United States)

    Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

    2012-12-01

    Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. The Galaxy Evolution Probe

    Science.gov (United States)

    Glenn, Jason; Galaxy Evolution Probe Team

    2018-01-01

    The Galaxy Evolution Probe (GEP) is a concept for a far-infrared observatory to survey large regions of sky for star-forming galaxies from z = 0 to beyond z = 3. Our knowledge of galaxy formation is incomplete and requires uniform surveys over a large range of redshifts and environments to accurately describe mass assembly, star formation, supermassive black hole growth, interactions between these processes, and what led to their decline from z ~ 2 to the present day. Infrared observations are sensitive to dusty, star-forming galaxies, which have bright polycyclic aromatic hydrocarbon (PAH) emission features and warm dust continuum in the rest-frame mid infrared and cooler thermal dust emission in the far infrared. Unlike previous far-infrared continuum surveys, the GEP will measure photometric redshifts commensurate with galaxy detections from PAH emission and Si absorption features, without the need for obtaining spectroscopic redshifts of faint counterparts at other wavelengths.The GEP design includes a 2 m diameter telescope actively cooled to 4 K and two instruments: (1) An imager covering 10 to 300 um with 25 spectral resolution R ~ 8 bands (with lower R at the longest wavelengths) to detect star-forming galaxies and measure their redshifts photometrically. (2) A 23 – 190 um, R ~ 250 dispersive spectrometer for redshift confirmation and identification of obscured AGN using atomic fine-structure lines. Lines including [Ne V], [O IV], [O III], [O I], and [C II] will probe gas physical conditions, radiation field hardness, and metallicity. Notionally, the GEP will have a two-year mission: galaxy surveys with photometric redshifts in the first year and a second year devoted to follow-up spectroscopy. A comprehensive picture of star formation in galaxies over the last 10 billion years will be assembled from cosmologically relevant volumes, spanning environments from field galaxies and groups, to protoclusters, to dense galaxy clusters.Commissioned by NASA, the

  5. Acute binge pattern cocaine administration induces region-specific effects in D1-r- and D2-r-expressing cells in eGFP transgenic mice.

    Science.gov (United States)

    Lawhorn, C; Edusei, E; Zhou, Y; Ho, A; Kreek, M J

    2013-12-03

    Cocaine addiction is driven by genetic, neurologic and environmental components. The D1-like (D1 and D5) and D2-like (D2, D3 and D4) families of dopamine receptors play an important role in modulating the effects of cocaine administration on drug-seeking behavior. The advent of bacterial artificial chromosome-eGFP (enhanced green fluorescent protein) transgenic mice that express eGFP driven by the endogenous D1-receptor (D1-r) or D2-receptor (D2-r) promoters provides a unique opportunity to distinguish between these subpopulations of cells. In an effort to identify cocaine-induced alterations in D1-r- versus D2-r-expressing cells during the initial stages of addiction, we examined cells that expressed D1-rs in Drd1-eGFP mice, or D2-rs in Drd2-eGFP mice, after an acute, 1-day binge pattern of cocaine administration. We used multiphoton confocal microscopy and Visiopharm© software, to conduct unbiased stereological counts of D1-r-labeled or D2-r-labeled cells in various striatal regions. Mice were sacrificed at 30 min and 24-h post cocaine or saline administration. Compared to saline controls, Drd1-eGFP mice that received cocaine had a higher count of D1-r-labeled cells in the dorsolateral (DL) striatum, at the 30-min and 24-h time-points. No changes in the nucleus accumbens (NAc) core or shell were observed in Drd1-eGFP mice. Drd2-eGFP mice that received cocaine had fewer D2-r-labeled cells in the DL striatum and NAc core compared to saline controls. This effect was observed at the 30-min time-point but not at 24h. Drd2-eGFP mice that received cocaine also had fewer numbers of D2-r-labeled cells in the NAc core compared to saline controls, but no significant differences in the number of D2-r-labeled cells in the NAc shell. These results suggest that acute binge pattern cocaine administration may induce region-specific alterations in D1-r or D2-receptor gene expression, and may help elucidate the differential role of dopamine receptors in the initial stages of the

  6. Dynamics of Transgenic Enterobacter cloacae Expressing Green Fluorescent Protein-Defensin (GFP-D in Anopheles stephensi under Laboratory Condition

    Directory of Open Access Journals (Sweden)

    Hossein Dehghan

    2017-12-01

    Full Text Available Background: Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium with­in An. stephensi and its introduction methods to the mosquitoes have not yet been explored.Methods: Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium.Results: The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadi­ally from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time.Conclusion: Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

  7. Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry

    Directory of Open Access Journals (Sweden)

    Michael Brauchle

    2014-11-01

    Full Text Available Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

  8. Cosmological Probes for Supersymmetry

    Directory of Open Access Journals (Sweden)

    Maxim Khlopov

    2015-05-01

    Full Text Available The multi-parameter character of supersymmetric dark-matter models implies the combination of their experimental studies with astrophysical and cosmological probes. The physics of the early Universe provides nontrivial effects of non-equilibrium particles and primordial cosmological structures. Primordial black holes (PBHs are a profound signature of such structures that may arise as a cosmological consequence of supersymmetric (SUSY models. SUSY-based mechanisms of baryosynthesis can lead to the possibility of antimatter domains in a baryon asymmetric Universe. In the context of cosmoparticle physics, which studies the fundamental relationship of the micro- and macro-worlds, the development of SUSY illustrates the main principles of this approach, as the physical basis of the modern cosmology provides cross-disciplinary tests in physical and astronomical studies.

  9. Convex probe endobronchial ultrasound.

    Science.gov (United States)

    Bade, Brett; Furukawa, Brian; Tanner, Nichole T

    2014-12-01

    Convex probe endobronchial ultrasound (EBUS) is a minimally invasive diagnostic technique that allows real-time sampling of mediastinal and hilar lymph nodes and central pulmonary lesions. Its utility in diagnosing both malignant and nonmalignant diseases has led to an increased uptake and use by pulmonologists over the past decade. Because of the robust evidence supporting its safety and diagnostic yield, EBUS is now the first guideline recommended test for staging in non-small cell lung cancer (NSCLC). It has also a role in providing tissue for molecular analysis, thereby guiding in the selection of agents in the new era of personalized chemotherapies in the treatment of NSCLC. The following review highlights the evidence for EBUS in diagnosing mediastinal pathology and addresses technique, training, and competency and future directions for this technology. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  10. Non-inductive current probe

    DEFF Research Database (Denmark)

    Bak, Christen Kjeldahl

    1977-01-01

    The current probe described is a low-cost, shunt resistor for monitoring current pulses in e.g., pulsed lasers. Rise time is......The current probe described is a low-cost, shunt resistor for monitoring current pulses in e.g., pulsed lasers. Rise time is...

  11. 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...

  12. Nanobits: customizable scanning probe tips

    DEFF Research Database (Denmark)

    Kumar, Rajendra; Shaik, Hassan Uddin; Sardan Sukas, Özlem

    2009-01-01

    We present here a proof-of-principle study of scanning probe tips defined by planar nanolithography and integrated with AFM probes using nanomanipulation. The so-called 'nanobits' are 2-4 mu m long and 120-150 nm thin flakes of Si3N4 or SiO2, fabricated by electron beam lithography and standard...... or dislocation of the tips of the nanobit after several scans. This approach allows an unprecedented freedom in adapting the shape and size of scanning probe tips to the surface topology or to the specific application....... silicon processing. Using a microgripper they were detached from an array and fixed to a standard pyramidal AFM probe or alternatively inserted into a tipless cantilever equipped with a narrow slit. The nanobit-enhanced probes were used for imaging of deep trenches, without visible deformation, wear...

  13. Water cooled static pressure probe

    Science.gov (United States)

    Lagen, Nicholas T. (Inventor); Eves, John W. (Inventor); Reece, Garland D. (Inventor); Geissinger, Steve L. (Inventor)

    1991-01-01

    An improved static pressure probe containing a water cooling mechanism is disclosed. This probe has a hollow interior containing a central coolant tube and multiple individual pressure measurement tubes connected to holes placed on the exterior. Coolant from the central tube symmetrically immerses the interior of the probe, allowing it to sustain high temperature (in the region of 2500 F) supersonic jet flow indefinitely, while still recording accurate pressure data. The coolant exits the probe body by way of a reservoir attached to the aft of the probe. The pressure measurement tubes are joined to a single, larger manifold in the reservoir. This manifold is attached to a pressure transducer that records the average static pressure.

  14. Electrophoresis-mass spectrometry probe

    Science.gov (United States)

    Andresen, Brian D.; Fought, Eric R.

    1987-01-01

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface.

  15. Mobile Probes in Mobile Learning

    DEFF Research Database (Denmark)

    Ørngreen, Rikke; Blomhøj, Ulla; Duvaa, Uffe

    In this paper experiences from using mobile probes in educational design of a mobile learning application is presented. The probing process stems from the cultural probe method, and was influenced by qualitative interview and inquiry approaches. In the project, the mobile phone was not only acting...... as an agent for acquiring empirical data (as the situation in hitherto mobile probe settings) but was also the technological medium for which data should say something about (mobile learning). Consequently, not only the content of the data but also the ways in which data was delivered and handled, provided...... a valuable dimension for investigating mobile use. The data was collected at the same time as design activities took place and the collective data was analysed based on user experience goals and cognitive processes from interaction design and mobile learning. The mobile probe increased the knowledge base...

  16. Wearable probes for service design

    DEFF Research Database (Denmark)

    Mullane, Aaron; Laaksolahti, Jarmo Matti; Svanæs, Dag

    2014-01-01

    by service employees in reflecting on the delivery of a service. In this paper, we present the ‘wearable probe’, a probe concept that captures sensor data without distracting service employees. Data captured by the probe can be used by the service employees to reflect and co-reflect on the service journey......Probes are used as a design method in user-centred design to allow end-users to inform design by collecting data from their lives. Probes are potentially useful in service innovation, but current probing methods require users to interrupt their activity and are consequently not ideal for use......, helping to identify opportunities for service evolution and innovation....

  17. Optimizing E. coli-based membrane protein production using Lemo21(DE3) or pReX and GFP-Fusions

    NARCIS (Netherlands)

    Kuipers, Grietje; Peschke, Markus; Ismail, Nurzian Bernsel; Hjelm, Anna; Schlegel, Susan; Vikström, David; Luirink, Joen; de Gier, Jan Willem

    2017-01-01

    Optimizing the conditions for the production of membrane proteins in E. coli is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain or the pReX T7-based expression vector with membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the

  18. GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies

    DEFF Research Database (Denmark)

    Lübeck, M.; Knudsen, I.M.B.; Jensen, B.

    2002-01-01

    Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The beta-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated...

  19. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  20. Locally accelerated growth is part of the innate immune response and repair mechanisms in reef-building corals as detected by green fluorescent protein (GFP)-like pigments

    Science.gov (United States)

    D'Angelo, C.; Smith, E. G.; Oswald, F.; Burt, J.; Tchernov, D.; Wiedenmann, J.

    2012-12-01

    Homologs of the green fluorescent protein (GFP) are a prevalent group of host pigments responsible for the green, red and purple-blue colours of many reef-building corals. They have been suggested to contribute to the striking coloration changes of different corals species in response to wounding and infestation with epibionts/parasites. In order to elucidate the physiological processes underlying the potentially disease-related colour changes, we have analysed spatial and temporal expression patterns of GFP-like proteins and other biomarkers in corals from the Red Sea, the Arabian/Persian Gulf and Fiji both in their natural habitat and under specific laboratory conditions. The expression of distinct GFP-like proteins and the growth marker proliferating cell nuclear antigen was upregulated in growing branch tips and margins of healthy coral colonies as well as in disturbed colony parts. Furthermore, phenoloxidase activity increased in these proliferating tissues. It is thus demonstrated that locally accelerated growth is part of the innate immune response and repair mechanisms in reef-building corals and, moreover, these processes can be detected utilizing the excellent biomarker properties of GFP-like proteins. Finally, the results of this work suggest an additional vulnerability of corals in predicted future scenarios of increased ocean acidification, warming and eutrophication that are anticipated to reduce coral growth capacity.

  1. Development of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system.

    Science.gov (United States)

    Martinez-Jaramillo, E; Garza-Morales, R; Loera-Arias, M J; Saucedo-Cardenas, O; Montes-de-Oca-Luna, R; McNally, L R; Gomez-Gutierrez, J G

    2017-01-01

    Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.

  2. A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea.

    Science.gov (United States)

    Ford, Kathryn L; Baumgartner, Kendra; Henricot, Béatrice; Bailey, Andy M; Foster, Gary D

    2016-07-07

    Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen.

  3. A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration.

    Science.gov (United States)

    Moore, Amy M; Borschel, Gregory H; Santosa, Katherine A; Flagg, Eric R; Tong, Alice Y; Kasukurthi, Rahul; Newton, Piyaraj; Yan, Ying; Hunter, Daniel A; Johnson, Philip J; Mackinnon, Susan E

    2012-02-15

    In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. LDL receptor-GFP fusion proteins: new tools for the characterization of disease-causing mutations in the LDL receptor gene

    DEFF Research Database (Denmark)

    Holst, Henrik Uffe; Dagnæs-Hansen, Frederik; Corydon, Thomas Juhl

    2001-01-01

    . In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-locallisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum....

  5. Transient Astrophysics Probe

    Science.gov (United States)

    Camp, Jordan; Transient Astrophysics Probe Team

    2018-01-01

    The Transient Astrophysics Probe (TAP) is a wide-field multi-wavelength transient mission proposed for flight starting in the late 2020s. The mission instruments include unique “Lobster-eye” imaging soft X-ray optics that allow a ~1600 deg2 FoV; a high sensitivity, 1 deg2 FoV soft X-ray telescope; a 1 deg2 FoV Infrared telescope with bandpass 0.6-3 micron; and a set of 8 NaI gamma-ray detectors. TAP’s most exciting capability will be the observation of tens per year of X-ray and IR counterparts of GWs involving stellar mass black holes and neutron stars detected by LIGO/Virgo/KAGRA/LIGO-India, and possibly several per year X-ray counterparts of GWs from supermassive black holes, detected by LISA and Pulsar Timing Arrays. TAP will also discover hundreds of X-ray transients related to compact objects, including tidal disruption events, supernova shock breakouts, and Gamma-Ray Bursts from the epoch of reionization.

  6. Steerable Doppler transducer probes

    International Nuclear Information System (INIS)

    Fidel, H.F.; Greenwood, D.L.

    1986-01-01

    An ultrasonic diagnostic probe is described which is capable of performing ultrasonic imaging and Doppler measurement consisting of: a hollow case having an acoustic window which passes ultrasonic energy and including chamber means for containing fluid located within the hollow case and adjacent to a portion of the acoustic window; imaging transducer means, located in the hollow case and outside the fluid chamber means, and oriented to direct ultrasonic energy through the acoustic window toward an area which is to be imaged; Doppler transducer means, located in the hollow case within the fluid chamber means, and movably oriented to direct Doppler signals through the acoustic window toward the imaged area; means located within the fluid chamber means and externally controlled for controllably moving the Doppler transducer means to select one of a plurality of axes in the imaged area along which the Doppler signals are to be directed; and means, located external to the fluid chamber means and responsive to the means for moving, for providing an indication signal for identifying the selected axis

  7. Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: A study of DNAJB6 chaperone

    Directory of Open Access Journals (Sweden)

    Rasha Mohamed Hussein

    2015-07-01

    Full Text Available Alzheimer’s disease is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly Q peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

  8. In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish challenged with GFP tagged Vibrio parahaemolyticus Dahv2.

    Science.gov (United States)

    Girija, Vairavan; Malaikozhundan, Balasubramanian; Vaseeharan, Baskaralingam; Vijayakumar, Sekar; Gobi, Narayanan; Del Valle Herrera, Marian; Chen, Jiann-Chu; Santhanam, Perumal

    2018-01-01

    In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish (Danio rerio) challenged with GFP tagged Vibrio parahaemolyticus Dahv2 was studied. The cell free extract of probiotic B. licheniformis Dahb1 at 100 μg mL -1 showed growth inhibition of V. parahaemolyticus Dahv2 in vitro. B. licheniformis Dahb1 also inhibited the biofilm growth of GFP tagged V. parahaemolyticus Dahv2 at 100 μg mL -1 in vitro. The growth and survival of zebrafish was tested using probiotic B. licheniformis Dahb1. Weight (1.28 g) of zebrafish that received the cell free extract was much higher than in control (1.04 g). The mortality of zebrafish infected with GFP tagged V. parahaemolyticus Dahv2 at 10 7 Cfu mL -1 (Group IV) was 100%, whereas a complete survival of zebrafish that received the cell free extract of B. licheniformis Dahb1 at 10 7 Cfu mL -1 (Group VII) was observed after 30 days. The number of GFP tagged V. parahaemolyticus Dahv2 colonies in the intestine and gills significantly reduced after treatment with the cell free extract of B. licheniformis Dahb1. Furthermore, a significant decrease in the fluorescent colonies of GFP tagged V. parahaemolyticus Dahv2 was observed after treatment with the cell free extract of B. licheniformis Dahb1 under confocal laser scanning microscopy (CLSM). In conclusion, the cell free extract of B. licheniformis Dahb1 could prevent Vibrio infection by enhancing the growth and survival of zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Simultaneous tracking of movement and gene expression in multiple Drosophila melanogaster flies using GFP and DsRED fluorescent reporter transgenes

    Directory of Open Access Journals (Sweden)

    Tavaré Simon

    2009-04-01

    Full Text Available Abstract Background Fluorescent proteins such as GFP (Green Fluorescent Protein and DsRED (Discosoma sp.Red Fluorescent Protein are often used as reporter molecules for transgene expression in Drosophila and other species. We have recently reported methods that allow simultaneous tracking of animal movement and GFP expression in real time, however the assay was limited to single animals and a single transgene. Numerous studies would be facilitated by methods that allow for assay of multiple animals and multiple transgenes. Findings Here we report an improved fly video tracking system that allows multiple transgenic flies to be tracked simultaneously using visible light, GFP fluorescence and DsRED fluorescence. The movement of multiple flies could be accurately tracked at real time rates, while simultaneously assaying the expression level of two different transgenes marked with GFP and DsRED. The individual flies could be accurately tracked and distinguished even during periods when transgene fluorescence was undetected. For example, characteristic patterns of hsp70 and hsp22 transgene induction could be simultaneously quantified and correlated with animal movement in aging flies, and as groups of flies died due to dessication/starvation. Conclusion The improved methods allow for more efficient assay of the correlation between gene expression, behavior, aging and mortality: multiple animals can be assayed with simultaneous quantification of multiple transgenes using GFP and DsRED fluorescence. These methods should allow for increased flexibility in experimental designs. For example, in the future it should be possible to use gene expression levels to predict remaining life span more accurately, and to quantify gene expression changes caused by interactions between animals in real time.

  10. Survival of GFP-tagged Rhodococcus sp. D310-1 in chlorimuron-ethyl-contaminated soil and its effects on the indigenous microbial community.

