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Sample records for aequorea victoria gfp

  1. Identifying and modeling motion primitives for the hydromedusae Sarsia tubulosa and Aequorea victoria.

    PubMed

    Sledge, Isaac; Krieg, Michael; Lipinski, Doug; Mohseni, Kamran

    2015-12-01

    The movements of organisms can be thought of as aggregations of motion primitives: motion segments containing one or more significant actions. Here, we present a means to identify and characterize motion primitives from recorded movement data. We address these problems by assuming that the motion sequences can be characterized as a series of dynamical-system-based pattern generators. By adopting a nonparametric, Bayesian formalism for learning and simplifying these pattern generators, we arrive at a purely data-driven model to automatically identify breakpoints in the movement sequences. We apply this model to swimming sequences from two hydromedusa. The first hydromedusa is the prolate Sarsia tubulosa, for which we obtain five motion primitives that correspond to bell cavity pressurization, jet formation, jetting, cavity fluid refill, and coasting. The second hydromedusa is the oblate Aequorea victoria, for which we obtain five motion primitives that correspond to bell compression, vortex separation, cavity fluid refill, vortex formation, and coasting. Our experimental results indicate that the breakpoints between primitives are correlated with transitions in the bell geometry, vortex formation and shedding, and changes in derived dynamical quantities. These dynamics quantities include terms like pressure, power, drag, and thrust. Such findings suggest that dynamics information is inherently present in the observed motions. PMID:26495992

  2. Identifying and modeling motion primitives for the hydromedusae Sarsia tubulosa and Aequorea victoria.

    PubMed

    Sledge, Isaac; Krieg, Michael; Lipinski, Doug; Mohseni, Kamran

    2015-10-23

    The movements of organisms can be thought of as aggregations of motion primitives: motion segments containing one or more significant actions. Here, we present a means to identify and characterize motion primitives from recorded movement data. We address these problems by assuming that the motion sequences can be characterized as a series of dynamical-system-based pattern generators. By adopting a nonparametric, Bayesian formalism for learning and simplifying these pattern generators, we arrive at a purely data-driven model to automatically identify breakpoints in the movement sequences. We apply this model to swimming sequences from two hydromedusa. The first hydromedusa is the prolate Sarsia tubulosa, for which we obtain five motion primitives that correspond to bell cavity pressurization, jet formation, jetting, cavity fluid refill, and coasting. The second hydromedusa is the oblate Aequorea victoria, for which we obtain five motion primitives that correspond to bell compression, vortex separation, cavity fluid refill, vortex formation, and coasting. Our experimental results indicate that the breakpoints between primitives are correlated with transitions in the bell geometry, vortex formation and shedding, and changes in derived dynamical quantities. These dynamics quantities include terms like pressure, power, drag, and thrust. Such findings suggest that dynamics information is inherently present in the observed motions.

  3. Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis.

    PubMed

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2009-12-01

    Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 A resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 A, alpha = 90.96, beta = 103.41, gamma = 101.79 degrees. The Matthews coefficient (V(M) = 2.10 A(3) Da(-1)) indicated that the crystal contained two mAG molecules per asymmetric unit.

  4. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

    Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.; Wolfgang-Kimball, D.

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.

  5. Prolongation of GFP-expressed skin graft after intrathymic injection of GFP positive splenocytes in adult rat

    NASA Astrophysics Data System (ADS)

    Hakamata, Yoji; Igarashi, Yuka; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    GFP is a fluorescent product of the jellyfish Aequorea victoria and has been used for a variety of biological experiments as a reporter molecule. While GFP possesses advantages for the non-invasive imaging of viable cells, GFP-positive cells are still considered potential xeno-antigens. It is difficult to observe the precise fate of transplanted cells/organs in recipients without immunological control. The aim of this study was to determine whether intrathymic injection of GFP to recipients and the depletion of peripheral lymphocytes could lead to donor-specific unresponsiveness to GFP-expressed cell. LEW rats were administered intraperitoneally with 0.2 ml of anti-rat lymphocyte serum (ALS) 1 day prior to intrathymic injection of donor splenocytes or adeno-GFP vector. Donor cells and vector were non-invasively inoculated into the thymus under high frequency ultrasound imaging using an echo-guide. All animals subsequently received a 7 days GFP-expressed skin graft from the same genetic background GFP LEW transgenic rat. Skin graft survival was greater in rats injected with donor splenocytes (23.6+/-9.1) compared with adeno-GFP (13.0+/-3.7) or untreated control rats (9.5+/-1.0). Intrathymic injection of donor antigen into adult rats can induce donor-specific unresponsiveness. Donor cells can be observed for a long-term in recipients with normal immunity using this strategy.

  6. The use of GFP to localize Rho GTPases in living cells.

    PubMed

    Michaelson, David; Philips, Mark

    2006-01-01

    The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems. PMID:16472666

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

    NASA Technical Reports Server (NTRS)

    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.

  8. Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain.

    PubMed

    Sarrocco, Sabrina; Mikkelsen, Lisbeth; Vergara, Mariarosaria; Jensen, Dan Funck; Lübeck, Mette; Vannacci, Giovanni

    2006-02-01

    The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry. PMID:16388938

  9. Application of GFP technique for cytoskeleton visualization onboard the International Space Station.

    PubMed

    Kordyum, E L; Shevchenko, G V; Yemets, A I; Nyporko, A I; Blume, Ya B

    2005-03-01

    Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensing and trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different modifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles, they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocytochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletal visualization onboard the ISS, namely, application of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. The creation of chimerical protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of the cytoskeleton functional status in microgravity.

  10. Expression of GFP in tumor cells and fluorescent examination by confocal microscope

    NASA Astrophysics Data System (ADS)

    Jin, Ying; Xing, Da; Xu, Chaoyang

    2002-04-01

    The green fluorescent protein (GFP), from the bioluminescent jellyfish Aequorea victoria, yields a bright green fluorescence when expressed in either eukaryotic or prokaryotic cells and illuminated by blue or UV light. The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the plasmid EGFP encoding GFP was used to transfect SWO cells (a cancer cell line of nerve gelatinous tissue) mediated by liposome: (1) The plasmid EGFP-C1, purchased from Clontech Co., propagated in suitable E. coli strain (JM 109), was extracted by Concert High Purity Plasmid Miniprep (Gibco). (2) SWO was cultured in RPMI 1640 (10% FCS and 25 mM HEPES), 37 degree(s)C, 5% CO2. Cancer cells were transfected in 6-cm tissue culture dishes by Lipofectin Reagent (Gibco) for 6-12 hr using 2 ug DNA. (3) Then, infected cells were collected in medium containing 800 ug/ml G418, and the resistant clones were harvested and subcloned in fresh culture medium maintaining 800 ug/ml G418. (4) The cells were examined by using Nikon fluorescent microscope (E600) and Bio-Rad confocal microscope (MRC 600). (5) Next step, the cancer cells, stably expressing GFP after in vivo transduction, were implanted by surgical orthotopic implantation (SOI) in nude mice. Tracking of these cancer cells will become more sensitive and rapid than the traditional procedure of histopathological examination or immunohistochemistry. This method demonstrates external, noninvasive, whole-body, real-time fluorescence optical imaging of internally growing tumors and metastases in transplanted animals.

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

    PubMed

    Xiong, Tou Cheu; Ronzier, Elsa; Sanchez, Frédéric; Corratgé-Faillie, Claire; Mazars, Christian; Thibaud, Jean-Baptiste

    2014-01-01

    Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-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 Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) 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 Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins. PMID:24600459

  12. Application of GFP-Technique for Cytoskeleton Visualization Onboard

    NASA Astrophysics Data System (ADS)

    Kordyum, E. L.; Shevchenko, G. V.; Yemets, A. I.; Nyporko, A. I.; Blume, Ya. B.

    2002-01-01

    gravity perception and transduction. Elucidation of cytoskeleton involvement in the above processes will be a great contribution into the fundamental biology, namely, in signaling mechanisms. One of the useful models for investigation of cell gravisensing are higher plants where the sites of gravity perception and reaction are spatially separated. From this point of view the localization of cytoskeletal elements at the different stages of plant development with the special emphasis on cytoskeleton dynamics during development of gravisensitive regions are of special interests. It should be noted that most of plant cytoskeletal researches are conducted by the means of immunohistochemical reactions with the application of monoclonal antibodies specific to specific cytoskeletal proteins. Different modifications of these methods are beneficial for the ground-based experiments, but for the performance onboard the latter represents quite routine and complicated technique, which requires time and special skills for astronauts. Besides, immunohistochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for better understanding of cytoskeleton function. In this connection we propose new approach for cytoskeleton visualization onboard in particular, use of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. Recently appeared this method helped to obtain significant data on the cytoskeleton dynamics in living cells, including plant cells. The creation of chimaeric protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of cytoskeleton functional status in microgravity. To realize this idea at present state of art it would be possible to produce a respective chimaeric plant tubulin gene

  13. Proton nuclear magnetic resonance and fluorescence spectroscopic studies of segmental mobility in aequorin and a green fluorescent protein from Aequorea forskalea

    SciTech Connect

    Nageswara Rao, B.D.; Kemple, M.D.; Prendergast, F.G.

    1980-10-01

    Aequorin is a protein of low molecular weight (20,000) isolated from the jellyfish Aequorea forskalea which emits blue light upon the binding of Ca/sup 2 +/ ions. This bioluminescence requires neither exogenous oxygen nor any other cofactors. The light emission occurs from an excited state of a chromophore (an imidazolopyrazinone) which is tightly and noncovalently bound to the protein. Apparently the binding of Ca/sup 2 +/ by the protein induces changes in the protein conformation which allow oxygen, already bound or otherwise held by the protein, to react with and therein oxidize the chromophore. The resulting discharged protein remains intact, with the Ca/sup 2 +/ and the chromophore still bound, but is incapable of further luminescence. The fluorescence spectrum of this discharged protein and the bioluminescence spectrum of the original charged aequorin are identical. A green fluorescent protein (GFP) of approx. 30,000 mol wt isolated from the same organism, functions in vivo as an acceptor of energy from aequorin and subsequently emits green light. We are applying proton nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy to examine structural details of, and fluctuations associated with the luminescent reaction of aequorin and the in vivo energy transfer from aequorin to the GFP.

  14. Two-color GFP expression system for C. elegans.

    PubMed

    Miller, D M; Desai, N S; Hardin, D C; Piston, D W; Patterson, G H; Fleenor, J; Xu, S; Fire, A

    1999-05-01

    We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.

  15. Marketing ACE in Victoria.

    ERIC Educational Resources Information Center

    2001

    This publication presents options raised through various forums for marketing adult and community education (ACE) in Victoria, Australia, and suggested strategies. After an introduction (chapter 1), chapters 2 and 3 provide a broad view of the current situation for marketing ACE. Chapter 2 discusses general issues in the current position--ACE…

  16. Big Spherules near 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This frame from the microscopic imager on NASA's Mars Exploration Rover Opportunity shows spherules up to about 5 millimeters (one-fifth of an inch) in diameter. The camera took this image during the 924th Martian day, or sol, of Opportunity's Mars-surface mission (Aug. 30, 2006), when the rover was about 200 meters (650 feet) north of 'Victoria Crater.'

    Opportunity discovered spherules like these, nicknamed 'blueberries,' at its landing site in 'Eagle Crater,' and investigations determined them to be iron-rich concretions that formed inside deposits soaked with groundwater. However, such concretions were much smaller or absent at the ground surface along much of the rover's trek of more than 5 kilometers (3 miles) southward to Victoria. The big ones showed up again when Opportunity got to the ring, or annulus, of material excavated and thrown outward by the impact that created Victoria Crater. Researchers hypothesize that some layer beneath the surface in Victoria's vicinity was once soaked with water long enough to form the concretions, that the crater-forming impact dispersed some material from that layer, and that Opportunity might encounter that layer in place if the rover drives down into the crater.

  17. Stretched View Showing 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Stretched View Showing 'Victoria'

    This pair of images from the panoramic camera on NASA's Mars Exploration Rover Opportunity served as initial confirmation that the two-year-old rover is within sight of 'Victoria Crater,' which it has been approaching for more than a year. Engineers on the rover team were unsure whether Opportunity would make it as far as Victoria, but scientists hoped for the chance to study such a large crater with their roving geologist. Victoria Crater is 800 meters (nearly half a mile) in diameter, about six times wider than 'Endurance Crater,' where Opportunity spent several months in 2004 examining rock layers affected by ancient water.

    When scientists using orbital data calculated that they should be able to detect Victoria's rim in rover images, they scrutinized frames taken in the direction of the crater by the panoramic camera. To positively characterize the subtle horizon profile of the crater and some of the features leading up to it, researchers created a vertically-stretched image (top) from a mosaic of regular frames from the panoramic camera (bottom), taken on Opportunity's 804th Martian day (April 29, 2006).

    The stretched image makes mild nearby dunes look like more threatening peaks, but that is only a result of the exaggerated vertical dimension. This vertical stretch technique was first applied to Viking Lander 2 panoramas by Philip Stooke, of the University of Western Ontario, Canada, to help locate the lander with respect to orbiter images. Vertically stretching the image allows features to be more readily identified by the Mars Exploration Rover science team.

    The bright white dot near the horizon to the right of center (barely visible without labeling or zoom-in) is thought to be a light-toned outcrop on the far wall of the crater, suggesting that the rover can see over the low rim of Victoria. In figure 1, the northeast and southeast rims are labeled

  18. GFP as potential cellular viscosimeter

    NASA Astrophysics Data System (ADS)

    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.

  19. GFP as potential cellular viscosimeter

    NASA Astrophysics Data System (ADS)

    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.

  20. 33 CFR 165.837 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... regulations in 33 CFR part 165.23, no person or vessel may enter or remain in the zone described in paragraph... Docks, Victoria Barge Canal, Victoria, Texas. 165.837 Section 165.837 Navigation and Navigable Waters... Guard District § 165.837 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria,...