    Science.gov (United States)

    Xiong, Minghua; Hu, Zhiguang; Zhang, Ying; Cheng, Xiaosong; Li, Chunyan

    2013-05-15

    The recently isolated bacterial strain Rhodococcus sp. D310-1 can degrade high concentrations of chlorimuron-ethyl (up to 1000 mg L(-1)), indicating its potential for the bioremediation of soil contaminated with high levels of chlorimuron-ethyl. In this study, Rhodococcus sp. D310-1 was tagged with green fluorescent protein gene (gfp) to track its survival in soil. Subsequently, degradation activity of the gfp-tagged strain and its effects on indigenous microbial community were analyzed. Results showed the cell numbers of Rhodococcus sp. D310-1::gfp in non-sterilized soil maintained at 8.5 × 10(4) cells g(-1) dry soil 45 days after inoculation of 7.74 × 10(6) cells g(-1) dry soil and approximately 49% of chlorimuron-ethyl was removed. However, The cell numbers of Rhodococcus sp. D310-1::gfp in sterilized samples increased gradually to 7.85 × 10(7) cells g(-1) dry soil and approximately 78% of chlorimuron-ethyl was removed. PCR-DGGE demonstrated that inoculation of this gfp-tagged strain in chlorimuron-ethyl-contaminated soil has negligible impact on the community structure of bacteria, actinomycetes and fungi. These results indicate that Rhodococcus sp. D310-1 is effective for the remediation of chlorimuron-ethyl-contaminated soil and also provides valuable information about the behavior of the inoculant population during bioremediation, which could be directly used in the risk assessment of inoculant population and optimization of bioremediation process. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Probing the Probes: Fitness Factors For Small Molecule Tools

    OpenAIRE

    Workman, Paul; Collins, Ian

    2010-01-01

    Chemical probes for interrogating biological processes are of considerable current interest. Cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. To be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. Although recognizing that...

  12. GFP transgenic medaka (Oryzias latipes under the inducible cyp1a promoter provide a sensitive and convenient biological indicator for the presence of TCDD and other persistent organic chemicals.

    Directory of Open Access Journals (Sweden)

    Grace Hwee Boon Ng

    Full Text Available Persistent organic pollutants (POPs are resistant to environmental degradation and can cause multitude of health problems. Cytochrome P450 1A (Cyp1a is often up-regulated by POPs through the activation of aryl hydrocarbon receptor (AhR pathway and is thus usually used as a biomarker for xenobiotics exposure. To develop a convenient in vivo tool to monitor xenobiotic contamination in the water, we have established GFP transgenic medaka using the inducible cyp1a promoter, Tg(cyp1a:gfp. Here we tested Tg(cyp1a:gfp medaka at three different stages, prehatching embryos, newly hatched fry and adult with 2,3,7,8-tetrachlorodiebnzo-p-dioxin (TCDD, a dioxin. While GFP induction was observed in all three stages, newly hatched fry were the most sensitive with the lowest observed effective concentration of 0.005 nM or 16.1 ng/L. The highly sensitive organs included the kidney, liver and intestine. With high concentrations of TCDD, several other organs such as the olfactory pit, tail fin, gills, lateral line neuromast cells and blood vessels also showed GFP expression. In addition, Tg(cyp1a:gfp medaka fry also responded to two other AhR agonists, 3-methylcholanthrene and benzo[a]pyrene, for GFP induction, but no significant GFP induction was observed towards several other chemicals tested, indicating the specificity of this transgenic line. The GFP inducibility of Tg(cyp1a:gfp medaka at both fry and adult stages may be useful for development of high-throughput assays as well as online water monitoring system to detect xenobiotic toxicity.

  13. A green fluorescent protein (GFP)-based plasmid system to study post-transcriptional control of gene expression in vivo.

    Science.gov (United States)

    Urban, Johannes H; Vogel, Jörg

    2009-01-01

    Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression, which mainly modulate the translation of trans-encoded mRNAs. Typically, these molecules are 50-200 nucleotides in size and do not contain expressed open reading frames (ORFs). In Escherichia coli, about 70 members of this group have been identified to date and further estimates assume hundreds of sRNAs per bacterial genome. Regulation of gene expression by sRNAs is predominantly mediated by physical sRNA/target mRNA interactions that are based on short and imperfect complementarity. Although the contribution of sRNAs to overall bacterial gene regulation is now being appreciated, the function of many sRNAs is still unknown and their targets await to be uncovered. We recently developed a modular two-plasmid system, based on the green fluorescent protein (GFP) as non-invasive reporter of gene expression, to rapidly monitor the regulatory potential of sRNA/target mRNA pairs under investigation in vivo. The specialized reporter plasmid series also provides a suitable platform to study the function of cis-encoded riboregulators such as natural riboswitches, thermosensors, or engineered aptamer-based regulatory switches.

  14. No Overt Deficits in Aged Tau-Deficient C57Bl/6.Mapttm1(EGFPKit GFP Knockin Mice.

    Directory of Open Access Journals (Sweden)

    Annika van Hummel

    Full Text Available Several mouse lines with knockout of the tau-encoding MAPT gene have been reported in the past; they received recent attention due to reports that tau reduction prevented Aβ-induced deficits in mouse models of Alzheimer's disease. However, the effects of long-term depletion of tau in vivo remained controversial. Here, we used the tau-deficient GFP knockin line Mapttm1(EGFPkit on a pure C57Bl/6 background and subjected a large cohort of males and females to a range of motor, memory and behavior tests and imaging analysis, at the advanced age of over 16 months. Neither heterozygous nor homozygous Mapttm1(EGFPkit mice presented with deficits or abnormalities compared to wild-type littermates. Differences to reports using other tau knockout models may be due to different genetic backgrounds, respective gene targeting strategies or other confounding factors, such as nutrition. To this end, we report no functional or morphological deficits upon tau reduction or depletion in aged mice.

  15. Therapeutic Effect and Location of GFP-Labeled Placental Mesenchymal Stem Cells on Hepatic Fibrosis in Rats

    Directory of Open Access Journals (Sweden)

    Jiong Yu

    2017-01-01

    Full Text Available Background. Liver fibrosis is a chronic progressive liver disease, but no established effective treatment exists except for liver transplantation. The present study was designed to investigate the effect of human placenta mesenchymal stem cells (hPMSCs expressing green fluorescent protein (GFP on carbon tetrachloride- (CCl4- induced liver fibrosis in rats. Methods. Liver fibrosis was induced by subcutaneous injection with CCl4; hPMSCs were directly transplanted into rats through the caudal vein. The therapeutic efficacy of hPMSCs on liver fibrosis was measured by liver function tests, liver elastography, histopathology, Masson’s trichrome and Sirius red staining, and immunohistochemical studies. The expression levels of fibrotic markers, transforming growth factor β1 (TGF-β1 and α-smooth muscle actin (α-SMA, were assessed using real-time polymerase chain reaction. Results. We demonstrated that liver fibrosis was significantly dampened in the hPMSC transplantation group according to the Laennec fibrosis scoring system and histological data. The Sirius red-stained collagen area and the elastography score were significantly reduced in the hPMSC-treated group. Meanwhile, hPMSC administration significantly decreased TGF-β1 and α-SMA expression and enhanced liver functions in CCl4-induced fibrotic rats. Conclusion. This study indicates that transplantation of hPMSCs could repair liver fibrosis induced by CCl4 in rats, which may serve as a valuable therapeutic approach to treat liver diseases.

  16. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

    Science.gov (United States)

    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  17. Potential utility of eGFP-expressing NOG mice (NOG-EGFP as a high purity cancer sampling system

    Directory of Open Access Journals (Sweden)

    Shima Kentaro

    2012-06-01

    Full Text Available Abstract Purpose It is still technically difficult to collect high purity cancer cells from tumor tissues, which contain noncancerous cells. We hypothesized that xenograft models of NOG mice expressing enhanced green fluorescent protein (eGFP, referred to as NOG-EGFP mice, may be useful for obtaining such high purity cancer cells for detailed molecular and cellular analyses. Methods Pancreato-biliary cancer cell lines were implanted subcutaneously to compare the tumorigenicity between NOG-EGFP mice and nonobese diabetic/severe combined immunodeficiency (NOD/SCID mice. To obtain high purity cancer cells, the subcutaneous tumors were harvested from the mice and enzymatically dissociated into single-cell suspensions. Then, the cells were sorted by fluorescence-activated cell sorting (FACS for separation of the host cells and the cancer cells. Thereafter, the contamination rate of host cells in collected cancer cells was quantified by using FACS analysis. The viability of cancer cells after FACS sorting was evaluated by cell culture and subsequent subcutaneous reimplantation in NOG-EGFP mice. Results The tumorigenicity of NOG-EGFP mice was significantly better than that of NOD/SCID mice in all of the analyzed cell lines (p  Conclusions This method provides a novel cancer sampling system for molecular and cellular analysis with high accuracy and should contribute to the development of personalized medicine.

  18. Monitoring probe for groundwater flow

    Science.gov (United States)

    Looney, B.B.; Ballard, S.

    1994-08-23

    A monitoring probe for detecting groundwater migration is disclosed. The monitor features a cylinder made of a permeable membrane carrying an array of electrical conductivity sensors on its outer surface. The cylinder is filled with a fluid that has a conductivity different than the groundwater. The probe is placed in the ground at an area of interest to be monitored. The fluid, typically saltwater, diffuses through the permeable membrane into the groundwater. The flow of groundwater passing around the permeable membrane walls of the cylinder carries the conductive fluid in the same general direction and distorts the conductivity field measured by the sensors. The degree of distortion from top to bottom and around the probe is precisely related to the vertical and horizontal flow rates, respectively. The electrical conductivities measured by the sensors about the outer surface of the probe are analyzed to determine the rate and direction of the groundwater flow. 4 figs.

  19. A three dimensional probe positioner

    International Nuclear Information System (INIS)

    Intrator, T.; Sun, X.; Furno, I.; Dorf, L.; Lapenta, G.

    2008-01-01

    In order to sort out the physics that is important in many plasma experiments, data in three dimensions (3D) are becoming necessary. Access to the usual cylindrical vacuum vessel is typically restricted to radially or axially insertable probes that can pivot. The space that can be explored usually has significant restrictions either because probe travel must be along a travel path, or a 'wobbly' probe positioner requires one to map between a moveable coordinate system and a preferred laboratory coordinate system. This could for example introduce errors in measurements of vector quantities such as magnetic field or flow. We describe the design and implementation of a 3D probe positioner that slides in two dimensions on a double O-ring seal and radially inserts along the third dimension. The net result is that a 3D space can be explored in a laboratory Cartesian reference frame.

  20. Pneumatic probe with laser interferometer

    International Nuclear Information System (INIS)

    Wilkens, P.H.

    1978-01-01

    Improvements to upgrade the accuracy of Rotacon probes by a complete redesign of probe to include a Michelson interferometer to replace the existing long-range capacity transducer are described. This has resulted in a compact and interchangeable probe cartridge with a 3 μin. resolution and accuracy; the cartridge can be installed and replaced in the Rotacon gauge with the minimum of realignment, which should reduce our dependence on operator skill. In addition, the stylus contact force can be reduced to 750 mg for the contacting types, but an alternative feature, which we are still developing, will use a gas jet cushion in place of the stylus to provide a noncontacting version of the same basic probe cartridge. This device is very sensitive to external vibration effects because it is virtually frictionless

  1. 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.

  2. 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

  3. DIGITAL CONTACT POTENTIAL DIFFERENCE PROBE

    Directory of Open Access Journals (Sweden)

    K. U. Pantsialeyeu

    2016-01-01

    Full Text Available Nowadays the technique of analog contact potential difference probes well developed. Due to the influence of various parasitic factors, analog probes has substantial errors. The integration time for automatic CPD compensation should be at least several seconds to achieve high accuracy measurements. The speed and the accuracy are essential, for example, for Scanning Kelvin Probes. The purpose of this paper is to develop a digital contact potential difference probe, with a higher accuracy and speed of measurements as compared to analog probe. The digital probe made on base of 32-bit microprocessor with a Cortex M4 core. Measuring cycle consists of at least two successive determinations of the output signal amplitude at different compensation voltage generated by the microcontroller. It allows synchronizing of the generated oscillations and reading of the measuring signals. Data arrays processed in real time of the Digital Signal Processing by microprocessor. In this case is possible computation of the root mean square value or determination of the desired spectral line of the signal after fast Fourier transformation. Both methods permit eliminate of random noise and spurious harmonics. The method provides the digital contact potential difference probe operation in large signal mode and with a large signal/noise ratio. This eliminates the error associated with the zero signal finding. Also the integration time for automatic CPD compensation of the measured value is not necessary, which significantly reduces the measurement time and eliminates errors of compensation and DAC. In addition, the microcontroller could control the movement of the probe during scanning and transfer data to the host computer on interface USB, etc.

  4. GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus.

    Science.gov (United States)

    Gobi, Narayanan; Malaikozhundan, Balasubramanian; Sekar, Vijayakumar; Shanthi, Sathappan; Vaseeharan, Baskaralingam; Jayakumar, Rengarajan; Khudus Nazar, Abdul

    2016-05-01

    In this study, the pathogenicity of GFP tagged Vibrio parahaemolyticus Dahv2 and the protective effect of the probiotic strain, Bacillus licheniformis Dahb1 was studied on the Asian catfish, Pangasius hypophthalmus. The experiment was carried out for 24 days with three groups and one group served as the control (without treatment). In the first group, P. hypophthalmus was orally infected with 1 mL of GFP tagged V. parahaemolyticus Dahv2 at two different doses (10(5) and 10(7) cfu mL(-1)). In the second group, P. hypophthalmus was orally administrated with 1 ml of the probiotic B. licheniformis Dahb1 at two different doses (10(5) and 10(7) cfu mL(-1)). In the third group, P. hypophthalmus was orally infected first with 1 mL of GFP tagged V. parahaemolyticus Dahv2 followed by the administration of 1 mL of B. licheniformis Dahb1 (combined treatment) at two different doses (10(5) and 10(7) cfu mL(-1)). The growth, immune (myeloperoxidase, respiratory burst, natural complement haemolytic and lysozyme activity) and antioxidant (glutathione-S-transferase, reduced glutathione and total glutathione) responses of P. hypophthalmus were reduced after post infection of GFP tagged V. parahaemolyticus Dahv2 compared to control. However, after administration with the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1), P. hypophthalmus showed significant increase in the growth, immune and antioxidant responses compared to 10(7) cfu mL(-1). On the otherhand, the growth, immune and antioxidant responses of P. hypophthalmus infected and administrated with combined GFP tagged Vibrio + Bacillus at 10(5) cfu mL(-1) were relatively higher than that of GFP tagged V. parahaemolyticus Dahv2 and control groups but lower than that of probiotic B. licheniformis Dahb1 groups. The results of the present study conclude that the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1) has the potential to protect the P. hypophthalmus against V. parahaemolyticus Dahv2 infection by enhancing the growth

  5. Microscopic Analysis of Corn Fiber Using Corn Starch- and Cellulose-Specific Molecular Probes

    Energy Technology Data Exchange (ETDEWEB)

    Porter, S. E.; Donohoe, B. S.; Beery, K. E.; Xu, Q.; Ding, S.-Y.; Vinzant, T. B.; Abbas, C. A.; Himmel, M. E.

    2007-09-01

    Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.

  6. IVVS probe mechanical concept design

    Energy Technology Data Exchange (ETDEWEB)

    Rossi, Paolo, E-mail: paolo.rossi@enea.it; Neri, Carlo; De Collibus, Mario Ferri; Mugnaini, Giampiero; Pollastrone, Fabio; Crescenzi, Fabio

    2015-10-15

    Highlights: • ENEA designed, developed and tested a laser based In Vessel Viewing System (IVVS). • IVVS mechanical design has been revised from 2011 to 2013 to meet ITER requirements. • Main improvements are piezoceramic actuators and a step focus system. • Successful qualification activities validated the concept design for ITER environment. - Abstract: ENEA has been deeply involved in the design, development and testing of a laser based In Vessel Viewing System (IVVS) required for the inspection of ITER plasma-facing components. The IVVS probe shall be deployed into the vacuum vessel, providing high resolution images and metrology measurements to detect damages and possible erosion. ENEA already designed and manufactured an IVVS probe prototype based on a rad-hard concept and driven by commercial micro-step motors, which demonstrated satisfying viewing and metrology performances at room conditions. The probe sends a laser beam through a reflective rotating prism. By rotating the axes of the prism, the probe can scan all the environment points except those present in a shadow cone and the backscattered light signal is then processed to measure the intensity level (viewing) and the distance from the probe (metrology). During the last years, in order to meet all the ITER environmental conditions, such as high vacuum, gamma radiation lifetime dose up to 5 MGy, cumulative neutron fluence of about 2.3 × 10{sup 17} n/cm{sup 2}, temperature of 120 °C and magnetic field of 8 T, the probe mechanical design was significantly revised introducing a new actuating system based on piezo-ceramic actuators and improved with a new step focus system. The optical and mechanical schemes have been then modified and refined to meet also the geometrical constraints. The paper describes the mechanical concept design solutions adopted in order to fulfill IVVS probe functional performance requirements considering ITER working environment and geometrical constraints.

  7. POTENTIAL APPLICATIONS OF SOS-GFP BIOSENSOR TO IN VITRO RAPID SCREENING OF CYTOTOXIC AND GENOTOXIC EFFECT OF ANTICANCER AND ANTIDIABETIC PHARMACIST RESIDUES IN SURFACE WATER

    Directory of Open Access Journals (Sweden)

    Marzena Matejczyk

    2014-12-01

    Full Text Available Escherichia coli K-12 GFP-based bacterial biosensors allowed the detection of cytotoxic and genotoxic effect of anticancer drug– cyclophosphamide and antidiabetic drug – metformin in PBS buffer and surface water. Experimental data indicated that recA::gfpmut2 genetic system was sensitive to drugs and drugs mixture applied in experiment. RecA promoter was a good bioindicator in cytotoxic and genotoxic effect screening of cyclophosphamide, metformin and the mixture of the both drugs in PBS buffer and surface water. The results indicated that E. coli K-12 recA::gfp mut2 strain could be potentially useful for first-step screening of cytotoxic and genotoxic effect of anticancer and antidiabetic pharmacist residues in water. Next steps in research will include more experimental analysis to validate recA::gfpmut2 genetic system in E. coli K-12 on different anticancer drugs.

  8. Antigen Uptake during Different Life Stages of Zebrafish (Danio rerio Using a GFP-Tagged Yersinia ruckeri.

    Directory of Open Access Journals (Sweden)

    Rozalia Korbut

    Full Text Available Immersion-vaccines (bacterins are routinely used for aquacultured rainbow trout to protect against Yersinia ruckeri (Yr. During immersion vaccination, rainbow trout take up and process the antigens, which induce protection. The zebrafish was used as a model organism to study uptake mechanisms and subsequent antigen transport in fish. A genetically modified Yr was developed to constitutively express green fluorescent protein (GFP and was used for bacterin production. Larval, juvenile and adult transparent zebrafish (tra:nac mutant received a bath in the bacterin for up to 30 minutes. Samples were taken after 1 min, 15 min, 30 min, 2 h, 12 h and 24 h. At each sampling point fish were used for live imaging of the uptake using a fluorescence stereomicroscope and for immunohistochemistry (IHC. In adult fish, the bacterin could be traced within 30 min in scale pockets, skin, oesophagus, intestine and fins. Within two hours post bath (pb Yr-antigens were visible in the spleen and at 24 h in liver and kidney. Bacteria were associated with the gills, but uptake at this location was limited. Antigens were rarely detected in the blood and never in the nares. In juvenile fish uptake of the bacterin was seen in the intestine 30 min pb and in the nares 2 hpb but never in scale pockets. Antigens were detected in the spleen 12 hpb. Zebrafish larvae exhibited major Yr uptake only in the mid-intestine enterocytes 24 hpb. The different life stages of zebrafish varied with regard to uptake locations, however the gut was consistently a major uptake site. Zebrafish and rainbow trout tend to have similar uptake mechanisms following immersion or bath vaccination, which points towards zebrafish as a suitable model organism for this aquacultured species.