  1. 33 CFR 165.837 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... regulations in 33 CFR part 165.23, no person or vessel may enter or remain in the zone described in paragraph... Docks, Victoria Barge Canal, Victoria, Texas. 165.837 Section 165.837 Navigation and Navigable Waters... Guard District § 165.837 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria,...

  2. 33 CFR 165.837 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... regulations in 33 CFR part 165.23, no person or vessel may enter or remain in the zone described in paragraph... Docks, Victoria Barge Canal, Victoria, Texas. 165.837 Section 165.837 Navigation and Navigable Waters... Guard District § 165.837 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria,...

  3. 33 CFR 165.837 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... regulations in 33 CFR part 165.23, no person or vessel may enter or remain in the zone described in paragraph... Docks, Victoria Barge Canal, Victoria, Texas. 165.837 Section 165.837 Navigation and Navigable Waters... Guard District § 165.837 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria,...

  4. 33 CFR 165.837 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... regulations in 33 CFR part 165.23, no person or vessel may enter or remain in the zone described in paragraph... Docks, Victoria Barge Canal, Victoria, Texas. 165.837 Section 165.837 Navigation and Navigable Waters... Guard District § 165.837 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria,...

  5. GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

    PubMed

    Ishag, Hassan Z A; Liu, Maojun; Yang, Ruosong; Xiong, Qiyan; Feng, Zhixin; Shao, Guoqing

    2016-01-01

    Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression. PMID:27386255

  6. Teaching Molecular Biology to Undergraduate Biology Students: An Illustration of Protein Expression and Purification

    ERIC Educational Resources Information Center

    Sommer, Cesar Adolfo; Silva, Flavio Henrique; Novo, Maria Teresa Marques

    2004-01-01

    Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of "Aequorea victoria" is an interesting system for didactic purposes because it can be viewed easily during…

  7. Monitoring transgenic plants using in vivo markers

    SciTech Connect

    Stewart, C.N. Jr.

    1996-06-01

    The gene coding for green fluorecent protein (GFP), isolated and cloned from the jellyfish Aequorea victoria, is an ideal transgene for the monitoring of any plant species. It has the ability to fluoresce without added substrate, enzyme, or cofactor; it does not introduce morphological or sexual aberrations when expressed. 7 refs., 1 fig.

  8. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-07

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  9. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2014-04-01

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  10. A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

    ERIC Educational Resources Information Center

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase chain…

  11. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2015-07-14

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  12. 'Victoria Crater' from 'Duck Bay'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    This view is presented as a cylindrical projection with geometric seam correction.

  13. Light-activated Reassembly of Split GFP

    PubMed Central

    Kent, Kevin P.; Boxer, Steven G.

    2011-01-01

    Truncated Green Fluorescent Protein (GFP) with the 11th β-strand removed is potentially interesting for bioconjugation, imaging, and the preparation of semi-synthetic proteins with novel spectroscopic or functional properties. Surprisingly, the truncated GFP generated by removing the 11th strand, once refolded, does not reassemble with a synthetic peptide corresponding to strand 11, but does reassemble following light activation. The mechanism of this process has been studied in detail by absorption, fluorescence and Raman spectroscopy. The chromophore in this refolded truncated GFP is found to be in the trans configuration. Upon exposure to light a photostationary state is formed between the trans and cis conformations of the chromophore, and only truncated GFP with the cis configuration of the chromophore binds the peptide. A kinetic model describing the light activated reassembly of this split GFP is discussed. This unique light-driven reassembly is potentially useful for controlling protein-protein interactions. PMID:21351768

  14. Degradation of Victoria crater, Mars

    USGS Publications Warehouse

    Grant, J. A.; Wilson, S.A.; Cohen, B. A.; Golombek, M.P.; Geissler, P.E.; Sullivan, R.J.; Kirk, R.L.; Parker, T.J.

    2008-01-01

    The ???750 m diameter and ???75 m deep Victoria crater in Meridiani Planum, Mars, is a degraded primary impact structure retaining a ???5 m raised rim consisting of 1-2 m of uplifted rocks overlain by ???3 m of ejecta at the rim crest. The rim is 120-220 m wide and is surrounded by a dark annulus reaching an average of 590 m beyond the raised rim. Comparison between observed morphology and that expected for pristine craters 500-750 m across indicates that the original, pristine crater was close to 600 m in diameter. Hence, the crater has been erosionally widened by ???150 m and infilled by ???50 m of sediments. Eolian processes are responsible for most crater modification, but lesser mass wasting or gully activity contributions cannot be ruled out. Erosion by prevailing winds is most significant along the exposed rim and upper walls and accounts for ???50 m widening across a WNW-ESE diameter. The volume of material eroded from the crater walls and rim is ???20% less than the volume of sediments partially filling the crater, indicating eolian infilling from sources outside the crater over time. The annulus formed when ???1 m deflation of the ejecta created a lag of more resistant hematite spherules that trapped <10-20 cm of darker, regional basaltic sands. Greater relief along the rim enabled meters of erosion. Comparison between Victoria and regional craters leads to definition of a crater degradation sequence dominated by eolian erosion and infilling over time. Copyright 2008 by the American Geophysical Union.

  15. Measles surveillance in Victoria, Australia.

    PubMed Central

    Wang, Yung-Hsuan J.; Andrews, Ross M.; Lambert, Stephen B.

    2006-01-01

    OBJECTIVE: Many countries are implementing measles elimination strategies. In Australia, the State of Victoria has conducted enhanced measles surveillance since 1997 using case interviews and home-based specimen collection for laboratory confirmation. We attempted to identify features of notified cases that would better target surveillance resources. METHODS: We retrospectively classified notifications received from 1998 to 2003 as having been received in an epidemic (one or more laboratory-confirmed cases) or an interepidemic period (no laboratory-confirmed cases). We labelled the first case notified in any epidemic period that was not laboratory-confirmed at the time of notification as a "sentinel case". To maximize detection of sentinel cases while minimizing the follow-up of eventually discarded notifications, we generated algorithms using sentinel cases and interepidemic notifications. FINDINGS: We identified 10 sentinel cases with 422 interepidemic notifications from 1281 Victorian notifications. Sentinel cases were more likely to report fever at rash onset (odds ratio (OR) 15.7, 95% confidence interval (CI) CI: 2.1-688.9), cough (OR 10.4, 95% CI: 1.4-456.7), conjunctivitis (OR 7.9, 95% CI: 1.8-39.1), or year of birth between 1968 and 1981 (OR 31.8, 95% CI: 6.7-162.3). Prospective application of an algorithm consisting of fever at rash onset or born between 1968 and 1981 in the review period would have detected all sentinel cases and avoided the need for enhanced follow-up of 162 of the 422 eventually discarded notifications. CONCLUSION: Elimination strategies should be refined to suit regional and local priorities. The prospective application of an algorithm in Victoria is likely to reduce enhanced measles surveillance resource use in interepidemic periods, while still detecting early cases during measles outbreaks. PMID:16501727

  16. 'Lyell' Panorama inside Victoria Crater

    NASA Technical Reports Server (NTRS)

    2008-01-01

    During four months prior to the fourth anniversary of its landing on Mars, NASA's Mars Exploration Rover Opportunity examined rocks inside an alcove called 'Duck Bay' in the western portion of Victoria Crater. The main body of the crater appears in the upper right of this stereo panorama, with the far side of the crater lying about 800 meters (half a mile) away. Bracketing that part of the view are two promontories on the crater's rim at either side of Duck Bay. They are 'Cape Verde,' about 6 meters (20 feet) tall, on the left, and 'Cabo Frio,' about 15 meters (50 feet) tall, on the right. The rest of the image, other than sky and portions of the rover, is ground within Duck Bay.