  9. Examining gender focal point (gfp roles to implement gender mainstreaming: The experiences of public sectors in malaysia

    Directory of Open Access Journals (Sweden)

    Nur Syakiran Akmal Ismail

    2013-07-01

    Full Text Available The participation of women in all spheres of life has been accelerated by strategies such as gender mainstreaming. Gender mainstreaming, which was launched in 1995 at the Fourth World Conference on Women in Beijing, is a global strategy used to promote gender equality. It refers to the process of assessing the implications for women and men of any planned action, including legislation, policies or programs, in all areas and at all levels. Hence, Malaysia has agreed to comply with GM procedure when the population of women in this country achieves 49 percent. Malaysian’s Gender Gap Index (MGGI was used to evaluate the achievement of gender equality. It was developed by the Organization C that responsibility to women and community development in Malaysia with the assistance of United Nations Development Programme (UNDP in 2004. Four dimensions are used as parameters to evaluate MGGI. They are (i women empowerment in politics, (ii activities in economics, (iii health and (iv education. This paper discusses the roles of gender focal point (GFP as a case study in two selected public sector organizations in implementation of Gender Mainstreaming in Malaysia. This study uses interview and content analysis. The results of this study show that the GFPs appointed have performed their roles based on the tasks listed by the Organization C. However the tasks were carried out based on the needs and interests of the respective GFPs organizations only.   Similar to other countries, the implementation of GM in the ministries also faces similar problems such as vague understanding of GM, and lack of commitment from the institutions’ leadership.

  10. TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

    Czech Academy of Sciences Publication Activity Database

    Brejchová, Jana; Sýkora, Jan; Ostašov, Pavel; Merta, Ladislav; Roubalová, Lenka; Janáček, Jiří; Hof, Martin; Svoboda, Petr

    2015-01-01

    Roč. 1848, č. 3 (2015), s. 781-796 ISSN 0005-2736 R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 ; RVO:61388955 Keywords : cholesterol * TRH-R-eGFP mobility * FRAP * RICS * DPH fluorescence * G protein coupling Subject RIV: CE - Biochemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.687, year: 2015

  11. Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2001-01-01

    Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

  12. Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy

    Directory of Open Access Journals (Sweden)

    Lara Marrone

    2018-02-01

    Full Text Available Summary: Perturbations in stress granule (SG dynamics may be at the core of amyotrophic lateral sclerosis (ALS. Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments. : Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS. Keywords: amyotrophic lateral sclerosis, induced pluripotent stem cells, FUS, stress granules, autophagy, gene editing, CRISPR/Cas9n

  13. Diphtheria Toxin- and GFP-Based Mouse Models of Acquired Hypoparathyroidism and Treatment With a Long-Acting Parathyroid Hormone Analog.

    Science.gov (United States)

    Bi, Ruiye; Fan, Yi; Lauter, Kelly; Hu, Jing; Watanabe, Tomoyuki; Cradock, Jim; Yuan, Quan; Gardella, Thomas; Mannstadt, Michael

    2016-05-01

    Hypoparathyroidism (HP) arises most commonly from parathyroid (PT) gland damage associated with neck surgery, and is typically treated with oral calcium and active vitamin D. Such treatment effectively increases levels of serum calcium (sCa), but also brings risk of hypercalciuria and renal damage. There is thus considerable interest in using PTH or PTH analogs to treat HP. To facilitate study of this disease and the assessment of new treatment options, we developed two mouse models of acquired HP, and used them to assess efficacy of PTH(1-34) as well as a long-acting PTH analog (LA-PTH) in regulating blood calcium levels. In one model, we used PTHcre-iDTR mice in which the diphtheria toxin (DT) receptor (DTR) is selectively expressed in PT glands, such that systemic DT administration selectively ablates parathyroid cells. For the second model, we generated GFP-PT mice in which green fluorescent protein (GFP) is selectively expressed in PT cells, such that parathyroidectomy (PTX) is facilitated by green fluorescence of the PT glands. In the PTHcre-iDTR mice, DT injection (2 × 5 μg/kg, i.p.) resulted in moderate yet consistent reductions in serum PTH and sCa levels. The more severe hypoparathyroid phenotype was observed in GFP-PT mice following GFP-guided PTX surgery. In each model, a single subcutaneous injection of LA-PTH increased sCa levels more effectively and for a longer duration (>24 hours) than did a 10-fold higher dose of PTH(1-34), without causing excessive urinary calcium excretion. These new mouse models thus faithfully replicate two degrees of acquired HP, moderate and severe, and may be useful for assessing potential new modes of therapy. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.

  14. Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts

    Czech Academy of Sciences Publication Activity Database

    Kolesová, H.; Čapek, Martin; Radochová, Barbora; Janáček, Jiří; Sedmera, David

    2016-01-01

    Roč. 146, č. 2 (2016), s. 142-152 ISSN 0948-6143 R&D Projects: GA ČR(CZ) GA13-12412S; GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : green fluorescent protein (GFP) * confocal microscopy * optical projection tomography * tissue transparency * heart * embryo Subject RIV: EA - Cell Biology Impact factor: 2.553, year: 2016

  15. Compound parabolic concentrator optical fiber tip for FRET-based fluorescent sensors

    DEFF Research Database (Denmark)

    Hassan, Hafeez Ul; Nielsen, Kristian; Aasmul, Soren

    2015-01-01

    polymer optical fiber for an excitation and emission wavelength of 550 nm and 650nm, respectively. The model suggests an increase of a factor of 1.6 to 4 in the collected fluorescent power for an ideal CPC tip, as compared to the plane-cut fiber tip for fiber lengths between 5 and 45mm......The Compound Parabolic Concentrator (CPC) optical fiber tip shape has been proposed for intensity based fluorescent sensors working on the principle of FRET (Förster Resonance Energy Transfer). A simple numerical Zemax model has been used to optimize the CPC tip geometry for a step-index multimode...

  16. Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

    International Nuclear Information System (INIS)

    Zekavati, Roya; Bayat, Mansour; Safi, Shahabeddin; Hashemi, Seyed Jamal; Rahmani-Cherati, Tavoos; Tabatabaei, Meisam; Mohsenifar, Afshin

    2013-01-01

    We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immuno reaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL −1 . The limit of detection is 2 × 10 −11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps. (author)

  17. Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library.

    Science.gov (United States)

    Ødum, Anders S R; Olesen, Kjeld; Østergaard, Søren; Thim, Lars; Nørby, Inga; Meldal, Morten

    2015-01-01

    Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1', and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.

  18. Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor

    OpenAIRE

    John, Scott A.; Ottolia, Michela; Weiss, James N.; Ribalet, Bernard

    2007-01-01

    To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600µM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (τ =5 to 15 s) in COS and C2C12 cells and slow (τ =20 to 40 s) in HEK and CHO cells. Upon ...

  19. Graphene quantum dots FRET based sensor for early detection of heart attack in human.

    Science.gov (United States)

    Bhatnagar, Deepika; Kumar, Vanish; Kumar, Ashok; Kaur, Inderpreet

    2016-05-15

    Cardiac immunosensor for early detection of heart attack (myocardial infarction) was developed using amine functionalized graphene quantum dots (afGQDs) conjugated with antibody anti-cardiac Troponin I (anti-cTnI) to detect cardiac marker antigen Troponin I (cTnI) in blood based on fluorescence resonance energy transfer (FRET) between conjugate and graphene (quencher) only in 10 min. The anti-cTnI was covalently conjugated to afGQDs through carbodiimide coupling reaction. The conjugate was characterized by zeta potential UV-vis spectroscopy and field emission scanning electron microscopy (FESEM). The sensing performance of the sensor was studied with respect to changes in the photon count and photoluminescence of GQDs based on interaction of target cTnI with its specific anti-cTnI antibody. The sensor is highly specific and shows negligible response to non-specific antigens. The sensor displayed a linear response to cTnI from 0.001 to 1000 ng mL(-1) with a limit of detection of 0.192 pg mL(-1). Copyright © 2015 Elsevier B.V. All rights reserved.

  20. [Transfection Efficiency of Ad5F11p-GFP on CIK and NK-92 Cells and Its Influence on Biological Characteristics].

    Science.gov (United States)

    Xu, Zan-Mei; Lu, Ying; Zhao, Lan-Jun; Liu, Jin; Hu, Xian-Wen; Wu, Chu-Tse; Duan, Hai-Feng

    2016-06-01

    To study transfection efficiency of Ad5F11p-GFP and its influence on biological characteristics of CIK and NK-92 cells in order to predict the application of Ad5F11p vector in immunotherapy. Two kinds of immune cells, cytokine-induced killer (CIK) cells and natural-killer (NK) cell line NK-92 cells, were transfected by Ad5F11p-GFP at different multiplicity of transfection (MOI), and untransfected immune cells were used as negative control. GFP expression was determined by flow cytometry, the cell morphology was observed with microscope, the cell proliferation was analyzed by trypan blue staining, specific cytotoxicity of NK-92 cells was determined by LDH assay. About 90% of transfection efficiency for NK-92 cells could be achieved at a MOI of 25, while the transfection efficiency for CIK was less than 40% at a MOI of 200. In addition, the transfection efficiency basically unchanged at the same MOI for 48 h and 96 h, and the immune cells transfected with the virus trended to form agglomeration, displaying slower proliferation, increase of IFN-γ release and enhancement of tumor killing activity. Ad5F11p- modified NK-92 shows a good prospect for adoptive immunotherapy.

  1. A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library.

    Science.gov (United States)

    Huang, Ziliang; Li, Gang; Zhang, Chong; Xing, Xin-Hui

    2016-02-01

    Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Construction of a green fluorescent protein (GFP)-marked multifunctional pesticide-degrading bacterium for simultaneous degradation of organophosphates and γ-hexachlorocyclohexane.

    Science.gov (United States)

    Yang, Chao; Liu, Ruihua; Yuan, Yulan; Liu, Jianli; Cao, Xiangyu; Qiao, Chuanling; Song, Cunjiang

    2013-02-13

    An autofluorescent whole-cell biocatalyst capable of simultaneously degrading organophosphates (OPs) and γ-hexachlorocyclohexane (γ-HCH) was constructed by display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion on the cell surface of a γ-HCH-degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC-OPH-GFP fusion was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibits cell growth nor affects cell viability. In sterile and nonsterile soil samples, a mixture of parathion (100 mg kg(-1)) and γ-HCH (10 mg kg(-1)) could be degraded completely within 15 days when inoculated with the engineered UT26, and the strain could be easily monitored by fluorescence during bioremediation. These results indicate that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of environments contaminated with multiple pesticides.

  3. Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

    International Nuclear Information System (INIS)

    Wan Haiyan; Korzh, Svitlana; Li Zhen; Mudumana, Sudha Puttur; Korzh, Vladimir; Jiang Yunjin; Lin Shuo; Gong Zhiyuan

    2006-01-01

    In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development

  4. Autophagy Monitoring Assay II: Imaging Autophagy Induction in LLC-PK1 Cells Using GFP-LC3 Protein Fusion Construct.

    Science.gov (United States)

    Adiseshaiah, Pavan P; Skoczen, Sarah L; Rodriguez, Jamie C; Potter, Timothy M; Kota, Krishna; Stern, Stephan T

    2018-01-01

    Autophagy is a catabolic process involved in the degradation and recycling of long-lived proteins and damaged organelles for maintenance of cellular homeostasis, and it has also been proposed as a type II cell death pathway. The cytoplasmic components targeted for catabolism are enclosed in a double-membrane autophagosome that merges with lysosomes, to form autophagosomes, and are finally degraded by lysosomal enzymes. There is substantial evidence that several nanomaterials can cause autophagy and lysosomal dysfunction, either by prevention of autophagolysosome formation, biopersistence or inhibition of lysosomal enzymes. Such effects have emerged as a potential mechanism of cellular toxicity, which is also associated with various pathological conditions. In this chapter, we describe a method to monitor autophagy by fusion of the modifier protein MAP LC3 with green fluorescent protein (GFP; GFP-LC3). This method enables imaging of autophagosome formation in real time by fluorescence microscopy without perturbing the MAP LC3 protein function and the process of autophagy. With the GFP-LC3 protein fusion construct, a longitudinal study of autophagy can be performed in cells after treatment with nanomaterials.

  5. All-Fiber Raman Probe

    DEFF Research Database (Denmark)

    Brunetti, Anna Chiara

    to perform real-time measurements with little or no sample preparation, Raman spectroscopy is now considered an invaluable analytical tool, finding application in several fields including medicine, defense and process control. When combined with fiber optics technology, Raman spectroscopy allows......The design and development of an all-in-fiber probe for Raman spectroscopy are presented in this Thesis. Raman spectroscopy is an optical technique able to probe a sample based on the inelastic scattering of monochromatic light. Due to its high specificity and reliability and to the possibility...... for the realization of flexible and minimally-invasive devices, able to reach remote or hardly accessible samples, and to perform in-situ analyses in hazardous environments. The work behind this Thesis focuses on the proof-of-principle demonstration of a truly in-fiber Raman probe, where all parts are realized...

  6. Gamma-ray imaging probes

    International Nuclear Information System (INIS)

    Wild, W.J.

    1988-01-01

    External nuclear medicine diagnostic imaging of early primary and metastatic lung cancer tumors is difficult due to the poor sensitivity and resolution of existing gamma cameras. Nonimaging counting detectors used for internal tumor detection give ambiguous results because distant background variations are difficult to discriminate from neighboring tumor sites. This suggests that an internal imaging nuclear medicine probe, particularly an esophageal probe, may be advantageously used to detect small tumors because of the ability to discriminate against background variations and the capability to get close to sites neighboring the esophagus. The design, theory of operation, preliminary bench tests, characterization of noise behavior and optimization of such an imaging probe is the central theme of this work

  7. 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.

  8. Radioactive Probes on Ferromagnetic Surfaces

    CERN Multimedia

    2002-01-01

    On the (broad) basis of our studies of nonmagnetic radioactive probe atoms on magnetic surfaces and at interfaces, we propose to investigate the magnetic interaction of magnetic probe atoms with their immediate environment, in particular of rare earth (RE) elements positioned on and in ferromagnetic surfaces. The preparation and analysis of the structural properties of such samples will be performed in the UHV chamber HYDRA at the HMI/Berlin. For the investigations of the magnetic properties of RE atoms on surfaces Perturbed Angular Correlation (PAC) measurements and Mössbauer Spectroscopy (MS) in the UHV chamber ASPIC (Apparatus for Surface Physics and Interfaces at CERN) are proposed.

  9. Probing nuclear matter with dileptons

    International Nuclear Information System (INIS)

    Schroeder, L.S.

    1986-06-01

    Dileptons are shown to be of interest in helping probe extreme conditions of temperature and density in nuclear matter. The current state of experimental knowledge about dileptons is briefly described, and their use in upcoming experiments with light ions at CERN SPS are reviewed, including possible signatures of quark matter formation. Use of dileptons in an upcoming experiment with a new spectrometer at Berkeley is also discussed. This experiment will probe the nuclear matter equation of state at high temperature and density. 16 refs., 8 figs

  10. SUB-SLAB PROBE INSTALLATION

    Science.gov (United States)

    Sub-slab sampling has become an integral part of vapor intrusion investigations. It is now recommended in guidance documents developed by EPA and most states. A method for sub-slab probe installation was devised in 2002, presented at conferences through 2005, and finally docume...

  11. Resolution analysis by random probing

    NARCIS (Netherlands)

    Fichtner, Andreas; van Leeuwen, T.

    2015-01-01

    We develop and apply methods for resolution analysis in tomography, based on stochastic probing of the Hessian or resolution operators. Key properties of our methods are (i) low algorithmic complexity and easy implementation, (ii) applicability to any tomographic technique, including full‐waveform

  12. Seismic probing of Fennoscandian lithosphere

    Czech Academy of Sciences Publication Activity Database

    Bock, G.; Achauer, U.; Alinaghi, A.; Ansorge, J.; Bruneton, M.; Friederich, W.; Grad, M.; Guterch, A.; Hjelt, S. E.; Plomerová, Jaroslava

    2001-01-01

    Roč. 82, č. 50 (2001), s. 621, 628-629 ISSN 0096-3941 R&D Projects: GA AV ČR IAA3012908 Institutional research plan: CEZ:AV0Z3012916 Keywords : seismic probing * lithosphere * Fennoscandia * SVEKALAPKO * Europrobe Subject RIV: DC - Siesmology, Volcanology, Earth Structure

  13. Evaluation of the effects of ethinylestradiol on sexual differentiation in the olvas-GFP/STII-YI medaka (transgenic Oryzias latipes) strain as estimated by proliferative activity of germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Hano, Takeshi [National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452 (Japan); Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan); Oshima, Yuji, E-mail: yoshima@agr.kyushu-u.ac.jp [Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan); Kinoshita, Masato [Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan); Tanaka, Minoru [Laboratory of Molecular Genetics for Reproduction, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585 Aichi (Japan); Mishima, Noriko; Wakamatsu, Yuko; Ozato, Kenjiro [Laboratory of Freshwater Fish Stocks, Bioscience and Biotechnology Center, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Shimasaki, Yohei; Honjo, Tsuneo [Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan)

    2011-08-15

    We evaluated the effects of 17(-ethinylestradiol (EE{sub 2}) on sexual differentiation in transgenic olvas-GFP/STII-YI medaka (Oryzias latipes) in terms of the proliferative activity of germ cells. This strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ cell-specific expression of GFP can be visualized in living (transparent) individuals. From 0 days post-hatch (0 dph) onwards, juveniles were exposed to graded concentrations of EE{sub 2} (25.2-1710 ng/L) for 35 days. The gonads of live specimens were monitored by measuring their size and calculating their GFP-fluorescence area. GFP-fluorescent area in control females was about 10 times that in control males at 10 days posthatch (dph) whereas the gonadal size of 10 dph males that had been exposed to 158 ng/L of EE{sub 2} significantly increased up to twice the size of control males, indicating that abnormal sexual differentiation towards female might occur in these individuals. Histological examination and identification of the sex-linked marker SL1 indicated that male to female sex reversal occurred at EE{sub 2} exposure {>=}45.1 ng/L at 35 dph. These results suggest that observation of proliferative activity of germ cells in the olvas-GFP/STII-YI strain could be applied to facilitated screening fish model to detect adverse effects on sexual differentiation as early as 10 dph juveniles.

  14. 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.

  15. Cantilevered probe detector with piezoelectric element

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Jesse D; Sulchek, Todd A; Feigin, Stuart C

    2014-04-29

    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.

  16. Where do pulse oximeter probes break?

    Science.gov (United States)

    Crede, S; Van der Merwe, G; Hutchinson, J; Woods, D; Karlen, W; Lawn, J

    2014-06-01

    Pulse oximetry, a non-invasive method for accurate assessment of blood oxygen saturation (SPO2), is an important monitoring tool in health care facilities. However, it is often not available in many low-resource settings, due to expense, overly sophisticated design, a lack of organised procurement systems and inadequate medical device management and maintenance structures. Furthermore medical devices are often fragile and not designed to withstand the conditions of low-resource settings. In order to design a probe, better suited to the needs of health care facilities in low-resource settings this study aimed to document the site and nature of pulse oximeter probe breakages in a range of different probe designs in a low to middle income country. A retrospective review of job cards relating to the assessment and repair of damaged or faulty pulse oximeter probes was conducted at a medical device repair company based in Cape Town, South Africa, specializing in pulse oximeter probe repairs. 1,840 job cards relating to the assessment and repair of pulse oximeter probes were reviewed. 60.2 % of probes sent for assessment were finger-clip probes. For all probes, excluding the neonatal wrap probes, the most common point of failure was the probe wiring (>50 %). The neonatal wrap most commonly failed at the strap (51.5 %). The total cost for quoting on the broken pulse oximeter probes and for the subsequent repair of devices, excluding replacement components, amounted to an estimated ZAR 738,810 (USD $98,508). Improving the probe wiring would increase the life span of pulse oximeter probes. Increasing the life span of probes will make pulse oximetry more affordable and accessible. This is of high priority in low-resource settings where frequent repair or replacement of probes is unaffordable or impossible.

  17. Effect-directed analysis for estrogenic compounds in a fluvial sediment sample using transgenic cyp19a1b-GFP zebrafish embryos.

    Science.gov (United States)

    Fetter, Eva; Krauss, Martin; Brion, François; Kah, Olivier; Scholz, Stefan; Brack, Werner

    2014-09-01

    Xenoestrogens may persist in the environment by binding to sediments or suspended particulate matter serving as long-term reservoir and source of exposure, particularly for organisms living in or in contact with sediments. In this study, we present for the first time an effect-directed analysis (EDA) for identifying estrogenic compounds in a sediment sample using embryos of a transgenic reporter fish strain. In the tg(cyp19a1b-GFP) transgenic zebrafish strain, the expression of GFP (green fluorescent protein) in the brain is driven by an oestrogen responsive element in the promoter of the cyp19a1b (aromatase) gene. The selected sediment sample of the Czech river Bilina had already been analysed in a previous EDA using the yeast oestrogen screening assay and had revealed fractions containing estrogenic compounds. When normal phase HPLC (high performance liquid chromatography) fractionation was used for the separation of the sediment sample, the biotest with transgenic fish embryos revealed two estrogenic fractions. Chemical analysis of candidate compounds in these sediment fractions suggested alkylphenols and estrone as candidate compounds responsible for the observed estrogenic effect. Alkylphenol concentrations could partially explain the estrogenicity of the fractions. However, xenoestrogens below the analytical detection limit or non-targeted estrogenic compounds have probably also contributed to the sample's estrogenic potency. The results indicated the suitability of the tg(cyp19a1b-GFP) fish embryo for an integrated chemical-biological analysis of estrogenic effects. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

    Science.gov (United States)

    Woodfint, Rachel M; Chen, Paula R; Ahn, Jinsoo; Suh, Yeunsu; Hwang, Seongsoo; Lee, Sang Suk; Lee, Kichoon

    2017-01-19

    Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 ( MUC2 ) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis -acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  19. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Rachel M. Woodfint

    2017-01-01

    Full Text Available Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2 expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2, GATA binding protein 4 (GATA4, hepatocyte nuclear factor 4 α (HNF4A, and transcription factor 4 (TCF4 that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  20. Unveiling how an archetypal fluorescent protein operates: theoretical perspective on the ultrafast excited state dynamics of GFP variant S65T/H148D.

    Science.gov (United States)

    Armengol, Pau; Gelabert, Ricard; Moreno, Miquel; Lluch, José M

    2015-02-12

    Green fluorescent protein variant S65T/H148D has been reported to host a photocycle involving the photoinduced proton transfer reaction between the chromophore and residue Asp148 under 50 fs and without a measurable kinetic isotope effect, and experimental evidence is suggestive of the existence of a highly delocalized proton between these residues. The blinding speed at which this biological system undergoes proton transfer has been ascribed to the extreme increase of acidity of the GFP chromophore in the electronic excited state where proton transfer takes place. This work strives to present a coherent, complete, and balanced description of the dynamics of this specific variant of GFP in which it will be shown that this increase of acidity is insufficient to explain the behavior observed. This study tracks the behavior of this photosystem to the delicate interplay between structure and dynamics shown in the presence of solvent. In this way, it has been found that the dynamics of this protein intertwines its structure with the intervening solvent to give rise to effectively degenerate situations in what concerns the reactants and products of the proton transfer reaction in ground and, most importantly, photoexcited state, in terms of potential energy profiles associated with the proton migration. Under these conditions, proton transfer can occur in accordance with the experimental data available. This set of characteristics is possibly common to a host of other proton transfer based fluorescent proteins, and helps promoting GFP S65T/H148D to a case of archetypal significance. Thus, our results can be useful to understand the way many fluorescent proteins work and, more generally, the molecular basis for proton transfer reactions in proteins.

  1. Probe-based recording technology

    International Nuclear Information System (INIS)

    Naberhuis, Steve

    2002-01-01

    The invention of the scanning tunneling microscope (STM) prompted researchers to contemplate whether such technology could be used as the basis for the storage and retrieval of information. With magnetic data storage technology facing limits in storage density due to the thermal instability of magnetic bits, the super-paramagnetic limit, the heir-apparent for information storage at higher densities appeared to be variants of the STM or similar probe-based storage techniques such as atomic force microscopy (AFM). Among these other techniques that could provide replacement technology for magnetic storage, near-field optical scanning optical microscopy (NSOM or SNOM) has also been investigated. Another alternative probe-based storage technology called atomic resolution storage (ARS) is also currently under development. An overview of these various technologies is herein presented, with an analysis of the advantages and disadvantages inherent in each particularly with respect to reduced device dimensions. The role of micro electro mechanical systems (MEMS) is emphasized

  2. Distance probes of dark energy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, A. G.; Padmanabhan, N.; Aldering, G.; Allen, S. W.; Baltay, C.; Cahn, R. N.; D’Andrea, C. B.; Dalal, N.; Dawson, K. S.; Denney, K. D.; Eisenstein, D. J.; Finley, D. A.; Freedman, W. L.; Ho, S.; Holz, D. E.; Kasen, D.; Kent, S. M.; Kessler, R.; Kuhlmann, S.; Linder, E. V.; Martini, P.; Nugent, P. E.; Perlmutter, S.; Peterson, B. M.; Riess, A. G.; Rubin, D.; Sako, M.; Suntzeff, N. V.; Suzuki, N.; Thomas, R. C.; Wood-Vasey, W. M.; Woosley, S. E.

    2015-03-01

    This document presents the results from the Distances subgroup of the Cosmic Frontier Community Planning Study (Snowmass 2013). We summarize the current state of the field as well as future prospects and challenges. In addition to the established probes using Type Ia supernovae and baryon acoustic oscillations, we also consider prospective methods based on clusters, active galactic nuclei, gravitational wave sirens and strong lensing time delays.

  3. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  4. Head-neck domain of Arabidopsis myosin XI, MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells

    Directory of Open Access Journals (Sweden)

    Holweg Carola L

    2008-07-01

    Full Text Available Abstract Background The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis. Results We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin. Conclusion The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of

  5. Preparation of Metallochelating Microbubbles and Study on Their Site-Specific Interaction with rGFP-HisTag as a Model Protein

    Czech Academy of Sciences Publication Activity Database

    Lukáč, R.; Kauerová, Z.; Mašek, J.; Bartheldyová, E.; Kulich, P.; Koudelka, Š.; Korvasová, Z.; Plocková, J.; Papoušek, František; Kolář, František; Schmidt, R.; Turánek, J.

    2011-01-01

    Roč. 27, č. 8 (2011), s. 4829-4837 ISSN 0743-7463 R&D Projects: GA AV ČR KAN200520703 Grant - others:GA ČR(CZ) GAP304/10/1951; GA AV ČR(CZ) KAN200100801 Program:GA; KA Institutional research plan: CEZ:AV0Z50110509 Keywords : microbubble * ultrasound imaging * metallochelating bond * rGFP * liposome * contrast echocardiography * static light scattering * flow * cytometry * confocal Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 4.186, year: 2011

  6. Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

    Science.gov (United States)

    Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

    2016-03-01

    Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

  7. The ML1Nx2 Phosphatidylinositol 3,5-Bisphosphate Probe Shows Poor Selectivity in Cells.

    Science.gov (United States)

    Hammond, Gerald R V; Takasuga, Shunsuke; Sasaki, Takehiko; Balla, Tamas

    2015-01-01

    Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.

  8. The Van Allen Probes mission

    CERN Document Server

    Burch, James

    2014-01-01

    This collection of articles provides broad and detailed information about NASA’s Van Allen Probes (formerly known as the Radiation Belt Storm Probes) twin-spacecraft Earth-orbiting mission. The mission has the objective of achieving predictive understanding of the dynamic, intense, energetic, dangerous, and presently unpredictable belts of energetic particles that are magnetically trapped in Earth’s space environment above the atmosphere. It documents the science of the radiation belts and the societal benefits of achieving predictive understanding. Detailed information is provided about the Van Allen Probes mission design, the spacecraft, the science investigations, and the onboard instrumentation that must all work together to make unprecedented measurements within a most unforgiving environment, the core of Earth’s most intense radiation regions.
 This volume is aimed at graduate students and researchers active in space science, solar-terrestrial interactions and studies of the up...

  9. A computerized Langmuir probe system

    Science.gov (United States)

    Pilling, L. S.; Bydder, E. L.; Carnegie, D. A.

    2003-07-01

    For low pressure plasmas it is important to record entire single or double Langmuir probe characteristics accurately. For plasmas with a depleted high energy tail, the accuracy of the recorded ion current plays a critical role in determining the electron temperature. Even for high density Maxwellian distributions, it is necessary to accurately model the ion current to obtain the correct electron density. Since the electron and ion current saturation values are, at best, orders of magnitude apart, a single current sensing resistor cannot provide the required resolution to accurately record these values. We present an automated, personal computer based data acquisition system for the determination of fundamental plasma properties in low pressure plasmas. The system is designed for single and double Langmuir probes, whose characteristics can be recorded over a bias voltage range of ±70 V with 12 bit resolution. The current flowing through the probes can be recorded within the range of 5 nA-100 mA. The use of a transimpedance amplifier for current sensing eliminates the requirement for traditional current sensing resistors and hence the need to correct the raw data. The large current recording range is realized through the use of a real time gain switching system in the negative feedback loop of the transimpedance amplifier.

  10. Influence of probe geometry on the response of an electrostatic probe

    DEFF Research Database (Denmark)

    Johansson, Torben; Crichton, George C; McAllister, Iain Wilson

    1999-01-01

    The response of an electrostatic probe is examined with reference to the probe geometry. The study involves the evaluation of the probe lambda function, from which response-related characteristic parameters can be derived. These parameters enable the probe detection sensitivity Se and spatial...

  11. Project Prometheus and Future Entry Probe Missions

    Science.gov (United States)

    Spilker, Thomas R.

    2005-01-01

    A viewgraph presentation on project Prometheus and future entry probe missions is shown. The topics include: 1) What Is Project Prometheus?; 2) What Capabilities Can Project Prometheus Offer? What Mission Types Are Being Considered?; 3) Jupiter Icy Moons Orbiter (JIMO); 4) How Are Mission Opportunities Changing?; 5) Missions Of Interest a Year Ago; 6) Missions Now Being Considered For Further Study; 7) Galileo-Style (Conventional) Probe Delivery; 8) Galileo-Style Probe Support; 9) Conventional Delivery and Support of Multiple Probes; 10) How Entry Probe Delivery From an NEP Vehicle Is Different; and 11) Concluding Remarks.

  12. [HPV contamination of endocavity vaginal ultrasound probes].

    Science.gov (United States)

    Heard, I; Favre, M

    2015-02-01

    While the use of endovaginal ultrasound probes is increasing, the risk of contamination of women with endocavity vaginal probes was not assessed. In particular, the clinical significance of detection of human papillomavirus (HPV) infection, the most common sexually transmitted viral infection, on endovaginal ultrasound probes is uncertain. The recommendations of good practice for decontamination of these probes developed by the High Council for Public Health and the Academy of Medicine have not been evaluated. The objective of this article was to review recent publications concluding to the detection of HPV and human cellular DNA after gynecological examination and disinfection of vaginal ultrasound probes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  13. Probe Selection in Multiprobe OTA Setups

    DEFF Research Database (Denmark)

    Fan, Wei; Sun, Fan; Nielsen, Jesper Ødum

    2014-01-01

    is costly, so finding ways to limit the number of probes while still reproducing the target channels accurately could make the test system both cheaper and simpler to implement. Several probe selection algorithms are presented in this paper to address this issue. The proposed techniques provide a probe...... selection framework for the channel emulation techniques published in the literature. Simulation results show that good channel emulation accuracy can be achieved with the selected subset of probes for the considered target channel models. The probe selection algorithm is further supported by measurement...... results in a practical multiprobe setup...

  14. Zero voltage mass spectrometry probes and systems

    Science.gov (United States)

    Cooks, Robert Graham; Wleklinski, Michael Stanley; Bag, Soumabha; Li, Yafeng

    2017-10-10

    The invention generally relates to zero volt mass spectrometry probes and systems. In certain embodiments, the invention provides a system including a mass spectrometry probe including a porous material, and a mass spectrometer (bench-top or miniature mass spectrometer). The system operates without an application of voltage to the probe. In certain embodiments, the probe is oriented such that a distal end faces an inlet of the mass spectrometer. In other embodiments, the distal end of the probe is 5 mm or less from an inlet of the mass spectrometer.

  15. GFP variants with alternative β-strands and their application as light-driven protease sensors: a tale of two tails.

    Science.gov (United States)

    Do, Keunbong; Boxer, Steven G

    2013-07-17

    Green fluorescent protein (GFP) variants that carry one extra strand 10 (s10) were created and characterized, and their possible applications were explored. These proteins can fold with either one or the other s10, and the ratio of the two folded forms, unambiguously distinguished by their resulting colors, can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. We have discovered robust empirical rules that accurately predict the product ratios of any given construct in both bacterial and mammalian expressions. Exploiting earlier studies on photodissociation of cut s10 from GFP (Do and Boxer, 2011), ratiometric protease sensors were designed from the construct by engineering a specific protease cleavage site into one of the inserted loops, where the bound s10 is replaced by the other strand upon protease cleavage and irradiation with light to switch its color. Since the conversion involves a large spectral shift, these genetically encoded sensors display a very high dynamic range. Further engineering of this class of proteins guided by mechanistic understanding of the light-driven process will enable interesting and useful application of the protein.

  16. Fine processes of Nestin-GFP-positive radial glia-like stem cells in the adult dentate gyrus ensheathe local synapses and vasculature.

    Science.gov (United States)

    Moss, Jonathan; Gebara, Elias; Bushong, Eric A; Sánchez-Pascual, Irene; O'Laoi, Ruadhan; El M'Ghari, Imane; Kocher-Braissant, Jacqueline; Ellisman, Mark H; Toni, Nicolas

    2016-05-03

    Adult hippocampal neurogenesis relies on the activation of neural stem cells in the dentate gyrus, their division, and differentiation of their progeny into mature granule neurons. The complex morphology of radial glia-like (RGL) stem cells suggests that these cells establish numerous contacts with the cellular components of the neurogenic niche that may play a crucial role in the regulation of RGL stem cell activity. However, the morphology of RGL stem cells remains poorly described. Here, we used light microscopy and electron microscopy to examine Nestin-GFP transgenic mice and provide a detailed ultrastructural reconstruction analysis of Nestin-GFP-positive RGL cells of the dentate gyrus. We show that their primary processes follow a tortuous path from the subgranular zone through the granule cell layer and ensheathe local synapses and vasculature in the inner molecular layer. They share the ensheathing of synapses and vasculature with astrocytic processes and adhere to the adjacent processes of astrocytes. This extensive interaction of processes with their local environment could allow them to be uniquely receptive to signals from local neurons, glia, and vasculature, which may regulate their fate.

  17. A High-Throughput Oxidative Stress Biosensor Based on Escherichia coli roGFP2 Cells Immobilized in a k-Carrageenan Matrix

    Directory of Open Access Journals (Sweden)

    Lia Ooi

    2015-01-01

    Full Text Available Biosensors fabricated with whole-cell bacteria appear to be suitable for detecting bioavailability and toxicity effects of the chemical(s of concern, but they are usually reported to have drawbacks like long response times (ranging from hours to days, narrow dynamic range and instability during long term storage. Our aim is to fabricate a sensitive whole-cell oxidative stress biosensor which has improved properties that address the mentioned weaknesses. In this paper, we report a novel high-throughput whole-cell biosensor fabricated by immobilizing roGFP2 expressing Escherichia coli cells in a k-carrageenan matrix, for the detection of oxidative stress challenged by metalloid compounds. The E. coli roGFP2 oxidative stress biosensor shows high sensitivity towards arsenite and selenite, with wide linear range and low detection limit (arsenite: 1.0 × 10−3–1.0 × 101 mg·L−1, LOD: 2.0 × 10−4 mg·L−1; selenite: 1.0 × 10−5–1.0 × 102 mg·L−1, LOD: 5.8 × 10−6 mg·L−1, short response times (0–9 min, high stability and reproducibility. This research is expected to provide a new direction in performing high-throughput environmental toxicity screening with living bacterial cells which is capable of measuring the bioavailability and toxicity of environmental stressors in a friction of a second.

  18. Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.

    Science.gov (United States)

    Norcross, Stevie; Trull, Keelan J; Snaider, Jordan; Doan, Sara; Tat, Kiet; Huang, Libai; Tantama, Mathew

    2017-11-22

    Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically encoded sensor. Here, we report a new family of genetically encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.

  19. Green Fluorescent Protein (GFP-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Shengdi Fan

    2013-09-01

    Full Text Available Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC and size exclusion chromatography (SEC at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23-lauryl-ether (Brij-35 was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

  20. Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue

    Directory of Open Access Journals (Sweden)

    Zahra Erami

    2016-01-01

    Full Text Available E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.

  1. Dynamics of TransgenicEnterobacter cloacaeExpressing Green Fluorescent Protein Defensin (GFP-D) inAnopheles stephensiUnder Laboratory Condition.

    Science.gov (United States)

    Dehghan, Hossein; Oshaghi, Mohammad Ali; Moosa-Kazemi, Seyed Hassan; Yakhchali, Bagher; Vatandoost, Hassan; Maleki-Ravasan, Naseh; Rassi, Yavar; Mohammadzadeh, Habib; Abai, Mohammad Reza; Mohtarami, Fatemeh

    2017-12-01

    Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium within An. stephensi and its introduction methods to the mosquitoes have not yet been explored. Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium. The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadially from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time. Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

  2. Green fluorescent protein (GFP)-based overexpression screening and characterization of AgrC, a Receptor protein of quorum sensing in Staphylococcus aureus.

    Science.gov (United States)

    Wang, Lina; Quan, Chunshan; Liu, Baoquan; Xu, Yongbin; Zhao, Pengchao; Xiong, Wen; Fan, Shengdi

    2013-09-06

    Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP) fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) at yields of ≥ 10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23)-lauryl-ether (Brij-35) was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD) spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

  3. Locally-excited (LE) versus charge-transfer (CT) excited state competition in a series of para-substituted neutral green fluorescent protein (GFP) chromophore models.

    Science.gov (United States)

    Olsen, Seth

    2015-02-12

    In this paper, I provide a characterization of the low-energy electronic structure of a series of para-substituted neutral green fluorescent protein (GFP) chromophore models using a theoretical approach that blends linear free energy relationships (LFERs) with state-averaged complete-active-space self-consistent field (SA-CASSCF) theory. The substituents are chosen to sample the Hammett σ(p) scale from R = F to NH2, and a model of the neutral GFP chromophore structure (R = OH) is included. I analyze the electronic structure for different members of the series in a common complete-active-space valence-bond (CASVB) representation, exploiting an isolobal analogy between active-space orbitals for different members of the series. I find that the electronic structure of the lowest adiabatic excited state is a strong mixture of weakly coupled states with charge-transfer (CT) or locally excited (LE) character and that the dominant character changes as the series is traversed. Chromophores with strongly electron-donating substituents have a CT-like excited state such as expected for a push-pull polyene or asymmetric cyanine. Chromophores with weakly electron-donating (or electron-withdrawing) substituents have an LE-like excited state with an ionic biradicaloid structure localized to the ground-state bridge π bond.

  4. Colonization of Vitis vinifera by a green fluorescence protein-labeled, gfp-marked strain of Xylophilus ampelinus, the causal agent of bacterial necrosis of grapevine.

    Science.gov (United States)

    Grall, Sophie; Manceau, Charles

    2003-04-01

    The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.

  5. Soft QGP probes with ALICE

    CERN Document Server

    Graczykowski, Łukasz Kamil

    2016-01-01

    In heavy-ion collisions at the LHC a hot and dense medium of deconfided partons, the Quark-Gluon Plasma (QGP), is created. Its global properties can be characterized by the measurements of particles in the low transverse momentum (or "soft") regime, which represent the majority of created particles. In this report we outline a selection of measurements of the soft probes by the ALICE experiment in pp, p--Pb, and Pb--Pb collisions. The paper focuses on recent flow measurements via angular correlations and femtoscopic studies. The first ever preliminary analysis of $\\mathrm{K}^0_{\\rm S}\\mathrm{K}^{\\pm}$ femtoscopy is also presented.