    Opportunity's targets of study during the last quarter of 2007 were rock layers within a band exposed around the interior of the crater, about 6 meters (20 feet) from the rim. Bright rocks within the band are visible in the foreground of the panorama. The rover science team assigned informal names to three subdivisions of the band: 'Steno,' 'Smith,' and 'Lyell.'

    This view combines many images taken by Opportunity's panoramic camera (Pancam) from the 1,332nd through 1,379th Martian days, or sols, of the mission (Oct. 23 to Dec. 11, 2007). Images taken through Pancam filters centered on wavelengths of 753 nanometers, 535 nanometers and 432 nanometers were mixed to produce an approximately true-color panorama. Some visible patterns in dark and light tones are the result of combining frames that were affected by dust on the front sapphire window of the rover's camera.

    Opportunity landed on Jan. 25, 2004, Universal Time, (Jan. 24, Pacific Time) inside a much smaller crater about 6 kilometers (4 miles) north of Victoria Crater, to begin a surface mission designed to last 3 months and drive about 600 meters (0.4 mile).

  17. Identification and Characterisation of a pH-stable GFP

    PubMed Central

    Roberts, Tania Michelle; Rudolf, Fabian; Meyer, Andreas; Pellaux, Rene; Whitehead, Ellis; Panke, Sven; Held, Martin

    2016-01-01

    Green fluorescent proteins (GFPs) are invaluable tools for modern cell biology. Even though many properties of GFP have been successfully engineered, a GFP retaining brightness at low pH has not emerged. This limits the use of GFP in quantitative studies performed in fluctuating or acidic conditions. We report the engineering and characterisation of tandem dimer GFP (pH-tdGFP), a bright and stable GFP that can be efficiently excited and maintains its fluorescence properties in acidic conditions. Therefore, pH-tdGFP could act as a quantitative marker for cellular processes that occur at low pH, such as endocytosis, autophagy or starvation. PMID:27324986

  18. Fluorescence imaging using synthetic GFP chromophores.

    PubMed

    Walker, Christopher L; Lukyanov, Konstantin A; Yampolsky, Ilia V; Mishin, Alexander S; Bommarius, Andreas S; Duraj-Thatte, Anna M; Azizi, Bahareh; Tolbert, Laren M; Solntsev, Kyril M

    2015-08-01

    Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility. PMID:26117808

  19. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2008-06-24

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  20. Circular permutant GFP insertion folding reporters

    SciTech Connect

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2013-04-16

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  1. Circular permutant GFP insertion folding reporters

    SciTech Connect

    Waldo, Geoffrey S; Cabantous, Stephanie

    2013-02-12

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  2. Circular permutant GFP insertion folding reporters

    SciTech Connect

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-14

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  3. 'Victoria' on Opportunity's Horizon (Orbital View)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This image from the Mars Orbiter Camera on NASA's Mars Global Surveyor highlights the Mars Exploration Rover Opportunity's approach toward 'Victoria Crater.'

    North is to the left. Opportunity's location at sol 804 (April 29, 2006) is marked, as are the left and right edges of Victoria's rim from the rover's point of view. The labeled 'promontory' is a bright spot that scientists believe is an outcrop on the far side of the crater. Marked in light purple is a small, 35-meter (115-foot) crater.

    Victoria Crater is 750 meters (nearly half a mile) in diameter, about six times wider than 'Endurance Crater,' where Opportunity spent several months in 2004 examining rock layers affected by ancient water.

    This image is an uncalibrated version that the rover team uses for planning. It has been reprojected and stretched in some places and isn't used for scientific purposes.

  4. Opportunity at Work Inside Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA Mars Exploration Rover Opportunity used its front hazard-identification camera to capture this wide-angle view of its robotic arm extended to a rock in a bright-toned layer inside Victoria Crater.

    The image was taken during the rover's 1,322nd Martian day, or sol (Oct. 13, 2007).

    Victoria Crater has a scalloped shape of alternating alcoves and promontories around the crater's circumference. Opportunity descended into the crater two weeks earlier, within an alcove called 'Duck Bay.' Counterclockwise around the rim, just to the right of the arm in this image, is a promontory called 'Cabo Frio.'

  5. Geography and Geographical Education in Victoria

    ERIC Educational Resources Information Center

    Kriewaldt, Jeana

    2006-01-01

    Victoria has just emerged from 10 years where Geography has been one of three strands in the key learning area of Studies of Society and Environment (SOSE). The overarching framework emerged from an attempt to develop a national curriculum. Whilst the national curriculum was rejected by Australian state and territories who each hold legislative…

  6. Lead accumulation potential in Acacia victoria.

    PubMed

    Mahdavi, Ali; Khermandar, Khadijeh; Asbchin, Salman Ahmady; Tabaraki, Reza

    2014-01-01

    To assess the potential of Pb+2 accumulation in different parts of Acacia victoria, one year old A. victoria seedlings were exposed to Pb2+(NO3)2 in 5 different concentrations: 0, 50, 250, 500 and 1000 (mg Pb2+ L(-1)) for 45 days. Subsequently, Pb2+ uptake was quantified in roots, shoots and leaves of the seedlings by Atomic Absorption Spectroscopy (AAS). In addition, some physiological parameters such as biomass production, shoots and roots length, plant appearance, tissue concentrations and chlorophyll content were examined. Tissue concentrations increased as Pb2+ concentration increased for A. victoria. The visible toxicity symptoms (chlorosis and necrosis) appeared only to the highest concentration (1000 mg Pb2+ L(-1)), resulting in photosynthesis decrease, plant height, root length and dry biomass reduction. Almost 70% (up to 3580 mg Kg(-1) of dry tissue) from the Pb2+ was accumulated in the entire plant tissues was retained in the roots in the seedlings exposed to 1000 mg Pb2+ L(-1). The seedlings accumulated between 403 to 913 mg Kg(-1) of Pb2+ in shoots and 286 to 650 mg Kg(-1) of Pb2+ in leaves at different treatments. Bioconcentration and translocation factors were determined 5.14 and 0.255, respectively. The results show that A. victoria is suitable for lead-phytostabilization in Pb(2+) -contaminated soil.