  6. Soil moisture calibration of TDR multilevel probes

    Directory of Open Access Journals (Sweden)

    Serrarens Daniel

    2000-01-01

    Full Text Available Time domain reflectometry (TDR probes are increasingly used for field estimation of soil water content. The objective of this study was to evaluate the accuracy of the multilevel TDR probe under field conditions. For this purpose, eight such TDR probes were installed in small plots that were seeded with beans and sorghum. Data collection from the probes was such that soil moisture readings were automated and logged using a standalone field unit. Neutron probe measurements were used to calibrate the TDR probes. Soil-probe contact and soil compaction were critical to the accuracy of the TDR, especially when a number of TDR probes are combined for a single calibration curve. If each probe is calibrated individually, approximate measurement errors were between 0.005 and 0.015 m³ m-3. However, measurement errors doubled to approximately 0.025 to 0.03 m³ m-3, when TDR probes were combined to yield a single calibration curve.

  7. Eddy current flaw detecting probe

    International Nuclear Information System (INIS)

    Hashimoto, Mitsuo; Harada, Yutaka; Shimone, Junri; Maeda, Kotaro

    1998-01-01

    The present invention provides an eddy current-flaw detection probe facilitating quantitative evaluation, which is used for maintenance and inspection of metal fine tubes of a heat exchanger of a nuclear power plant. Namely, the probe comprises a substantially cylindrical or columnar flow detection main body to be inserted to a metal tube. Wires are wound on the circumferential surface of the flaw detection main body substantially uniformly and in parallel to form a solenoid portion having a predetermined width. Magnetic sensors are disposed on the lateral center of the solenoid portion. With such a constitution, the solenoid portion forms eddy current in the circumferential direction. The eddy current is substantially in parallel having the same intensity at the lateral central portion of the solenoid. Accordingly, the quantitative evaluation for the shape and the size of cracks in the axial direction of the tube can be conducted by the magnetic sensors disposed to the portion. In addition, since the eddy current is substantially uniform, parameters upon reverse analysis can be reduced upon determination of the shape of flaws. (I.S.)

  8. The Gravity Probe B Experiment

    Science.gov (United States)

    Kolodziejczak, Jeffrey

    2008-01-01

    This presentation briefly describes the Gravity Probe B (GP-B) Experiment which is designed to measure parts of Einstein's general theory of relativity by monitoring gyroscope orientation relative to a distant guide star. To measure the miniscule angles predicted by Einstein's theory, it was necessary to build near-perfect gyroscopes that were approximately 50 million times more precise than the best navigational gyroscopes. A telescope mounted along the central axis of the dewar and spacecraft provided the experiment's pointing reference to a guide star. The telescope's image divide precisely split the star's beam into x-axis and y-axis components whose brightness could be compared. GP-B's 650-gallon dewar, kept the science instrument inside the probe at a cryogenic temperature for 17.3 months and also provided the thruster propellant for precision attitude and translation control. Built around the dewar, the GP-B spacecraft was a total-integrated system, comprising both the space vehicle and payload, dedicated as a single entity to experimentally testing predictions of Einstein's theory.

  9. Portal monitor incorporating smart probes

    International Nuclear Information System (INIS)

    Bartos, D.; Constantin, F.; Guta, T.

    2003-01-01

    Portal monitors are intended for detection of radioactive and special nuclear materials in vehicles, pedestrians, luggage, as well as for prevention of illegal traffic of radioactive sources. Monitors provide audio and visual alarms when radioactive and/or special nuclear materials are detected. They can be recommended to officers of customs, border guard and emergency services, civil defense, fire brigades, police and military departments or nuclear research or energetic facilities. The portal monitor developed by us consists in a portal frame, which sustains five intelligent probes having long plastic scintillator (0.5 liters each). The probes communicate, by serial transmission, with a Central Unit constructed on the basis of the 80552 microcontroller. This one manages the handshake, calculates the background, establishes the measuring time, starts and stops each measurement and makes all the other decisions. Sound signals and an infrared sensor monitor the passing through the portal and the measuring procedure. For each measurement the result is displayed on a LCD device contaminated/uncontaminated; for the contaminated case a loud and long sound signal is also issued. An RS 232 serial interface is provided in order to further developments or custom made devices. As a result, the portal monitor detects 1 μ Ci 137 Cs, spread all over a human body, in a 20 μR/h gamma background for a measuring time of 1.5 or 10 seconds giving a 99% confidence factor. (authors)

  10. Influence of probe motion on laser probe temperature in circulating blood.

    Science.gov (United States)

    Hehrlein, C; Splinter, R; Littmann, L; Tuntelder, J R; Tatsis, G P; Svenson, R H

    1991-01-01

    The purpose of this study was to evaluate the effect of probe motion on laser probe temperature in various blood flow conditions. Laser probe temperatures were measured in an in vitro blood circulation model consisting of 3.2 nm-diameter plastic tubes. A 2.0 mm-diameter metal probe attached to a 300 microns optical quartz fiber was coupled to an argon laser. Continuous wave 4 watts and 8 watts of laser power were delivered to the fiber tip corresponding to a 6.7 +/- 0.5 and 13.2 +/- 0.7 watts power setting at the laser generator. The laser probe was either moved with constant velocity or kept stationary. A thermocouple inserted in the lateral portion of the probe was used to record probe temperatures. Probe temperature changes were found with the variation of laser power, probe velocity, blood flow, and duration of laser exposure. Probe motion significantly reduced probe temperatures. After 10 seconds of 4 watts laser power the probe temperature in stagnant blood decreased from 303 +/- 18 degrees C to 113 +/- 17 degrees C (63%) by moving the probe with a velocity of 5 cm/sec. Blood flow rates of 170 ml/min further decreased the probe temperature from 113 +/- 17 degrees C to 50 +/- 8 degrees C (56%). At 8 watts of laser power a probe temperature reduction from 591 +/- 25 degrees C to 534 +/- 36 degrees C (10%) due to 5 cm/sec probe velocity was noted. Probe temperatures were reduced to 130 +/- 30 degrees C (78%) under the combined influence of 5 cm/sec probe velocity and 170 ml/min blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils.

    Science.gov (United States)

    Anbalagan, Charumathi; Lafayette, Ivan; Antoniou-Kourounioti, Melissa; Gutierrez, Carmen; Martin, Jose Rodriguez; Chowdhuri, Debapratim K; De Pomerai, David I

    2013-01-01

    As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439-455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We

  12. High throughput generation of promoter reporter (GFP transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

    Directory of Open Access Journals (Sweden)

    Xiao Yong-Li

    2010-08-01

    Full Text Available Abstract Background Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. Results A high throughput pipeline was developed to generate promoter-reporter (GFP transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP constructs were successfully transferred into Agrobacterium (GV3101 by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO codes. Conclusions Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study

  13. Directional neutronometric probe; Kierunkowa sonda neutronometryczna

    Energy Technology Data Exchange (ETDEWEB)

    Strzelecki, M.; Owczarczyk, A.; Wieclaw, B.; Szpilowwski, S.

    1991-11-04

    The neutronometric probe for direction and rate of groundwater flow measurements has been designed. The probe consists in collimated proportional neutron counter and also collimated and shielded neutron source. The neutron source can be disconnected from the detection part of the probe and transported separately in especially designed container. In the place of measurement the source can be easily joined to the detection part. 1 fig.

  14. Gravity Probe B spacecraft description

    International Nuclear Information System (INIS)

    Bennett, Norman R; Burns, Kevin; Katz, Russell; Kirschenbaum, Jon; Mason, Gary; Shehata, Shawky

    2015-01-01

    The Gravity Probe B spacecraft, developed, integrated, and tested by Lockheed Missiles and Space Company and later Lockheed Martin Corporation, consisted of structures, mechanisms, command and data handling, attitude and translation control, electrical power, thermal control, flight software, and communications. When integrated with the payload elements, the integrated system became the space vehicle. Key requirements shaping the design of the spacecraft were: (1) the tight mission timeline (17 months, 9 days of on-orbit operation), (2) precise attitude and translational control, (3) thermal protection of science hardware, (4) minimizing aerodynamic, magnetic, and eddy current effects, and (5) the need to provide a robust, low risk spacecraft. The spacecraft met all mission requirements, as demonstrated by dewar lifetime meeting specification, positive power and thermal margins, precision attitude control and drag-free performance, reliable communications, and the collection of more than 97% of the available science data. (paper)

  15. Gravity Probe B orbit determination

    International Nuclear Information System (INIS)

    Shestople, P; Ndili, A; Parkinson, B W; Small, H; Hanuschak, G

    2015-01-01

    The Gravity Probe B (GP-B) satellite was equipped with a pair of redundant Global Positioning System (GPS) receivers used to provide navigation solutions for real-time and post-processed orbit determination (OD), as well as to establish the relation between vehicle time and coordinated universal time. The receivers performed better than the real-time position requirement of 100 m rms per axis. Post-processed solutions indicated an rms position error of 2.5 m and an rms velocity error of 2.2 mm s −1 . Satellite laser ranging measurements provided independent verification of the GPS-derived GP-B orbit. We discuss the modifications and performance of the Trimble Advance Navigation System Vector III GPS receivers. We describe the GP-B precision orbit and detail the OD methodology, including ephemeris errors and the laser ranging measurements. (paper)

  16. Nuclear probes of fundamental symmetries

    International Nuclear Information System (INIS)

    Adelberger, E.G.

    1983-01-01

    Nuclear experiments which probe the parity (P) and time-reversal (T) symmetries and lepton-number conservation are reviewed. The P-violating NN interaction, studied in the NN system and in light nuclei, provides an unique window on ΔS=0 hadronic weak processes. Results are in accord with expectations. Sensitive searches for T-violation via detailed balance, T-odd correlations in γ and β-decay, and a possible neutron electric dipole moment (EDM) are discussed. No T-violation is observed. The EDM limit is almost good enough to eliminate one of the leading theoretical explanations for CP violation. Experimental studies of double β-decay are reviewed. Although ββ nu nu decay has been convincingly detected in geochemical experiments there is no evidence for the lepton number violating ββ decay mode

  17. Nuclear reactions as structure probes

    International Nuclear Information System (INIS)

    Fernandez, Bernard; Cugnon, Joseph; Roussel-Chomaz, Patricia; Sparenberg, Jean-Marc; Oliveira Santos, Francois de; Bauge, Eric; Poves, Alfredo; Keeley, Nicholas; Simenel, Cedric; Avez, Benoit; Lacroix, Denis; Baye, Daniel; Cortina-Gil, Dolores; Pons, Alexandre

    2007-09-01

    This publication gathers courses which aim at giving a view on new experiments which are performed by using radioactive beams, notably low intensity beams, in different accelerators, and allow the structure of very exotic nuclei to be characterized. Experimental as well as theoretical aspects are thus addressed. The contributions propose: a brief history of nuclear reactions and of instruments used to study them from the discovery of nucleus to the DWBA (Distorted Wave Born Approximation); an overview of nuclear reactions; experimental techniques; the theory of collisions at low energy; resonant elastic scattering, inelastic scattering and astrophysical reactions; to probe nuclear structure with nucleons; shell model and spectroscopic factors; analysis of transfer reactions and determination of spectroscopic factors; microscopic approaches of nuclear dynamics; theoretical aspects of dissociation reactions; experimental aspects of knockout reactions; research in oenology with the chemical characterisation of defective ageing of dry white wines

  18. Pitfalls in Kelvin probe measurements

    International Nuclear Information System (INIS)

    Ottinger, Oliver M.; Melzer, Christian; Seggern, Heinz von

    2009-01-01

    We report on the interpretation of thickness-dependent surface potential profiles in insulators on metal substrates measured by Kelvin probe method. The electrical potentials are calculated within a self-consistent model taking both the conductive substrate and the insulator into account. It is shown that interpreting the Kelvin potentials for different layer thicknesses as the prevailing potential profile of a thick insulator film is generally wrong. Even more controversially, the reconstruction of the potential profile in a thick insulator layer on the basis of layer-thickness-dependent Kelvin measurements alone is per se impossible. This will be demonstrated exemplarily on the basis of doped and undoped organic films on conductive substrates.

  19. A novel thermal decomposition approach to synthesize hydroxyapatite-silver nanocomposites and their antibacterial action against GFP-expressing antibiotic resistant E. coli.

    Science.gov (United States)

    Sahni, Geetika; Gopinath, P; Jeevanandam, P

    2013-03-01

    A novel thermal decomposition approach to synthesize hydroxyapatite-silver (Hap-Ag) nanocomposites has been reported. The nanocomposites were characterized by X-ray diffraction, field emission scanning electron microscopy coupled with energy dispersive X-ray analysis, transmission electron microscopy and diffuse reflectance spectroscopy techniques. Antibacterial activity studies for the nanocomposites were explored using a new rapid access method employing recombinant green fluorescent protein (GFP) expressing antibiotic resistant Escherichia coli (E. coli). The antibacterial activity was studied by visual turbidity analysis, optical density analysis, fluorescence spectroscopy and microscopy. The mechanism of bactericidal action of the nanocomposites on E. coli was investigated using atomic force microscopy, and TEM analysis. Excellent bactericidal activity at low concentration of the nanocomposites was observed which may allow their use in the production of microbial contamination free prosthetics. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. The Oxford Probe: an open access five-hole probe for aerodynamic measurements

    International Nuclear Information System (INIS)

    Hall, B F; Povey, T

    2017-01-01

    The Oxford Probe is an open access five-hole probe designed for experimental aerodynamic measurements. The open access probe can be manufactured by the end user via additive manufacturing (metal or plastic). The probe geometry, drawings, calibration maps, and software are available under a creative commons license. The purpose is to widen access to aerodynamic measurement techniques in education and research environments. There are many situations in which the open access probe will allow results of comparable accuracy to a well-calibrated commercial probe. We discuss the applications and limitations of the probe, and compare the calibration maps for 16 probes manufactured in different materials and at different scales, but with the same geometrical design. (paper)

  1. Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events.

    Science.gov (United States)

    Majzoub, Ramsey N; Chan, Chia-Ling; Ewert, Kai K; Silva, Bruno F B; Liang, Keng S; Safinya, Cyrus R

    2015-06-01

    Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t<1h), only a fraction (≈35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP's membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs. Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Internalization of Escherichia coli O157:H7 gfp+ in rocket and Swiss chard baby leaves as affected by abiotic and biotic damage.

    Science.gov (United States)

    Hartmann, R; Fricke, A; Stützel, H; Mansourian, S; Dekker, T; Wohanka, W; Alsanius, B

    2017-07-01

    Internalization of human pathogens in edible parts of vegetables eaten raw is a major concern, since once internalized they are protected from sanitizing treatments. In this study, we examined the invasion of gfp-labelled Escherichia coli O157:H7 into intact and biotically (infection with Xanthomonas campestris/Pseudomonas syringae) and abiotically (grating with silicon carbide) damaged leaves of wild rocket (Diplotaxis tenuifolia) and Swiss chard (Beta vulgaris subsp. cicla) using laser scanning confocal microscopy. Bacterial cells were found in internal locations of the tissue, irrespective of tissue health status. Contaminated leaf sections of biotically and abiotically damaged wild rocket leaves showed higher susceptibility to microbial invasion, while the pathogen was internalized in greater numbers into intact Swiss chard leaf sections when abiotically, but not biotically, damaged. The greatest differences were observed between the plant species; after surface sanitization, E. coli O157:H7 was still detected in wild rocket leaves, but not in Swiss chard leaves. Contamination of leafy vegetables with Escherichia coli O157:H7 is a growing problem, as reported outbreaks are increasing. However, establishment of this human pathogen in the phyllosphere is not completely understood. Using laser scanning confocal microscopy, we demonstrated that E. coli O157:H7gfp+ can invade plant tissue of Swiss chard and wild rocket leaves and that the bacterium is more sensitive to surface sanitization of Swiss chard leaves. Damage to leaf tissue promoted leaf invasion, but the nature of the damage (abiotic or biotic) and plant species had an impact. © 2017 The Society for Applied Microbiology.

  3. The TORE SUPRA fast reciprocating RF probe

    International Nuclear Information System (INIS)

    Thomas, C.E. Jr.; Harris, J.H.; Haste, G.R.

    1994-01-01

    A fast reciprocating ICRF (Ion Cyclotron Range of Frequencies) probe was installed and operated on TORE SUPRA during 1992/1993. The body of the probe was originally used on the ATF experiment at ORNL. The probe was adapted for use on TORE SUPRA, and mounted on one of the two fast reciprocating probe mounts. The probe consists of two orthogonal single-turn wire loops, mounted so that one loop senses toroidal RF magnetic fields and the other senses poloidal RF magnetic fields. The probe began operation in June, 1993. The probe active area is approximately 5 cm long by 2 cm, and the reciprocating mount has a slow stroke (5 cm/sec) of 30 cm by 2 cm, and the reciprocating mount has a slow stroke (5 cm/sec) of 30 cm and a fast stroke (1.5 m/sec) of about 10 cm. The probe was operated at distances from the plasma edge ranging from 30 cm to -5 cm (i.e., inside the last closed flux surface). The probe design, electronics, calibration, data acquisition and data processing are discussed. First data from the probe are presented as a function of ICRF power, distance from the plasma, loop orientation, and other plasma parameters. Initial data shows parametric instabilities do not play an important role for ICRF in the TORE SUPRA edge and scrape-off-layer (SOL) plasmas. Additionally it is observed that the probe signal has little or no dependence on position in the SOL/plasma edge

  4. A probe for Eddy current inspection devices

    International Nuclear Information System (INIS)

    1974-01-01

    The invention relates to a surface probe for Eddy current inspection devices. According to the invention, said probe comprises two magnetic core windings, with their axes in parallel relationship and at right angles to the surface of the part to be inspected. This can be applied to the nondestructive inspection of reactor components [fr

  5. Inspecting Friction Stir Welding using Electromagnetic Probes

    Science.gov (United States)

    Kinchen, David G.

    2004-01-01

    A report describes the use of advanced electromagnetic probes to measure the dimensions, the spatial distribution of electrical conductivity, and related other properties of friction stir welds (FSWs) between parts made of the same or different aluminum alloy(s). The probes are of the type described in in another Tech Brief. To recapitulate: A probe of this type is essentially an eddy-current probe that includes a primary (driver) winding that meanders and multiple secondary (sensing) windings that meander along the primary winding. Electrical conductivity is commonly used as a measure of heat treatment and tempering of aluminum alloys, but prior to the development of these probes, the inadequate sensitivity and limited accuracy of electrical-conductivity probes precluded such use on FSWs between different aluminum alloys, and the resolution of those probes was inadequate for measurement of FSW dimensions with positions and metallurgical properties. In contrast, the present probes afford adequate accuracy and spatial resolution for the purposes of measuring the dimensions of FSW welds and correlating spatially varying electrical conductivities with metallurgical properties, including surface defects.

  6. Automatic kelvin probe compatible with ultrahigh vacuum

    NARCIS (Netherlands)

    Baikie, I.D.; van der Werf, Kees; Oerbekke, H.; Broeze, J.; van Silfhout, Arend

    1989-01-01

    This article describes a new type of in situ ultrahigh‐vacuum compatible kelvin probe based on a voice‐coil driving mechanism. This design exhibits several advantages over conventional mechanical feed‐through and (in situ) piezoelectric devices in regard to the possibility of multiple probe

  7. Surface charge measurement using an electrostatic probe

    DEFF Research Database (Denmark)

    Crichton, George C; McAllister, Iain Wilson

    1998-01-01

    During the 1960s, the first measurements of charge on dielectric surfaces using simple electrostatic probes were reported. However it is only within the last 10 years that a proper understanding of the probe response has been developed. This situation arose as a consequence of the earlier studies...

  8. Quality of the neutron probe calibration curve

    International Nuclear Information System (INIS)

    Libardi, Paulo Leonel; Moraes, Sergio Oliveira

    1997-01-01

    An experiment of neutron probe calibration has been performed, involving various volume size samples and collected at various distances from the access tubes. The experiment aimed to give some answers to questions such as suitable sample physical volume, always use of the same volume and sample distance from the neutron probe access tube

  9. Neutron-based portable drug probe

    International Nuclear Information System (INIS)

    Womble, P. C.; Vourvopoulos, G.; Ball Howard, J.; Paschal, J.

    1999-01-01

    Based on previous measurements, a probe prototype for contraband detection utilizing the neutron technique of Pulsed Fast-Thermal Neutron Analysis (PFTNA) is being constructed. The prototype weighs less than 45 kg and is composed of a probe (5 cm diameter), a power pack and a data acquisition and display system. The probe is designed to be inserted in confined spaces such as the boiler of a ship or a tanker truck filled with liquid. The probe provides information on a) the elemental content, and b) the density variations of the interrogated object. By measuring elemental content, the probe can differentiate between innocuous materials and drugs. Density variations can be found through fast neutron transmission. In all cases, hidden drugs are identified through the measurement of the elemental content of the object, and the comparison of expected and measured elemental ratios

  10. Molecular Imaging Probe Development using Microfluidics

    Science.gov (United States)

    Liu, Kan; Wang, Ming-Wei; Lin, Wei-Yu; Phung, Duy Linh; Girgis, Mark D.; Wu, Anna M.; Tomlinson, James S.; Shen, Clifton K.-F.

    2012-01-01

    In this manuscript, we review the latest advancement of microfluidics in molecular imaging probe development. Due to increasing needs for medical imaging, high demand for many types of molecular imaging probes will have to be met by exploiting novel chemistry/radiochemistry and engineering technologies to improve the production and development of suitable probes. The microfluidic-based probe synthesis is currently attracting a great deal of interest because of their potential to deliver many advantages over conventional systems. Numerous chemical reactions have been successfully performed in micro-reactors and the results convincingly demonstrate with great benefits to aid synthetic procedures, such as purer products, higher yields, shorter reaction times compared to the corresponding batch/macroscale reactions, and more benign reaction conditions. Several ‘proof-of-principle’ examples of molecular imaging probe syntheses using microfluidics, along with basics of device architecture and operation, and their potential limitations are discussed here. PMID:22977436

  11. Recent status of the Kiev nuclear probe

    Science.gov (United States)

    Lebed, S.; Tolmachov, M.; Kukharenko, O.; Veselov, O.

    2009-06-01

    The modified Van de Graaff accelerator with proton beam energy W ⩽ 3 MeV has been installed and put into operation at the TMM laboratory in Kiev. The laboratory incorporates the nuclear probe (NP) beam line, coupled to this accelerator. A short version of an optimized probe-forming system (PFS) has been developed for the Kiev NP. The system is based on divided triplet of the magnetic quadrupole lenses (MQLs). This PFS has two working regimes for the probe operations. The results of numerical calculations of the geometrical and ion-optical parameters of the PFS are presented. It is shown that this versatile PFS is a promising design for a modern nuclear nano-probe. A new precision adjustable MQL has been designed. Three lenses, the slit systems and target chamber are manufactured and installed at the Kiev probe beam line. Also a new data acquisition system for the Kiev NP is being developed.

  12. Recent status of the Kiev nuclear probe

    Energy Technology Data Exchange (ETDEWEB)

    Lebed, S. [Research Laboratory ' Spectra' , TMM Ltd., ul. Chaadaeva 2b, 03148, Kiev (Ukraine)], E-mail: salmp1@ukr.net; Tolmachov, M.; Kukharenko, O.; Veselov, O. [Research Laboratory ' Spectra' , TMM Ltd., ul. Chaadaeva 2b, 03148, Kiev (Ukraine)

    2009-06-15

    The modified Van de Graaff accelerator with proton beam energy W {<=} 3 MeV has been installed and put into operation at the TMM laboratory in Kiev. The laboratory incorporates the nuclear probe (NP) beam line, coupled to this accelerator. A short version of an optimized probe-forming system (PFS) has been developed for the Kiev NP. The system is based on divided triplet of the magnetic quadrupole lenses (MQLs). This PFS has two working regimes for the probe operations. The results of numerical calculations of the geometrical and ion-optical parameters of the PFS are presented. It is shown that this versatile PFS is a promising design for a modern nuclear nano-probe. A new precision adjustable MQL has been designed. Three lenses, the slit systems and target chamber are manufactured and installed at the Kiev probe beam line. Also a new data acquisition system for the Kiev NP is being developed.

  13. Imaging probe for tumor malignancy

    Science.gov (United States)

    Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Hiraoka, Hasahiro

    2009-02-01

    Solid tumors possess unique microenvironments that are exposed to chronic hypoxic conditions ("tumor hypoxia"). Although more than half a century has passed since it was suggested that tumor hypoxia correlated with poor treatment outcomes and contributed to cancer recurrence, a fundamental solution to this problem has yet to be found. Hypoxia-inducible factor (HIF-1) is the main transcription factor that regulates the cellular response to hypoxia. It induces various genes whose functions are strongly associated with malignant alteration of the entire tumor. The cellular changes induced by HIF-1 are extremely important targets of cancer therapy, particularly in therapy against refractory cancers. Imaging of the HIF-1-active microenvironment is therefore important for cancer therapy. To image HIF-1activity in vivo, we developed a PTD-ODD fusion protein, POHA, which was uniquely labeled with near-infrared fluorescent dye at the C-terminal. POHA has two functional domains: protein transduction domain (PTD) and VHL-mediated protein destruction motif in oxygen-dependent degradation (ODD) domain of the alpha subunit of HIF-1 (HIF-1α). It can therefore be delivered to the entire body and remain stabilized in the HIF-1-active cells. When it was intravenously injected into tumor-bearing mice, a tumor-specific fluorescence signal was detected in the tumor 6 h after the injection. These results suggest that POHA can be used an imaging probe for tumor malignancy.

  14. The Gravity Probe B gyroscope

    International Nuclear Information System (INIS)

    Buchman, S; Lipa, J A; Keiser, G M; Muhlfelder, B; Turneaure, J P

    2015-01-01

    The Gravity Probe B (GP-B) gyroscope, a unique cryogenically operated mechanical sensor, was used on-orbit to independently test two predictions of general relativity (GR). Here, we describe the development and performance of the GP-B gyroscope, its geometry and fabrication, spin-up and vacuum approach, magnetic considerations, and static charge management. The history of electrically suspended gyroscopes puts the current work in context. Fabrication and ground testing of the GP-B gyroscope are detailed, followed by a review of on-orbit initialization, calibration, operation, and performance. We find that the performance was degraded relative to the mission goals, but was still sufficient to provide excellent new tests of GR. The degradation is partially due to the existence of gyroscope torques due to an unanticipated interaction between patch potentials on the rotor and the housing. We discuss these patch potentials and describe the effect of related torques on gyro drift. It was essential to include models for the effects due to the patch potentials in the complete data analysis model to yield determinations of the two GR effects. (paper)

  15. Probing the string winding sector

    Energy Technology Data Exchange (ETDEWEB)

    Aldazabal, Gerardo; Mayo, Martín [G. Física CAB-CNEA and CONICET, Centro Atómico Bariloche,Av. Bustillo 9500, Bariloche (Argentina); Instituto Balseiro, Centro Atómico Bariloche,Av. Bustillo 9500, Bariloche (Argentina); Nuñez, Carmen [Instituto de Astronomía y Física del Espacio (CONICET-UBA),C.C. 67 - Suc. 28, 1428 Buenos Aires (Argentina); Departamento de Física, FCEN, Universidad de Buenos Aires,C.C. 67 - Suc. 28, 1428 Buenos Aires (Argentina)

    2017-03-17

    We probe a slice of the massive winding sector of bosonic string theory from toroidal compactifications of Double Field Theory (DFT). This string subsector corresponds to states containing one left and one right moving oscillators. We perform a generalized Kaluza Klein compactification of DFT on generic 2n-dimensional toroidal constant backgrounds and show that, up to third order in fluctuations, the theory coincides with the corresponding effective theory of the bosonic string compactified on n-dimensional toroidal constant backgrounds, obtained from three-point amplitudes. The comparison between both theories is facilitated by noticing that generalized diffeomorphisms in DFT allow to fix generalized harmonic gauge conditions that help in identifying the physical degrees of freedom. These conditions manifest as conformal anomaly cancellation requirements on the string theory side. The explicit expression for the gauge invariant effective action containing the physical massless sector (gravity+antisymmetric+gauge+ scalar fields) coupled to towers of generalized Kaluza Klein massive states (corresponding to compact momentum and winding modes) is found. The action acquires a very compact form when written in terms of fields carrying O(n,n) indices, and is explicitly T-duality invariant. The global algebra associated to the generalized Kaluza Klein compactification is discussed.

  16. Probing Light Stops with Stoponium

    CERN Document Server

    Batell, Brian

    2015-01-01

    We derive new limits on light stops from diboson resonance searches in the $\\gamma\\gamma$, $Z \\gamma$, $ZZ$, $WW$ and $hh$ channels from the first run of the LHC. If the two-body decays of the light stop are mildly suppressed or kinematically forbidden, stoponium bound states will form in $pp$ collisions and subsequently decay via the pair annihilation of the constituent stops to diboson final states, yielding striking resonance signatures. Remarkably, we find that stoponium searches are highly complementary to direct collider searches and indirect probes of light stops such as Higgs coupling measurements. Using an empirical quarkonia potential model and including the first two $S$-wave stoponium states, we find that in the decoupling limit $m_{\\widetilde t_1} \\lesssim 130$ GeV is excluded for any value of the stop mixing angle and heavy stop mass by the combination of the latest resonance searches and the indirect constraints. The $\\gamma \\gamma$ searches are the most complementary to the indirect constraint...

  17. Integrated cosmological probes: concordance quantified

    Energy Technology Data Exchange (ETDEWEB)

    Nicola, Andrina; Amara, Adam; Refregier, Alexandre, E-mail: andrina.nicola@phys.ethz.ch, E-mail: adam.amara@phys.ethz.ch, E-mail: alexandre.refregier@phys.ethz.ch [Department of Physics, ETH Zürich, Wolfgang-Pauli-Strasse 27, CH-8093 Zürich (Switzerland)

    2017-10-01

    Assessing the consistency of parameter constraints derived from different cosmological probes is an important way to test the validity of the underlying cosmological model. In an earlier work [1], we computed constraints on cosmological parameters for ΛCDM from an integrated analysis of CMB temperature anisotropies and CMB lensing from Planck, galaxy clustering and weak lensing from SDSS, weak lensing from DES SV as well as Type Ia supernovae and Hubble parameter measurements. In this work, we extend this analysis and quantify the concordance between the derived constraints and those derived by the Planck Collaboration as well as WMAP9, SPT and ACT. As a measure for consistency, we use the Surprise statistic [2], which is based on the relative entropy. In the framework of a flat ΛCDM cosmological model, we find all data sets to be consistent with one another at a level of less than 1σ. We highlight that the relative entropy is sensitive to inconsistencies in the models that are used in different parts of the analysis. In particular, inconsistent assumptions for the neutrino mass break its invariance on the parameter choice. When consistent model assumptions are used, the data sets considered in this work all agree with each other and ΛCDM, without evidence for tensions.

  18. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier

    2014-01-01

    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  19. Development and application of DNA molecular probes

    Directory of Open Access Journals (Sweden)

    Priya Vizzini

    2017-02-01

    Full Text Available The development of DNA probes started from 1950's for diagnostic purposes and it is still growing. DNA probes are applied in several fields such as food, medical, veterinary, environment and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits microorganism identification, including pathogen detection, and their quantification when used in specific systems. Various techniques obtained success by the utilization of specific DNA probes, that allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon resonance (SPR, electrochemical, and quartz crystal microbalance (QCM biosensors that use different transduction systems. This review describes i the design and production of primers and probes, and their utilization from the traditional techniques to the new emerging techniques like biosensors used in current applications; ii the possibility to use labelled-free probes and probes labelled with an enzyme/fluorophore, etc.; iii the different sensitivity obtained by using specific systems; and iv the advantage obtained by using biosensors.

  20. Overview of Key Saturn Probe Mission Trades

    Science.gov (United States)

    Balint, Tibor S.; Kowalkowski, Theresa; Folkner, Bill

    2007-01-01

    Ongoing studies, performed at NASA/JPL over the past two years in support of NASA's SSE Roadmap activities, proved the feasibility of a NF class Saturn probe mission. I. This proposed mission could also provide a good opportunity for international collaboration with the proposed Cosmic Vision KRONOS mission: a) With ESA contributed probes (descent modules) on a NASA lead mission; b) Early 2017 launch could be a good programmatic option for ESA-CV/NASA-NF. II. A number of mission architectures could be suitable for this mission: a) Probe Relay based architecture with short flight time (approx. 6.3-7 years); b) DTE probe telecom based architecture with long flight time (-11 years), and low probe data rate, but with the probes decoupled from the carrier, allowing for polar trajectories I orbiter. This option may need technology development for telecom; c) Orbiter would likely impact mission cost over flyby, but would provide significantly higher science return. The Saturn probes mission is expected to be identified in NASA's New Frontiers AO. Thus, further studies are recommended to refine the most suitable architecture. International collaboration is started through the KRONOS proposal work; further collaborated studies will follow once KRONOS is selected in October under ESA's Cosmic Vision Program.

  1. Transmit-receive eddy current probes

    International Nuclear Information System (INIS)

    Obrutsky, L.S.; Sullivan, S.P.; Cecco, V.S.

    1997-01-01

    In the last two decades, due to increased inspection demands, eddy current instrumentation has advanced from single-frequency, single-output instruments to multifrequency, computer-aided systems. This has significantly increased the scope of eddy current testing, but, unfortunately, it has also increased the cost and complexity of inspections. In addition, this approach has not always improved defect detectability or signal-to-noise. Most eddy current testing applications are still performed with impedance probes, which have well known limitations. However, recent research at AECL has led to improved eddy current inspections through the design and development of transmit-receive (T/R) probes. T/R eddy current probes, with laterally displaced transmit and receive coils, present a number of advantages over impedance probes. They have improved signal-to-noise ratio in the presence of variable lift-off compared to impedance probes. They have strong directional properties, permitting probe optimization for circumferential or axial crack detection, and possess good phase discrimination to surface defects. They can significantly increase the scope of eddy current testing permitting reliable detection and sizing of cracks in heat exchanger tubing as well as in welded areas of both ferritic and non-ferromagnetic components. This presentation will describe the operating principles of T/R probes with the help of computer-derived normalized voltage diagrams. We will discuss their directional properties and analyze the advantages of using single and multiple T/R probes over impedance probes for specific inspection cases. Current applications to surface and tube testing and some typical inspection results will be described. (author)

  2. Nuclear borehole probes - theory and experiments

    International Nuclear Information System (INIS)

    Joergensen, J.L.; Korsbech, U.; Gynther Nielsen, K.; Oelgaard, P.L.

    1985-06-01

    The report gives a summary of the theoretical and expeimental work on borehole probes that has been performed since 1971 at The Department of Electrophysics, The Technical University of Denmark. The first part of the report concerns the use of a spectral natural gamma-ray probe (SNG-probe), which is used for measurements of the spectral distribution of the gamma-rays of the geological strata around a borehole. In general the spectrum is divided into three parts - the gamma-rays from potassium-40, from thorium-232 and daughters, and from uranium-238 and daughters. A set of curves showing the intensities of the gamm-radiation from K, Th, and U versus depth is called a SNG-log. If proper calibrated, the SNG-log gives the concentration of Th, U, and K in the formation surrounding the borehole. Initially the basis for an interpretation of SNG-logs is discussed. Then follows a description og some SNG-problems designed and built by The Department of Electrophysics, and a discussion of the calibration of SNG-probes. Some examples of SNG-logs are presented, and some general comments on the use of SNG-logs are given. The second part of the report concerns mainly the development of theoretical models for neutron-neutron probes, gamma-gamma probes, and pulsed-neutron probes. The purpose of this work has been to examine how well the models correlate with measured results and - where reasonable agreement is found - to use the models in studies of the factors that affect the probe responses in interpretation of experimental results and in probe design. (author)

  3. The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo.

    Science.gov (United States)

    Lewis, Jo E; Brameld, John M; Hill, Phil; Barrett, Perry; Ebling, Francis J P; Jethwa, Preeti H

    2015-12-30

    The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

    Science.gov (United States)

    Nagashima, Shunta; Maruyama, Junichi; Kawano, Shodai; Iwasa, Hiroaki; Nakagawa, Kentaro; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Nishina, Hiroshi; Hata, Yutaka

    2016-06-01

    Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  5. A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria.

    Science.gov (United States)

    Karunakaran, R; Mauchline, T H; Hosie, A H F; Poole, P S

    2005-10-01

    A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

  6. Cone penetrometer moisture probe acceptance test report

    Energy Technology Data Exchange (ETDEWEB)

    Barnes, G.A.

    1996-04-23

    This Acceptance Test Report (ATR) documents the results of WHC-SD-WM-ATP-146 (Prototype Cone Penetrometer Moisture Probe Acceptance Test Procedure) and WHC-SD-WM-ATP-145 (Cone Penetrometer Moisture Probe Acceptance Test Procedure). The master copy of WHC-SD-WM-ATP-145 can be found in Appendix A and the master copy of WHC-SD-WM-ATP-146 can be found in Appendix B. Also included with this report is a matrix showing design criteria of the cone penetrometer moisture probe and the verification method used (Appendix C).

  7. Advanced ultrasound probes for medical imaging

    Science.gov (United States)

    Wildes, Douglas G.; Smith, L. Scott

    2012-05-01

    New medical ultrasound probe architectures and materials build upon established 1D phased array technology and provide improved imaging performance and clinical value. Technologies reviewed include 1.25D and 1.5D arrays for elevation slice thickness control; electro-mechanical and 2D array probes for real-time 3D imaging; catheter probes for imaging during minimally-invasive procedures; single-crystal piezoelectric materials for greater frequency bandwidth; and cMUT arrays using silicon MEMS in place of piezo materials.

  8. Electrostatic Probe with Shielded Probe Insulator Tube for Low Disturbing Plasma Measurements in Hall Thrusters

    International Nuclear Information System (INIS)

    Staack, D.; Raitses, Y.; Fisch, N.J.