  7. Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

    PubMed

    Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

    2015-01-01

    The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in

  8. Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

    PubMed

    Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

    2015-01-01

    The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in

  9. Pancam Peek into 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Left-eye view of a stereo pair for PIA08776

    [figure removed for brevity, see original site] Right-eye view of a stereo pair for PIA08776

    A drive of about 60 meters (about 200 feet) on the 943rd Martian day, or sol, of Opportunity's exploration of Mars' Meridiani Planum region (Sept. 18, 2006) brought the NASA rover to within about 50 meters (about 160 feet) of the rim of 'Victoria Crater.' This crater has been the mission's long-term destination for the past 21 Earth months. Opportunity reached a location from which the cameras on top of the rover's mast could begin to see into the interior of Victoria. This stereo anaglyph was made from frames taken on sol 943 by the panoramic camera (Pancam) to offer a three-dimensional view when seen through red-blue glasses. It shows the upper portion of interior crater walls facing toward Opportunity from up to about 850 meters (half a mile) away. The amount of vertical relief visible at the top of the interior walls from this angle is about 15 meters (about 50 feet). The exposures were taken through a Pancam filter selecting wavelengths centered on 750 nanometers.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is the expectation that a thick stack of geological layers will be exposed in the crater walls, potentially several times the thickness that was previously studied at Endurance and therefore, potentially preserving several times the historical record.

  10. Hazardous thunderstorm intensification over Lake Victoria

    PubMed Central

    Thiery, Wim; Davin, Edouard L.; Seneviratne, Sonia I.; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole P. M.

    2016-01-01

    Weather extremes have harmful impacts on communities around Lake Victoria, where thousands of fishermen die every year because of intense night-time thunderstorms. Yet how these thunderstorms will evolve in a future warmer climate is still unknown. Here we show that Lake Victoria is projected to be a hotspot of future extreme precipitation intensification by using new satellite-based observations, a high-resolution climate projection for the African Great Lakes and coarser-scale ensemble projections. Land precipitation on the previous day exerts a control on night-time occurrence of extremes on the lake by enhancing atmospheric convergence (74%) and moisture availability (26%). The future increase in extremes over Lake Victoria is about twice as large relative to surrounding land under a high-emission scenario, as only over-lake moisture advection is high enough to sustain Clausius–Clapeyron scaling. Our results highlight a major hazard associated with climate change over East Africa and underline the need for high-resolution projections to assess local climate change. PMID:27658848

  11. Hazardous thunderstorm intensification over Lake Victoria

    NASA Astrophysics Data System (ADS)

    Thiery, Wim; Davin, Edouard L.; Seneviratne, Sonia I.; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole P. M.

    2016-09-01

    Weather extremes have harmful impacts on communities around Lake Victoria, where thousands of fishermen die every year because of intense night-time thunderstorms. Yet how these thunderstorms will evolve in a future warmer climate is still unknown. Here we show that Lake Victoria is projected to be a hotspot of future extreme precipitation intensification by using new satellite-based observations, a high-resolution climate projection for the African Great Lakes and coarser-scale ensemble projections. Land precipitation on the previous day exerts a control on night-time occurrence of extremes on the lake by enhancing atmospheric convergence (74%) and moisture availability (26%). The future increase in extremes over Lake Victoria is about twice as large relative to surrounding land under a high-emission scenario, as only over-lake moisture advection is high enough to sustain Clausius-Clapeyron scaling. Our results highlight a major hazard associated with climate change over East Africa and underline the need for high-resolution projections to assess local climate change.

  12. Insertion of a GFP reporter gene in influenza virus.

    PubMed

    Perez, Jasmine T; García-Sastre, Adolfo; Manicassamy, Balaji

    2013-01-01

    The incorporation of a fluorescent reporter gene into a replication-competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of nonstructural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then cloned into the IAV rescue system backbone. Upon construction of the GFP-encoding segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP IAV can subsequently be used to visualize infected cells, both in vitro and in vivo.

  13. 'Victoria Crater' from 'Duck Bay' (Vertical Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    This view is presented as a vertical projection with geometric seam correction.

  14. 'Victoria Crater' from 'Duck Bay' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Figure 1

    [figure removed for brevity, see original site] Figure 2

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    The stereo-anaglyph view presented here is a cylindrical projection with geometric seam correction.

  15. 'Victoria Crater' from 'Duck Bay' (Polar Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    This view is presented as a polar projection with geometric seam correction.

  16. Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration

    PubMed Central

    Sliogeryte, Kristina; Thorpe, Stephen D.; Wang, Zhao; Thompson, Clare L.; Gavara, Nuria; Knight, Martin M.

    2016-01-01

    The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. PMID:26792287

  17. Uranium mineralization in southern Victoria Land, Antarctica

    SciTech Connect

    Dreschhoff, G.A.M.; Zeller, E.J.

    1986-01-01

    For the past 10 antarctic field seasons, an airborne gamma-ray spectrometric survey has been conducted over widely separated parts of the continent. Localized accumulations of both primary and secondary uranium minerals have been discovered at several localities scattered along the Transantarctic Mountains from the Scott Glacier to northern Victoria Land. A number of highly significant radiation anomalies have been discovered in the area between the Koettlitz Glacier and the Pyramid Trough. The occurrences consist of pegmatite vein complexes which contain an association of primary uranium and thorium minerals. Of still greater significance is the fact that abundant secondary uranium minerals were found in association with the primary deposits, and they indicate clearly that uranium is geochemically mobile under the conditions imposed by the arid polar climate that now exists in southern Victoria Land. Preliminary results of a uranium analysis performed by neutron activation indicate a concentration of 0.12% uranium in a composite sample from the two veins. Even higher levels of thorium are present. The nature of the primary uranium mineralization is currently under investigation. Preliminary results are discussed.

  18. On the Rim of 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a cylindrical projection with geometric seam correction.

  19. At Bright Band Inside Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    A layer of light-toned rock exposed inside Victoria Crater in the Meridiani Planum region of Mars appears to mark where the surface was at the time, many millions of years ago, when an impact excavated the crater. NASA's Mars Exploration Rover Opportunity drove to this bright band as the science team's first destination for the rover during investigations inside the crater.

    Opportunity's left front hazard-identification camera took this image just after the rover finished a drive of 2.25 meters (7 feet, 5 inches) during the rover's 1,305th Martian day, or sol, (Sept. 25, 2007). The rocks beneath the rover and its extended robotic arm are part of the bright band.

    Victoria Crater has a scalloped shape of alternating alcoves and promontories around the crater's circumference. Opportunity descended into the crater two weeks earlier, within an alcove called 'Duck Bay.' Counterclockwise around the rim, just to the right of the arm in this image, is a promontory called 'Cabo Frio.'

  20. Glycosylatable GFP as a compartment-specific membrane topology reporter

    SciTech Connect

    Lee, Hunsang; Min, Jisoo; Heijne, Gunnar von; Kim, Hyun

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.