    2003-01-01

    Electrostatic probes are widely used to measure spatial plasma parameters of the quasi-neutral plasma in Hall thrusters and similar ExB electric discharge devices. Significant perturbations of the plasma, induced by such probes, can mask the actual physics involved in operation of these devices. In Hall thrusters, probe-induced perturbations can produce changes in the discharge current and plasma parameters on the order of their steady state values. These perturbations are explored by varying the material, penetration distance, and residence time of various probe designs. A possible cause of these perturbations appears to be the secondary electron emission, induced by energetic plasma electrons, from insulator ceramic tubes in which the probe wire is inserted. A new probe in which a low secondary electron emission material, such as metal, shields the probe ceramic tube, is shown to function without producing such large perturbations. A segmentation of this shield further prevents probe -induced perturbations, by not shortening the plasma through the conductive shield. In a set of experiments with a segmented shield probe, the thruster was operated in the input power range of 500-2.5 kW and discharge voltages of 200-500 V, while the probe-induced perturbations of the discharge current were below 4% of its steady state value in the region in which 90% of the voltage drop takes place

  9. Combination therapy and evaluation of therapeutic effect in hepatocellular carcinoma cell using triple reporter genes; containing for NIS, HSV1-sr39tk and GFP

    Energy Technology Data Exchange (ETDEWEB)

    Lee, You La; Lee, Yong Jin; Ahn, Sohn Joo; Ahn, Byeong Cheol; Lee, Sang Woo; Yoo, Jeong Soo; Lee, Jae Tae [Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To identify therapeutic effect after combine Sodium Iodine Symporter (NIS) and Mutant Herpes-simplex virus type 1 sr39tk (HSV1-sr39tk) expression in hepatocellular carcinoma cell, we transfected triple gene and investigated the properties of these gene ability in hepatocellular carcinoma cell line. After making vector with gene encoding a fusion protein comprised of HSV1-sr39tk and green florescence protein (GFP), to make triple reporter genes NIS gene was further fused to the vector using IRES vector. The vector expressing triple reporter gene was transfected to the Huh-7 cell line using liposome. Functions of hNIS and HSV1-sr39tk expression were confirmed by radio iodine uptake with and without perchlorate and [3H]-penciclovir (3-H PCV) uptake, respectively. To evaluate therapeutic effect in vitro, GCV and I-131 was treated in Huh-7/NTG cell and dual therapy performed. An animal imaging acquired using Optix and microPET in vivo. I-125 uptake was increased up to 100-fold compare to that of non-transfected cells. The transfected cell accumulated H-3 PCV up to 53 times higher at 2 hour than that of non-transfected cells. With fluorescence microscopy, green fluorescence was detected in the transfected cell. In cytotoxic studies, the cell viability of Huh-7/NTG cell was decreased to 41 % of control cell at 10ug/ml GCV concentrations. The survival rate of the Huh-7/NTG cell treated with I-131 decreased up to 16%. In I-131 and GCV dual therapy, Huh-7/NTG cell survival rate decreased up to 4%. In animal studies, Huh-7/NTG tumors showed higher uptake of 18F-FHBG and I-124 than Huh-7 tumors. GFP signal is also higher in Huh-7/NTG tumor than control. We successfully constructed a vector with delivery two therapeutic genes and one reporter gene and transfected the vector to a Huh-7 cell. The hepatocellular carcinoma cell transfected with the vector can be treated with GCV and I-131. The effect of dual gene therapy could be easily assessed by the optical reporter gene imaging.

  10. Full information acquisition in scanning probe microscopy and spectroscopy

    Science.gov (United States)

    Jesse, Stephen; Belianinov, Alex; Kalinin, Sergei V.; Somnath, Suhas

    2017-04-04

    Apparatus and methods are described for scanning probe microscopy and spectroscopy based on acquisition of full probe response. The full probe response contains valuable information about the probe-sample interaction that is lost in traditional scanning probe microscopy and spectroscopy methods. The full probe response is analyzed post data acquisition using fast Fourier transform and adaptive filtering, as well as multivariate analysis. The full response data is further compressed to retain only statistically significant components before being permanently stored.

  11. Peptide nucleic acid probes with charged photocleavable mass markers

    Science.gov (United States)

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  12. 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.

  13. Intrauterine photoacoustic and ultrasound imaging probe.

    Science.gov (United States)

    Miranda, Christopher; Barkley, Joel; Smith, Barbara

    2018-04-01

    Intrauterine photoacoustic and ultrasound imaging are probe-based imaging modalities with translational potential for use in detecting endometrial diseases. This deep-tissue imaging probe design allows for the retrofitting of commercially available endometrial sampling curettes. The imaging probe presented here has a 2.92-mm diameter and approximate length of 26 cm, which allows for entry into the human endometrial cavity, making it possible to use photoacoustic imaging and high-resolution ultrasound to characterize the uterus. We demonstrate the imaging probes' ability to provide structural information of an excised pig uterus using ultrasound imaging and detect photoacoustic signals at a radial depth of 1 cm. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  14. Pneumatic Proboscis Heat Flow Probe Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The heat flow probe directly answers requirements in the topic: S1.11 Lunar Science Instruments and Technology: "Geophysical Measurements: Systems, subsystems, and...

  15. 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

  16. Flow cytometry, fluorescent probes, and flashing bacteria

    NARCIS (Netherlands)

    Bunthof, C.J.

    2002-01-01


    Key words: fluorescent probes, flow cytometry, CSLM, viability, survival, microbial physiology, lactic acid bacteria, Lactococcus lactis , Lactobacillus plantarum , cheese, milk,

  17. Software Risk Identification for Interplanetary Probes

    Science.gov (United States)

    Dougherty, Robert J.; Papadopoulos, Periklis E.

    2005-01-01

    The need for a systematic and effective software risk identification methodology is critical for interplanetary probes that are using increasingly complex and critical software. Several probe failures are examined that suggest more attention and resources need to be dedicated to identifying software risks. The direct causes of these failures can often be traced to systemic problems in all phases of the software engineering process. These failures have lead to the development of a practical methodology to identify risks for interplanetary probes. The proposed methodology is based upon the tailoring of the Software Engineering Institute's (SEI) method of taxonomy-based risk identification. The use of this methodology will ensure a more consistent and complete identification of software risks in these probes.

  18. Tools for Ultraspecific Probe/Primer Design

    National Research Council Canada - National Science Library

    Fofanov, Yurly

    2006-01-01

    .... Our approach will deliver DNA probes and PCR primers that have an unprecedentedly low probability of false positives or confusion by environmental background, and which resist evasion by threat agent engineering...

  19. Scanning microscopic four-point conductivity probes

    DEFF Research Database (Denmark)

    Petersen, Christian Leth; Hansen, Torben Mikael; Bøggild, Peter

    2002-01-01

    A method for fabricating microscopic four-point probes is presented. The method uses silicon-based microfabrication technology involving only two patterning steps. The last step in the fabrication process is an unmasked deposition of the conducting probe material, and it is thus possible to select...... the conducting material either for a silicon wafer or a single probe unit. Using shadow masking photolithography an electrode spacing (pitch) down to 1.1 mum was obtained, with cantilever separation down to 200 run. Characterisation measurements have shown the microscopic probes to be mechanically very flexible...... and robust. Repeated conductivity measurements on polythiophene films in the same surface area are reproduced within an accuracy of 3%. Automated nanoresolution position control allows scanning across millimetre sized areas, in order to create high spatial resolution maps of the in-plane conductivity....

  20. Modulated microwave microscopy and probes used therewith

    Science.gov (United States)

    Lai, Keji; Kelly, Michael; Shen, Zhi-Xun

    2012-09-11

    A microwave microscope including a probe tip electrode vertically positionable over a sample and projecting downwardly from the end of a cantilever. A transmission line connecting the tip electrode to the electronic control system extends along the cantilever and is separated from a ground plane at the bottom of the cantilever by a dielectric layer. The probe tip may be vertically tapped near or at the sample surface at a low frequency and the microwave signal reflected from the tip/sample interaction is demodulated at the low frequency. Alternatively, a low-frequency electrical signal is also a non-linear electrical element associated with the probe tip to non-linearly interact with the applied microwave signal and the reflected non-linear microwave signal is detected at the low frequency. The non-linear element may be semiconductor junction formed near the apex of the probe tip or be an FET formed at the base of a semiconducting tip.

  1. Surface sampling concentration and reaction probe

    Science.gov (United States)

    Van Berkel, Gary J; Elnaggar, Mariam S

    2013-07-16

    A method of analyzing a chemical composition of a specimen is described. The method can include providing a probe comprising an outer capillary tube and an inner capillary tube disposed co-axially within the outer capillary tube, where the inner and outer capillary tubes define a solvent capillary and a sampling capillary in fluid communication with one another at a distal end of the probe; contacting a target site on a surface of a specimen with a solvent in fluid communication with the probe; maintaining a plug volume proximate a solvent-specimen interface, wherein the plug volume is in fluid communication with the probe; draining plug sampling fluid from the plug volume through the sampling capillary; and analyzing a chemical composition of the plug sampling fluid with an analytical instrument. A system for performing the method is also described.

  2. Calibration models for high enthalpy calorimetric probes.

    Science.gov (United States)

    Kannel, A

    1978-07-01

    The accuracy of gas-aspirated liquid-cooled calorimetric probes used for measuring the enthalpy of high-temperature gas streams is studied. The error in the differential temperature measurements caused by internal and external heat transfer interactions is considered and quantified by mathematical models. The analysis suggests calibration methods for the evaluation of dimensionless heat transfer parameters in the models, which then can give a more accurate value for the enthalpy of the sample. Calibration models for four types of calorimeters are applied to results from the literature and from our own experiments: a circular slit calorimeter developed by the author, single-cooling jacket probe, double-cooling jacket probe, and split-flow cooling jacket probe. The results show that the models are useful for describing and correcting the temperature measurements.

  3. Multipartite entanglement detection with nonsymmetric probing

    DEFF Research Database (Denmark)

    Dellantonio, Luca; Das, Sumanta; Appel, Jürgen

    2017-01-01

    We show that spin-squeezing criteria commonly used for entanglement detection can be erroneous if the probe is not symmetric. We then derive a lower bound on squeezing for separable states in spin systems probed asymmetrically. Using this we further develop a procedure that allows us to verify th...... the degree of entanglement of a quantum state in the spin system. Finally, we apply our method for entanglement verification to existing experimental data, and use it to prove the existence of tripartite entanglement in a spin-squeezed atomic ensemble.......We show that spin-squeezing criteria commonly used for entanglement detection can be erroneous if the probe is not symmetric. We then derive a lower bound on squeezing for separable states in spin systems probed asymmetrically. Using this we further develop a procedure that allows us to verify...

  4. Probing plasmonic nanostructures by photons and electrons

    DEFF Research Database (Denmark)

    Kneipp, Katrin; Kneipp, Harald; Kneipp, Janina

    2015-01-01

    We discuss recent developments for studying plasmonic metal nanostructures. Exploiting photons and electrons opens up new capabilities to probe the complete plasmon spectrum including bright and dark modes and related local optical fields at subnanometer spatial resolution. This comprehensive...

  5. Urethral alarm probe for permanent prostate implants

    International Nuclear Information System (INIS)

    Cutajar, D.; Lerch, M.; Takacs, G.

    2008-01-01

    We have developed a urethral dosimetry system for real time dose verification along the urethra during permanent implant prostate brachytherapy. The urethral alarm uses 'spectroscopic dosimetry' to calculate the dose rate along the urethra in real time. The application of spectroscopic dosimetry for the urethral alarm probe was verified using Monte Carlo calculations. In phantom depth dose measurements as well as isotropy measurements were performed to verify the usefulness of the urethra alarm probe as an in vivo real time dosimeter. (author)

  6. Reactive Chemical Probes: Beyond the Kinase Cysteinome.

    Science.gov (United States)

    Jones, Lyn H

    2018-04-12

    The reaction of small molecule chemical probes with proteins has been harnessed to develop covalent inhibitor drugs and protein profiling technologies. This Essay discusses some of the recent enhancements to the chemical biology toolkit that are enabling the study of previously unchartered areas of chemoproteomic space. An analysis of the kinome is used to illustrate the potential for these approaches to pursue new targets using reactive chemical probes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Plasma diagnostics by means of electric probes

    International Nuclear Information System (INIS)

    Colunga S, S.

    1991-04-01

    In this work a summary of the classical theoretical models to interpret the characteristic curve of a Langmuir electric probe placed in a plasma without magnetic field and with the one is made. The methodology for the electron temperature calculation and the density of the plasma in both cases is given, starting from the characteristic curve of the probe, as well as the approaches for the correct application of this diagnostic method of the plasma. (Author)

  8. Accuracy of micro four-point probe measurements on inhomogeneous samples: A probe spacing dependence study

    DEFF Research Database (Denmark)

    Wang, Fei; Petersen, Dirch Hjorth; Østerberg, Frederik Westergaard

    2009-01-01

    In this paper, we discuss a probe spacing dependence study in order to estimate the accuracy of micro four-point probe measurements on inhomogeneous samples. Based on sensitivity calculations, both sheet resistance and Hall effect measurements are studied for samples (e.g. laser annealed samples...... the probe spacing is smaller than 1/40 of the variation wavelength, micro four-point probes can provide an accurate record of local properties with less than 1% measurement error. All the calculations agree well with previous experimental results.......) with periodic variations of sheet resistance, sheet carrier density, and carrier mobility. With a variation wavelength of ¿, probe spacings from 0.0012 to 1002 have been applied to characterize the local variations. The calculations show that the measurement error is highly dependent on the probe spacing. When...

  9. Characterization of coating probe with Ti-DLC for electrical scanning probe microscope

    International Nuclear Information System (INIS)

    Shia Xiaolei; Guo Liqiu; Bai Yang; Qiao Lijie

    2011-01-01

    In electrical scanning probe microscope (ESPM) applications, the wear and conductivity of the probe are undoubtedly serious concerns since they affect the integrity of the measurements. This study investigates the characterization of Ti doped diamond-like-carbon (DLC) as coating material on a silicon cantilever for ESPM. We deposited a layer of Ti-DLC thin film on the surface of Si cantilever by magnetron sputtering. The morphology and composition of the Ti-DLC films were characterized by scanning electron microscopy and Raman spectroscopy, respectively. We also compared the wear resistance, electric conductivity and scanning image quality of the Ti-DLC-coated probes with those of commercially available conductive probes. The results showed that the electric conductivity and the scanning image quality of the Ti-DLC-coated probes were the same as the commercial conductive probes, while the wear resistance and service life was significantly better.

  10. Characterization of coating probe with Ti-DLC for electrical scanning probe microscope

    Energy Technology Data Exchange (ETDEWEB)

    Shia Xiaolei [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (Ministry of Education), University of Science and Technology Beijing, Beijing 100083 (China); Guo Liqiu, E-mail: glq@mater.ustb.edu.cn [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (Ministry of Education), University of Science and Technology Beijing, Beijing 100083 (China); Bai Yang; Qiao Lijie [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (Ministry of Education), University of Science and Technology Beijing, Beijing 100083 (China)

    2011-06-01

    In electrical scanning probe microscope (ESPM) applications, the wear and conductivity of the probe are undoubtedly serious concerns since they affect the integrity of the measurements. This study investigates the characterization of Ti doped diamond-like-carbon (DLC) as coating material on a silicon cantilever for ESPM. We deposited a layer of Ti-DLC thin film on the surface of Si cantilever by magnetron sputtering. The morphology and composition of the Ti-DLC films were characterized by scanning electron microscopy and Raman spectroscopy, respectively. We also compared the wear resistance, electric conductivity and scanning image quality of the Ti-DLC-coated probes with those of commercially available conductive probes. The results showed that the electric conductivity and the scanning image quality of the Ti-DLC-coated probes were the same as the commercial conductive probes, while the wear resistance and service life was significantly better.

  11. Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin.

    Science.gov (United States)

    Sano, Toshio; Higaki, Takumi; Oda, Yoshihisa; Hayashi, Tomomi; Hasezawa, Seiichiro

    2005-11-01

    The actin cytoskeleton of higher plants plays an essential role in plant morphogenesis and in maintaining various cellular activities. In this study we established a tobacco BY-2 cell line, stably transformed with a GFP-fimbrin actin-binding domain (ABD) 2 construct, that allows visualization of actin microfilaments (MFs) in living cells. Using this cell line, designated BY-GF11, we performed time-sequential observations of MF dynamics during cell-cycle progression. Detailed analyses revealed the appearance of a broad MF band in the late G2 phase that separated to form a structure corresponding to the so-called actin-depleted zone (ADZ) in mitosis. In BY-GF11, the MF structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ. Measurements of fluorescent intensities of the cell cortex indicated an MF distribution that resembled two peaks, and we therefore named the structure MF 'twin peaks' (MFTP). The cell plate formed exactly within the valley between the MFTP at cytokinesis, and this cell-plate guidance was distorted by disruption of the MFTP by an inhibitor of actin polymerization. These results suggest that the MFTP originates from the broad MF band in the G2 phase and functions as a marker of cytokinesis.

  12. FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons

    Directory of Open Access Journals (Sweden)

    Carles eBosch

    2015-05-01

    Full Text Available The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs in mice. 3D reconstruction of spines in GCs aged 3–4 and 8–9 weeks revealed two different stages of spine development and unexpected features of synapse formation, including vacant and branched spines and presynaptic terminals establishing synapses with up to 10 spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.

  13. FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons.

    Science.gov (United States)

    Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2015-01-01

    The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3-4 and 8-9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.

  14. Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

    Science.gov (United States)

    George, Steve; Jauhar, Altamash M; Mackenzie, Jennifer; Kieβlich, Sascha; Aucoin, Marc G

    2015-09-01

    The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system. © 2015 Wiley Periodicals, Inc.

  15. Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

    Science.gov (United States)

    König, Alexander; Glebe, Dieter

    2017-01-01

    To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

  16. Preventing probe induced topography correlated artifacts in Kelvin Probe Force Microscopy.

    Science.gov (United States)

    Polak, Leo; Wijngaarden, Rinke J

    2016-12-01

    Kelvin Probe Force Microscopy (KPFM) on samples with rough surface topography can be hindered by topography correlated artifacts. We show that, with the proper experimental configuration and using homogeneously metal coated probes, we are able to obtain amplitude modulation (AM) KPFM results on a gold coated sample with rough topography that are free from such artifacts. By inducing tip inhomogeneity through contact with the sample, clear potential variations appear in the KPFM image, which correlate with the surface topography and, thus, are probe induced artifacts. We find that switching to frequency modulation (FM) KPFM with such altered probes does not remove these artifacts. We also find that the induced tip inhomogeneity causes a lift height dependence of the KPFM measurement, which can therefore be used as a check for the presence of probe induced topography correlated artifacts. We attribute the observed effects to a work function difference between the tip and the rest of the probe and describe a model for such inhomogeneous probes that predicts lift height dependence and topography correlated artifacts for both AM and FM-KPFM methods. This work demonstrates that using a probe with a homogeneous work function and preventing tip changes is essential for KPFM on non-flat samples. From the three investigated probe coatings, PtIr, Au and TiN, the latter appears to be the most suitable, because of its better resistance against coating damage. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Bunker probe: A plasma potential probe almost insensitive to its orientation with the magnetic field.

    Science.gov (United States)

    Costea, S; Fonda, B; Kovačič, J; Gyergyek, T; Schneider, B S; Schrittwieser, R; Ionita, C

    2016-05-01

    Due to their ability to suppress a large part of the electron current and thus measuring directly the plasma potential, ion sensitive probes have begun to be widely tested and used in fusion devices. For these probes to work, almost perfect alignment with the total magnetic field is necessary. This condition cannot always be fulfilled due to the curvature of magnetic fields, complex magnetic structure, or magnetic field reconnection. In this perspective, we have developed a plasma potential probe (named Bunker probe) based on the principle of the ion sensitive probe but almost insensitive to its orientation with the total magnetic field. Therefore it can be used to measure the plasma potential inside fusion devices, especially in regions with complex magnetic field topology. Experimental results are presented and compared with Ball-Pen probe measurements taken under identical conditions. We have observed that the floating potential of the Bunker probe is indeed little affected by its orientation with the magnetic field for angles ranging from 90° to 30°, in contrast to the Ball-Pen probe whose floating potential decreases towards that of a Langmuir probe if not properly aligned with the magnetic field.

  18. Bunker probe: A plasma potential probe almost insensitive to its orientation with the magnetic field

    International Nuclear Information System (INIS)

    Costea, S.; Schneider, B. S.; Schrittwieser, R.; Ionita, C.; Fonda, B.; Kovačič, J.; Gyergyek, T.

    2016-01-01

    Due to their ability to suppress a large part of the electron current and thus measuring directly the plasma potential, ion sensitive probes have begun to be widely tested and used in fusion devices. For these probes to work, almost perfect alignment with the total magnetic field is necessary. This condition cannot always be fulfilled due to the curvature of magnetic fields, complex magnetic structure, or magnetic field reconnection. In this perspective, we have developed a plasma potential probe (named Bunker probe) based on the principle of the ion sensitive probe but almost insensitive to its orientation with the total magnetic field. Therefore it can be used to measure the plasma potential inside fusion devices, especially in regions with complex magnetic field topology. Experimental results are presented and compared with Ball-Pen probe measurements taken under identical conditions. We have observed that the floating potential of the Bunker probe is indeed little affected by its orientation with the magnetic field for angles ranging from 90° to 30°, in contrast to the Ball-Pen probe whose floating potential decreases towards that of a Langmuir probe if not properly aligned with the magnetic field.