  1. The Bands Culture in Victoria, Australia: Live Music Benefits Career Paths, Employment and Community

    ERIC Educational Resources Information Center

    Watson, Amanda; Forrest, David

    2012-01-01

    This study explores the career paths, employment, business opportunities and community contributions made available through the provision and development of the contemporary performance bands' culture in the State of Victoria. It is framed with the support given to live music performers by Arts Victoria, Small Business Victoria and Music Victoria.…

  2. Layers of 'Cabo Frio' in 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of 'Victoria crater' is looking southeast from 'Duck Bay' towards the dramatic promontory called 'Cabo Frio.' The small crater in the right foreground, informally known as 'Sputnik,' is about 20 meters (about 65 feet) away from the rover, the tip of the spectacular, layered, Cabo Frio promontory itself is about 200 meters (about 650 feet) away from the rover, and the exposed rock layers are about 15 meters (about 50 feet) tall. This is an approximately true color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  3. Layers of 'Cape Verde' in 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is an approximately true color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  4. Forty Meters from Entry to Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view is presented as a cylindrical projection with geometric seam correction.

  5. Satellite View of Opportunity's Journey around 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2007-01-01

    Three years after embarking on a historic exploration of the red planet and six miles away from its landing site, NASA's Mars Exploration Rover Opportunity is traversing 'Victoria Crater' ridge by ridge, peering at layered cliffs in the interior. To identify various alcoves and cliffs along the way, science team members are using names of places visited by the 16th-century Earth explorer Ferdinand Magellan and his crew aboard the ship Victoria, who proved the Earth is round. (All names are unofficial unless approved by the International Astronomical Union.) This orbital view of 'Victoria Crater' was taken by NASA's Mars Reconnaissance Orbiter.

  6. Space Radar Image of Victoria, Canada

    NASA Technical Reports Server (NTRS)

    1994-01-01

    This three-frequency spaceborne radar image shows the southern end of Vancouver Island on the west coast of Canada. The white area in the lower right is the city of Victoria, the capital of the province of British Columbia. The three radar frequencies help to distinguish different land use patterns. The bright pink areas are suburban regions, the brownish areas are forested regions, and blue areas are agricultural fields or forest clear-cuts. Founded in 1843 as a fur trading post, Victoria has grown to become one of western Canada's largest commercial centers. In the upper right is San Juan Island, in the state of Washington. The Canada/U.S. border runs through Haro Strait, on the right side of the image, between San Juan Island and Vancouver Island. The image was acquired by the Spaceborne Imaging Radar-C/X-band Synthetic Aperture Radar (SIR-C/X-SAR) on October 6, 1994, onboard the space shuttle Endeavour. The area shown is 37 kilometers by 42 kilometers (23 miles by 26 miles) and is centered at 48.5 degrees north latitude, 123.3 degrees west longitude. North is toward the upper left. The colors are assigned to different radar frequencies and polarizations as follows: red is L-band horizontally transmitted and received; green is C-band, vertically transmitted and received; and blue is X-band, vertically transmitted and received. SIR-C/X-SAR, a joint mission of the German, Italian and United States space agencies, is part of NASA's Mission to Planet Earth program.

  7. On the Rim of 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Left-eye view of a stereo pair for PIA08780

    [figure removed for brevity, see original site] Right-eye view of a stereo pair for PIA08780

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a cylindrical-perspective projection with geometric seam correction.

  8. 'Lyell' Panorama inside Victoria Crater (Stereo)

    NASA Technical Reports Server (NTRS)

    2008-01-01

    During four months prior to the fourth anniversary of its landing on Mars, NASA's Mars Exploration Rover Opportunity examined rocks inside an alcove called 'Duck Bay' in the western portion of Victoria Crater. The main body of the crater appears in the upper right of this stereo panorama, with the far side of the crater lying about 800 meters (half a mile) away. Bracketing that part of the view are two promontories on the crater's rim at either side of Duck Bay. They are 'Cape Verde,' about 6 meters (20 feet) tall, on the left, and 'Cabo Frio,' about 15 meters (50 feet) tall, on the right. The rest of the image, other than sky and portions of the rover, is ground within Duck Bay.

    Opportunity's targets of study during the last quarter of 2007 were rock layers within a band exposed around the interior of the crater, about 6 meters (20 feet) from the rim. Bright rocks within the band are visible in the foreground of the panorama. The rover science team assigned informal names to three subdivisions of the band: 'Steno,' 'Smith,' and 'Lyell.'

    This view incorporates many images taken by Opportunity's panoramic camera (Pancam) from the 1,332nd through 1,379th Martian days, or sols, of the mission (Oct. 23 to Dec. 11, 2007). It combines a stereo pair so that it appears three-dimensional when seen through blue-red glasses. Some visible patterns in dark and light tones are the result of combining frames that were affected by dust on the front sapphire window of the rover's camera.

    Opportunity landed on Jan. 25, 2004, Universal Time, (Jan. 24, Pacific Time) inside a much smaller crater about 6 kilometers (4 miles) north of Victoria Crater, to begin a surface mission designed to last 3 months and drive about 600 meters (0.4 mile).

  9. Ecological Biogeography of the Terrestrial Nematodes of Victoria Land, Antarctica

    PubMed Central

    Adams, Byron J.; Wall, Diana H.; Virginia, Ross A.; Broos, Emma; Knox, Matthew A.

    2014-01-01

    Abstract The terrestrial ecosystems of Victoria Land, Antarctica are characteristically simple in terms of biological diversity and ecological functioning. Nematodes are the most commonly encountered and abundant metazoans of Victoria Land soils, yet little is known of their diversity and distribution. Herein we present a summary of the geographic distribution, habitats and ecology of the terrestrial nematodes of Victoria Land from published and unpublished sources. All Victoria Land nematodes are endemic to Antarctica, and many are common and widely distributed at landscape scales. However, at smaller spatial scales, populations can have patchy distributions, with the presence or absence of each species strongly influenced by specific habitat requirements. As the frequency of nematode introductions to Antarctica increases, and soil habitats are altered in response to climate change, our current understanding of the environmental parameters associated with the biogeography of Antarctic nematofauna will be crucial to monitoring and possibly mitigating changes to these unique soil ecosystems. PMID:25061360

  10. Energy profile of nanobody-GFP complex under force

    NASA Astrophysics Data System (ADS)

    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. The secretory membrane system studied in real-time. Robert Feulgen Prize Lecture, 2001.

    PubMed

    Lippincott-Schwartz, J

    2001-08-01

    The discovery and development of green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has revolutionized studies on protein localization and dynamics by allowing direct observation of a protein's life history and pathway in living cells, previously only deduced from genetic, biochemical, or immunolabeling studies. Applied to the secretory membrane system, which regulates delivery of newly synthesized proteins and lipids to the cell surface, GFP-based studies are providing important new insights into the maintenance and biogenesis of organelles, as well as the origin, pathway, and fate of secretory transport intermediates.