  19. Detection of gfp expression from gfp-labelled bacteria spot ...

    African Journals Online (AJOL)

    SERVER

    Joyce Prisca NJOLOMA1*, Moriya OOTA2, Yuichi SAEKI1 and Shoichiro AKAO1. 1Department of Biochemistry and Applied Bioscience, Faculty of Agriculture, University of Miyazaki, Miyazaki, 889-2192. Japan. 2Okinawa Prefecture Agricultural Experimental station, Nishizato, Hirara. 906-0012, Japan. Accepted 27 October ...

  20. Soil Properties from Low-Velocity Probe Penetration

    Directory of Open Access Journals (Sweden)

    Jerome B. Johnson

    2008-01-01

    Full Text Available A physical model of low-velocity probe penetration is developed to characterize soil by type, strength, maximum compaction, and initial density using Newton's second law to describe the processes controlling probe momentum loss. The probe loses momentum by causing soil failure (strength, accelerating and compacting soil around the probe (inertia, and through frictional sliding at the probe/soil interface (friction. Probe geometry, mass, and impact velocity influences are incorporated into the model. Model predictions of probe deceleration history and depth of penetration agree well with experiments, without the need for free variables or complex numerical simulations.

  1. Probing Zeolite Crystal Architecture and Structural Imperfections using Differently Sized Fluorescent Organic Probe Molecules

    NARCIS (Netherlands)

    Hendriks, Frank C.; Schmidt, Joel E.; Rombouts, Jeroen A.; Lammertsma, Koop; Bruijnincx, Pieter C.A.; Weckhuysen, Bert M.

    2017-01-01

    A micro-spectroscopic method has been developed to probe the accessibility of zeolite crystals using a series of fluorescent 4-(4-diethylaminostyryl)-1-methylpyridinium iodide (DAMPI) probes of increasing molecular size. Staining large zeolite crystals with MFI (ZSM-5) topology and subsequent

  2. Preventing probe induced topography correlated artifacts in Kelvin Probe Force Microscopy

    NARCIS (Netherlands)

    Polak, L.; Wijngaarden, Rinke J.

    2016-01-01

    Kelvin Probe Force Microscopy (KPFM) on samples with rough surface topography can be hindered by topography correlated artifacts. We show that, with the proper experimental configuration and using homogeneously metal coated probes, we are able to obtain amplitude modulation (AM) KPFM results on a

  3. In-vitro accuracy and reproducibility evaluation of probing depth measurements of selected periodontal probes

    Directory of Open Access Journals (Sweden)

    K.N. Al Shayeb

    2014-01-01

    Conclusion: Depth measurements with the Chapple UB-CF-15 probe were more accurate and reproducible compared to measurements with the Vivacare TPS and Williams 14 W probes. This in vitro model may be useful for intra-examiner calibration or clinician training prior to the clinical evaluation of patients or in longitudinal studies involving periodontal evaluation.

  4. Chromosome-specific DNA Repeat Probes

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  5. Donated chemical probes for open science.

    Science.gov (United States)

    Müller, Susanne; Ackloo, Suzanne; Arrowsmith, Cheryl H; Bauser, Marcus; Baryza, Jeremy L; Blagg, Julian; Böttcher, Jark; Bountra, Chas; Brown, Peter J; Bunnage, Mark E; Carter, Adrian J; Damerell, David; Dötsch, Volker; Drewry, David H; Edwards, Aled M; Edwards, James; Elkins, Jon M; Fischer, Christian; Frye, Stephen V; Gollner, Andreas; Grimshaw, Charles E; IJzerman, Adriaan; Hanke, Thomas; Hartung, Ingo V; Hitchcock, Steve; Howe, Trevor; Hughes, Terry V; Laufer, Stefan; Li, Volkhart Mj; Liras, Spiros; Marsden, Brian D; Matsui, Hisanori; Mathias, John; O'Hagan, Ronan C; Owen, Dafydd R; Pande, Vineet; Rauh, Daniel; Rosenberg, Saul H; Roth, Bryan L; Schneider, Natalie S; Scholten, Cora; Singh Saikatendu, Kumar; Simeonov, Anton; Takizawa, Masayuki; Tse, Chris; Thompson, Paul R; Treiber, Daniel K; Viana, Amélia Yi; Wells, Carrow I; Willson, Timothy M; Zuercher, William J; Knapp, Stefan; Mueller-Fahrnow, Anke

    2018-04-20

    Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de">https://openscienceprobes.sgc-frankfurt.dehttps://openscienceprobes.sgc-frankfurt.de/">/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project. © 2018, Müller et al.

  6. Configurations of polymers attached to probes

    Science.gov (United States)

    Bubis, Roy; Kantor, Yacov; Kardar, Mehran

    2009-11-01

    We study polymers attached to spherical (circular) or paraboloidal (parabolic) probes in three (two) dimensions. Both self-avoiding and random walks are examined numerically. The behavior of a polymer of size R0 attached to the tip of a probe with radius of curvature R, differs qualitatively for large and small values of the ratio s=R0/R. We demonstrate that the scaled compliance (inverse force constant) S/R02, and scaled mean position of the polymer end-point langx⊥rang/R can be expressed as a function of s. Scaled compliance is anisotropic, and quite large in the direction parallel to the surface when R0~R. The exponent γ, characterizing the number of polymer configurations, crosses over from a value of γ1 - characteristic of a planar boundary - at small s to one reflecting the overall shape of the probe at large s. For a spherical probe the crossover is to an unencumbered polymer, while for a parabolic probe we cannot rule out a new exponent.

  7. Luminescent probes and sensors for temperature.

    Science.gov (United States)

    Wang, Xu-dong; Wolfbeis, Otto S; Meier, Robert J

    2013-10-07

    Temperature (T) is probably the most fundamental parameter in all kinds of science. Respective sensors are widely used in daily life. Besides conventional thermometers, optical sensors are considered to be attractive alternatives for sensing and on-line monitoring of T. This Review article focuses on all kinds of luminescent probes and sensors for measurement of T, and summarizes the recent progress in their design and application formats. The introduction covers the importance of optical probes for T, the origin of their T-dependent spectra, and the various detection modes. This is followed by a survey on (a) molecular probes, (b) nanomaterials, and (c) bulk materials for sensing T. This section will be completed by a discussion of (d) polymeric matrices for immobilizing T-sensitive probes and (e) an overview of the various application formats of T-sensors. The review ends with a discussion on the prospects, challenges, and new directions in the design of optical T-sensitive probes and sensors.

  8. Outsourced Probe Data Effectiveness on Signalized Arterials

    Energy Technology Data Exchange (ETDEWEB)

    Young, Stanley E [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Sharifi, Elham [University of Maryland; Eshragh, Sepideh [University of Maryland; Hamedi, Masoud [University of Maryland; Juster, Reuben M. [University of Maryland; Kaushik, Kartik [University of Maryland

    2017-07-31

    This paper presents results of an I-95 Corridor Coalition sponsored project to assess the ability of outsourced vehicle probe data to provide accurate travel time on signalized roadways for the purposes of real-time operations as well as performance measures. The quality of outsourced probe data on freeways has led many departments of transportation to consider such data for arterial performance monitoring. From April 2013 through June of 2014, the University of Maryland Center for Advanced Transportation Technology gathered travel times from several arterial corridors within the mid-Atlantic region using Bluetooth traffic monitoring (BTM) equipment, and compared these travel times with the data reported to the I95 Vehicle Probe Project (VPP) from an outsourced probe data vendor. The analysis consisted of several methodologies: (1) a traditional analysis that used precision and bias speed metrics; (2) a slowdown analysis that quantified the percentage of significant traffic disruptions accurately captured in the VPP data; (3) a sampled distribution method that uses overlay methods to enhance and analyze recurring congestion patterns. (4) Last, the BTM and VPP data from each 24-hour period of data collection were reviewed by the research team to assess the extent to which VPP captured the nature of the traffic flow. Based on the analysis, probe data is recommended only on arterial roadways with signal densities (measured in signals per mile) up to one, and it should be tested and used with caution for signal densities between one and two, and is not recommended when signal density exceeds two.

  9. A fluorescent glycolipid-binding peptide probe traces cholesterol dependent microdomain-derived trafficking pathways.

    Directory of Open Access Journals (Sweden)

    Steffen Steinert

    Full Text Available BACKGROUND: The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Abeta, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB. Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol

  10. Cellular organization and spectral diversity of GFP-like proteins in live coral cells studied by single and multiphoton imaging and microspectroscopy

    Science.gov (United States)

    Salih, Anya; Cox, Guy C.; Larkum, Anthony W.

    2003-07-01

    Tissues of many marine invertebrates of class Anthozoa contain intensely fluorescent or brightly coloured pigments. These pigments belong to a family of photoactive proteins closely related to Green Fluorescent Protein (GFP), and their emissions range from blue to red wavelengths. The great diversity of these pigments has only recently been realised. To investigate the role of these proteins in corals, we have performed an in vivo fluorescent pigment (FP) spectral and cellular distribution analyses in live coral cells using single and multi-photon laser scanning imaging and microspectroscopy. These analyses revealed that even single colour corals contain spectroscopically heterogeneous pigment mixtures, with 2-5 major colour types in the same area of tissue. They were typically arranged in step-wise light emission energy gradients (e.g. blue, green, yellow, red). The successive overlapping emission-excitation spectral profiles of differently coloured FPs suggested that they were suited for sequential energy coupling. Traces of red FPs (emission = 570-660 nm) were present, even in non-red corals. We confirmed that radiative energy transfer could occur between separate granules of blue and green FPs and that energy transfer was inversely proportional to the square of the distance between them. Multi-photon micro-spectrofluorometric analysis gave significantly improved spectral resolution by restricting FP excitation to a single point in the focal plane of the sample. Pigment heterogeneity at small scales within granules suggested that fluorescence resonance energy transfer (FRET) might be occurring, and we confirmed that this was the case. Thus, energy transfer can take place both radiatively and by FRET, probably functioning in photoprotection by dissipation of excessive solar radiation.

  11. Antibacterial activity and mechanism of Ag-ZnO nanocomposite on S. aureus and GFP-expressing antibiotic resistant E. coli.

    Science.gov (United States)

    Matai, Ishita; Sachdev, Abhay; Dubey, Poornima; Kumar, S Uday; Bhushan, Bharat; Gopinath, P

    2014-03-01

    Emergence of multi-resistant organisms (MROs) leads to ineffective treatment with the currently available medications which pose a great threat to public health and food technology sectors. In this regard, there is an urgent need to strengthen the present therapies or to look over for other potential alternatives like use of "metal nanocomposites". Thus, the present study focuses on synthesis of silver-zinc oxide (Ag-ZnO) nanocomposites which will have a broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria. Ag-ZnO nanocomposites of varied molar ratios were synthesized by simple microwave assisted reactions in the absence of surfactants. The crystalline behavior, composition and morphological analysis of the prepared powders were evaluated by X-ray diffraction, infrared spectroscopy, field emission scanning electron microscopy (FE-SEM) and atomic absorption spectrophotometry (AAS). Particle size measurements were carried out by transmission electron microscopy (TEM). Staphylococcus aureus and recombinant green fluorescent protein (GFP) expressing antibiotic resistant Escherichia coli were selected as Gram-positive and Gram-negative model systems respectively and the bactericidal activity of Ag-ZnO nanocomposite was studied. The minimum inhibitory concentration (MIC) and minimum killing concentration (MKC) of the nanocomposite against the model systems were determined by visual turbidity analysis and optical density analysis. Qualitative and quantitative assessments of its antibacterial effects were performed by fluorescent microscopy, fluorescent spectroscopy and Gram staining measurements. Changes in cellular morphology were examined by atomic force microscopy (AFM), FE-SEM and TEM. Finally, on the basis of the present investigation and previously published reports, a plausible antibacterial mechanism of Ag-ZnO nanocomposites was proposed. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Pablo Tsukayama

    Full Text Available In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL. The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V. braziliensis, L. (V. panamensis, L. (V. guyanensis, L. (V. peruviana and L. (V. lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST. In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

  13. Development of FRET-Based Assays in the Far-Red Using CdTe Quantum Dots

    Directory of Open Access Journals (Sweden)

    E. Z. Chong

    2007-01-01

    Full Text Available Colloidal quantum dots (QDs are now commercially available in a biofunctionalized form, and Förster resonance energy transfer (FRET between bioconjugated dots and fluorophores within the visible range has been observed. We are particularly interested in the far-red region, as from a biological perspective there are benefits in pushing to ∼700 nm to minimize optical absorption (ABS within tissue and to avoid cell autofluorescence. We report on FRET between streptavidin- (STV- conjugated CdTe quantum dots, Qdot705-STV, with biotinylated DY731-Bio fluorophores in a donor-acceptor assay. We also highlight the changes in DY731-Bio absorptivity during the streptavidin-biotin binding process which can be attributed to the structural reorientation. For fluorescence beyond 700 nm, different alloy compositions are required for the QD core and these changes directly affect the fluorescence decay dynamics producing a marked biexponential decay with a long-lifetime component in excess of 100 nanoseconds. We compare the influence of the two QD relaxation routes upon FRET dynamics in the presence of DY731-Bio.

  14. Single organelle fret-based analysis of intracellular calcium: different effects of presinilis carrying Familial Alzheimer's Disease mutations

    OpenAIRE

    wong, andrea kuan cie

    2014-01-01

    Calcium (Ca2+) is one of the major intracellular messengers that impacts nearly every aspect of cell life. In particular, it plays essential roles in neuronal development, synaptic transmission and plasticity, as well as in the regulation of metabolic pathways and cell fate decisions. The first goal of my work was to study more in details the Golgi apparatus (GA), as an intracellular Ca2+ store. The Golgi complex may store up to 5% of the total cellular Ca2+ at higher concentrations an...

  15. FRET-based glucose imaging identifies glucose signalling in response to biotic and abiotic stresses in rice roots.

    Science.gov (United States)

    Zhu, Qingdong; Wang, Li; Dong, Qianli; Chang, Shu; Wen, Kexin; Jia, Shenghua; Chu, Zhilin; Wang, Hanmeng; Gao, Ping; Zhao, Heping; Han, Shengcheng; Wang, Yingdian

    2017-08-01

    Glucose is the primary energy provider and the most important sugar-signalling molecule, regulating metabolites and modulating gene expression from unicellular yeast to multicellular plants and animals. Therefore, monitoring intracellular glucose levels temporally and spatially in living cells is an essential step for decoding the glucose signalling in response to biotic and abiotic stresses. In this study, the genetically encoded FRET (Förster resonance energy transfer) nanosensors, FLIPglu-2μ∆13 and FLIPglu-600μΔ13, were used to measure cytosolic glucose dynamics in rice plants. First, we found that the FRET signal decreased in response to external glucose in a concentration-dependent manner. The glucose concentration at which the cytosolic level corresponded to the K 0.5 value for FLIPglu-2μΔ13 was approximately 10.05μM, and that for FLIPglu-600μΔ13 was 0.9mM, respectively. The substrate selectivity of nanosensors for glucose and its analogues is D-Glucose>2-deoxyglucose>3-O-methylglucose>L-Glucose. We further showed that the biotic elicitors (flg22 and chitin) and the abiotic elicitors (osmotic stress, salinity and extreme temperature) induce the intracellular glucose increases in the detached root segments of transgenic rice containing FLIPglu-2μΔ13 in a stimulus-specific manner, but not in FLIPglu-600μΔ13 transgenic lines. These results demonstrated that FRET nanosensors can be used to detect increases in intracellular glucose within the physiological range of 0.2-20μM in response to various stimuli in transgenic rice root cells, which indicated that intracellular glucose may act as a potential secondary messenger to connect extracellular stimuli with cellular physiological responses in plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. Gravity and On-Shell Probe Actions

    CERN Document Server

    Ferrari, Frank

    2016-08-08

    In any gravitational theory and in a wide class of background space-times, we argue that there exists a simple, yet profound, relation between the on-shell Euclidean gravitational action and the on-shell Euclidean action of probes. The probes can be, for instance, charged particles or branes. The relation is tightly related to the thermodynamic nature of gravity. We provide precise checks of the relation in several examples, which include both asymptotically flat and asymptotically AdS space-times, with particle, D-brane and M-brane probes. Perfect consistency is found in all cases, including in a highly non-trivial example including \\alpha'-corrections.

  17. Nanobits - exchangable and customisable scanning probe tips

    DEFF Research Database (Denmark)

    Yildiz, Izzet

    Invention of atomic force microscopy (AFM) pioneered a novel aspect for the surface metrology concept. A range of scanning probe methods have been developed over the years based on different sorts of tip-surface interaction: electrical, optical, thermal, force. Reproducible and fast fabrication...... miniaturisation requires the scanning probes to adapt into finer geometries to provide higher lateral resolution. To meet these needs critical dimension AFM (CD-AFM) and deep trench AFM (DT-AFM) were invented, which use different types of AFM tips: high-aspect-ratio tips for DT-AFM and CD tips for CD...... replacement could greatly increase the efficiency and adaptability of a CD system. In this PhD study, NanoBits – nano-sized customisable and exchangeable scanning probe tips – were developed to meet the demands of current AFM applications. Two different methods were followed for the fabrication of Nano...

  18. Small molecule probes for cellular death machines.

    Science.gov (United States)

    Li, Ying; Qian, Lihui; Yuan, Junying

    2017-08-01

    The past decade has witnessed a significant expansion of our understanding about the regulated cell death mechanisms beyond apoptosis. The application of chemical biological approaches had played a major role in driving these exciting discoveries. The discovery and use of small molecule probes in cell death research has not only revealed significant insights into the regulatory mechanism of cell death but also provided new drug targets and lead drug candidates for developing therapeutics of human diseases with huge unmet need. Here, we provide an overview of small molecule modulators for necroptosis and ferroptosis, two non-apoptotic cell death mechanisms, and discuss the molecular pathways and relevant pathophysiological mechanisms revealed by the judicial applications of such small molecule probes. We suggest that the development and applications of small molecule probes for non-apoptotic cell death mechanisms provide an outstanding example showcasing the power of chemical biology in exploring novel biological mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Remote tuning of NMR probe circuits.

    Science.gov (United States)

    Kodibagkar, V D; Conradi, M S

    2000-05-01

    There are many circumstances in which the probe tuning adjustments cannot be located near the rf NMR coil. These may occur in high-temperature NMR, low-temperature NMR, and in the use of magnets with small diameter access bores. We address here circuitry for connecting a fixed-tuned probe circuit by a transmission line to a remotely located tuning network. In particular, the bandwidth over which the probe may be remotely tuned while keeping the losses in the transmission line acceptably low is considered. The results show that for all resonant circuit geometries (series, parallel, series-parallel), overcoupling of the line to the tuned circuit is key to obtaining a large tuning bandwidth. At equivalent extents of overcoupling, all resonant circuit geometries have nearly equal remote tuning bandwidths. Particularly for the case of low-loss transmission line, the tuning bandwidth can be many times the tuned circuit's bandwidth, f(o)/Q. Copyright 2000 Academic Press.

  20. Redesign of a Low Energy Probe Head

    CERN Document Server

    Rao, Yi-Nong; Ries, Thomas

    2005-01-01

    The present situation of the low energy probe LE2 in TRIUMF cyclotron is that the thickness of the finger 5 is uniform over a radial length of 3.25 inch and its weight which amounts to ~447 g is affecting its re-circulating ball mechanism and causing it to fall below the median plane over its range of movement. We therefore re-design it in order to reduce its weight. First, we made simulations and determined the optimum thickness of the probe head vs its radial length. These simulation results are found to be in good agreement with experimental measurements made. Finally, we calculated the temperature rise caused by the beam power dumped on the probe, and figured out the maximum current of beam that can be dumped on the finger.