  12. A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

    PubMed

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Cruz Schneider, Maria Paula; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

  13. The secretory membrane system studied in real-time. Robert Feulgen Prize Lecture, 2001.

    PubMed

    Lippincott-Schwartz, J

    2001-08-01

    The discovery and development of green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has revolutionized studies on protein localization and dynamics by allowing direct observation of a protein's life history and pathway in living cells, previously only deduced from genetic, biochemical, or immunolabeling studies. Applied to the secretory membrane system, which regulates delivery of newly synthesized proteins and lipids to the cell surface, GFP-based studies are providing important new insights into the maintenance and biogenesis of organelles, as well as the origin, pathway, and fate of secretory transport intermediates. PMID:11685538

  14. Victoria Land, Ross Sea, and Ross Ice Shelf, Antarctica

    NASA Technical Reports Server (NTRS)

    2002-01-01

    On December 19, 2001, MODIS acquired data that produced this image of Antarctica's Victoria Land, Ross Ice Shelf, and the Ross Sea. The coastline that runs up and down along the left side of the image denotes where Victoria Land (left) meets the Ross Ice Shelf (right). The Ross Ice Shelf is the world's largest floating body of ice, approximately the same size as France. Credit: Jacques Descloitres, MODIS Land Rapid Response Team, NASA/GSFC

  15. Cuprizone-induced demyelination in CNP::GFP transgenic mice.

    PubMed

    Silvestroff, Lucas; Bartucci, Sandra; Soto, Eduardo; Gallo, Vittorio; Pasquini, Juana; Franco, Paula

    2010-06-15

    Cuprizone (bis-cyclohexanone oxaldihydrazone) was previously shown to induce demyelination in white matter enriched brain structures. In the present study we used the cuprizone demyelination model in transgenic mice expressing the enhanced green fluorescent protein (GFP) under the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) promoter. The use of these particular transgenic mice allows easy detection of cells belonging to the entire oligodendroglial (OLG) lineage, ranging from OLG precursors to mature myelinating OLGs. We were able to evaluate the precise extent of oligodendroglial cell damage and recovery within the murine adult central nervous system (CNS) after inducing demyelination by acute cuprizone intoxication. A generalized loss of GFP+ cells was observed after cuprizone exposure and correlated with a decline in myelin basic protein (MBP) expression. OLGs were depleted in many brain areas that were previously thought to be unaffected by cuprizone treatment. Thus, in addition to the well-known cuprizone effects on the medial corpus callosum, we also found a loss of GFP+ cells in most brain structures, particularly in the caudatus putamen, cortex, anterior commissure, olfactory bulb, hippocampus, optic chiasm, brainstem, and cingulum. Loss of GFP+ cells was accompanied by extensive astrogliosis and microglial activation, although neurons were not affected. Interestingly, cuprizone-treated animals showed both activation of GFAP expression and a higher proliferation rate in subventricular zone cells. A week after cuprizone removal from the diet, GFP+ oligodendroglial cells began repopulating the damaged structures. GFP expression precedes that of MBP and allows OLG detection before myelin restoration.

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

    PubMed

    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.

  17. Recent plant studies using Victoria 2.0

    SciTech Connect

    BIXLER,NATHAN E.; GASSER,RONALD D.

    2000-03-08

    VICTORIA 2.0 is a mechanistic computer code designed to analyze fission product behavior within the reactor coolant system (RCS) during a severe nuclear reactor accident. It provides detailed predictions of the release of radioactive and nonradioactive materials from the reactor core and transport and deposition of these materials within the RCS and secondary circuits. These predictions account for the chemical and aerosol processes that affect radionuclide behavior. VICTORIA 2.0 was released in early 1999; a new version VICTORIA 2.1, is now under development. The largest improvements in VICTORIA 2.1 are connected with the thermochemical database, which is being revised and expanded following the recommendations of a peer review. Three risk-significant severe accident sequences have recently been investigated using the VICTORIA 2.0 code. The focus here is on how various chemistry options affect the predictions. Additionally, the VICTORIA predictions are compared with ones made using the MELCOR code. The three sequences are a station blackout in a GE BWR and steam generator tube rupture (SGTR) and pump-seal LOCA sequences in a 3-loop Westinghouse PWR. These sequences cover a range of system pressures, from fully depressurized to full system pressure. The chief results of this study are the fission product fractions that are retained in the core, RCS, secondary, and containment and the fractions that are released into the environment.

  18. 'Lyell' Panorama inside Victoria Crater (False Color)

    NASA Technical Reports Server (NTRS)

    2008-01-01

    During four months prior to the fourth anniversary of its landing on Mars, NASA's Mars Exploration Rover Opportunity examined rocks inside an alcove called 'Duck Bay' in the western portion of Victoria Crater. The main body of the crater appears in the upper right of this stereo panorama, with the far side of the crater lying about 800 meters (half a mile) away. Bracketing that part of the view are two promontories on the crater's rim at either side of Duck Bay. They are 'Cape Verde,' about 6 meters (20 feet) tall, on the left, and 'Cabo Frio,' about 15 meters (50 feet) tall, on the right. The rest of the image, other than sky and portions of the rover, is ground within Duck Bay.

    Opportunity's targets of study during the last quarter of 2007 were rock layers within a band exposed around the interior of the crater, about 6 meters (20 feet) from the rim. Bright rocks within the band are visible in the foreground of the panorama. The rover science team assigned informal names to three subdivisions of the band: 'Steno,' 'Smith,' and 'Lyell.'

    This view combines many images taken by Opportunity's panoramic camera (Pancam) from the 1,332nd through 1,379th Martian days, or sols, of the mission (Oct. 23 to Dec. 11, 2007). Images taken through Pancam filters centered on wavelengths of 753 nanometers, 535 nanometers and 432 nanometers were mixed to produce this view, which is presented in a false-color stretch to bring out subtle color differences in the scene. Some visible patterns in dark and light tones are the result of combining frames that were affected by dust on the front sapphire window of the rover's camera.

  19. Are Victoria West cores "proto-Levallois"? A phylogenetic assessment.

    PubMed

    Lycett, Stephen J

    2009-02-01

    Cores from South Africa assigned to the "Victoria West" industry have long been purported as a "proto-Levallois" core form, and thus regarded as ancestral to the Levallois prepared core technologies of the Middle Paleolithic and African Middle Stone Age. Similarities in form between Victoria West cores, in terms of surface morphology and the removal of large flakes from a prepared surface, led to hypothesized schemes of technological evolution from Victoria West cores through to fully developed Levallois cores. However, the phylogenetic basis of this Victoria West "proto-Levallois" hypothesis, and the assumptions of phylogenetic homology upon which it rests, have never been tested formally. In recent years, archaeologists have begun to use phylogenetic methods drawn from biology to test hypotheses of technological and cultural evolution. Here, the phylogenetic assumptions of the Victoria West "proto-Levallois" hypothesis are tested directly using a cladistic (maximum parsimony) protocol. The cladistic analyses indicate that Victoria West cores are not the basal sister taxon of a Levallois clade, as predicted by the proto-Levallois hypothesis. Moreover, character analyses demonstrate that several characters relating to core surfaces and flake scar morphology are not phylogenetically homologous, but result from convergent technological evolution within the Acheulean techno-complex. Post hoc analyses further determine that these results are not confounded by choice of outgroup or raw material factors. The results were also shown to be robust on the basis of the ensemble retention index statistic, bootstrap analyses, and permutation tests. Hence, it is concluded that Victoria West cores do not represent a "proto-Levallois" core form, and that the term "para-Levallois" should more correctly be applied on phylogenetic grounds. It is further argued that even in cases where different technologies are found to share phylogenetically homologous features, use of the term

  20. Fluorescent labeling of antibody fragments using split GFP.

    PubMed

    Ferrara, Fortunato; Listwan, Pawel; Waldo, Geoffrey S; Bradbury, Andrew R M

    2011-01-01

    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. PMID:21998685

  1. Biosafety assessment of GFP transplastomic tobacco to rhizosphere microbial community.

    PubMed

    Lv, Yueping; Cai, Hongsheng; Yu, Jianping; Liu, Jiali; Liu, Qingguo; Guo, Changhong

    2014-05-01

    Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry, and has many applications as a marker, especially in plant transformation system. Although a number of studies have been conducted to assess the toxify of this protein to specific organisms, little is known about GFP on rhizosphere microbial community, which is regarded as good indicator for environmental risk assessment. Chloroplast genetic engineering has shown superiority over traditional nuclear genetic engineering, and has been used in many aspects of plant genetic engineering. High levels of chloroplast-based protein accumulation make this technology as an ideal strategy to evaluate biosafety of transgenes. In the present study, the effects of field-released GFP transplastomic tobacco (Nicotiana tabacum) on rhizosphere microbes over a whole growth cycle were investigated by using both culture-dependent and culture-independent methods. Compared to wild-type control, transplastomic tobacco had no significant influence on the microbial population at the seedling, vegetative, flowering and senescing stages. However, developmental stages had more influence than ecotypes (GFP-transformed and wild-type). This was confirmed by colony forming unit, Biolog Eco(TM) and PCR-DGGE analysis. Thus, these results suggest chloroplast transformation with a GFP reporter gene has no significant influence on rhizosphere microbial community, and will be potential platform for plant biotechnology in future. PMID:24429672

  2. A suite of engineered GFP molecules for oligomeric scaffolding

    SciTech Connect

    Leibly, David J.; Arbing, Mark A.; Pashkov, Inna; DeVore, Natasha; Waldo, Geoffrey S.; Terwilliger, Thomas C.; Yeates, Todd O.

    2015-08-13

    Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition. We generated a wide range of spatial arrangements of GFP subunits from 11 different oligomeric variants, and determined their X-ray structures in a total of 33 distinct crystal forms. Furthermore, some of the oligomeric GFP variants show geometric polymorphism depending on conditions, while others show considerable geometric rigidity. Potential future applications of this system are discussed.

  3. A suite of engineered GFP molecules for oligomeric scaffolding

    DOE PAGES

    Leibly, David J.; Arbing, Mark A.; Pashkov, Inna; DeVore, Natasha; Waldo, Geoffrey S.; Terwilliger, Thomas C.; Yeates, Todd O.

    2015-08-13

    Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition. We generated a wide range of spatial arrangements of GFP subunits from 11 different oligomeric variants, and determined their X-ray structuresmore » in a total of 33 distinct crystal forms. Furthermore, some of the oligomeric GFP variants show geometric polymorphism depending on conditions, while others show considerable geometric rigidity. Potential future applications of this system are discussed.« less

  4. Coherent Neutron Scattering and Collective Dynamics in the Protein, GFP

    PubMed Central

    Nickels, Jonathan D.; Perticaroli, Stefania; O’Neill, Hugh; Zhang, Qiu; Ehlers, Georg; Sokolov, Alexei P.

    2013-01-01

    Collective dynamics are considered to be one of the major properties of soft materials, including biological macromolecules. We present coherent neutron scattering studies of the low-frequency vibrations, the so-called boson peak, in fully deuterated green fluorescent protein (GFP). Our analysis revealed unexpectedly low coherence of the atomic motions in GFP. This result implies a low amount of in-phase collective motion of the secondary structural units contributing to the boson peak vibrations and fast conformational fluctuations on the picosecond timescale. These observations are in contrast to earlier studies of polymers and glass-forming systems, and suggest that random or out-of-phase motions of the β-strands contribute greater than two-thirds of the intensity to the low-frequency vibrational spectra of GFP. PMID:24209864

  5. Funding Victoria's public hospitals: the casemix policy of 2000-2001.

    PubMed

    McNair, Peter; Duckett, Stephen

    2002-01-01

    On 1 July 1993 Victoria became the first Australian state to use casemix information to set budgets for its public hospitals commencing with casemix funding for inpatient services. Victoria's casemix funding approach now embraces inpatient, outpatient and rehabilitation services.

  6. Hazardous thunderstorms over Lake Victoria: climate change and early warnings

    NASA Astrophysics Data System (ADS)

    Thiery, Wim; Davin, Edouard L.; Seneviratne, Sonia I.; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole

    2016-04-01

    Severe thunderstorms and associated high waves represent a constant threat to the 200,000 fishermen operating on Lake Victoria. According to the International Red Cross, presumably 3000 to 5000 fishermen die every year on the lake, thereby substantially contributing to the global death toll from natural disasters. Despite the long-known bad reputation of Lake Victoria, operational early warning systems are lacking and possible future changes of these extreme thunderstorms are unknown. Here we present the first dedicated high-resolution, coupled lake-land-atmosphere climate projection for the African Great Lakes region and analyse it in combination with new satellite data and coarser-scale ensemble projections. Our model projections for the end-of-the-century indicate that Lake Victoria amplifies the future intensification of extreme precipitation seen over the surrounding land. Under a high-emission scenario (RCP8.5), the 1% most extreme over-lake precipitation may intensify up to four times faster compared to surrounding land. Our findings are consistent with an ensemble of coarser-scale climate projections for Africa, but the lower skill of the ensemble over Lake Victoria constrains its applicability. Interestingly, the change in extremes contrasts to the change in average over-lake precipitation, which is projected to decrease by -6% for the same period. By further analyzing the high-resolution output we are able to explain this different response: while mesoscale circulation changes cause the average precipitation decline, the response of extremes is essentially thermodynamic. Finally, the study of the satellite-based detection of severe thunderstorms revealed a strong dependency of the nighttime storm intensity over Lake Victoria on the antecedent daytime land storm activity. This highlights the potential of this new satellite product for predicting intense storms over Lake Victoria. Overall, our results indicate a new major hazard associated with climate

  7. Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

    PubMed Central

    Stepanenko, Olesya V.; Verkhusha, Vladislav V.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, K.K.

    2010-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology. PMID:18691124

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

    ERIC Educational Resources Information Center

    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 final…

  9. Imaging and tracking of single GFP molecules in solution.

    PubMed Central

    Kubitscheck, U; Kückmann, O; Kues, T; Peters, R

    2000-01-01

    Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible. PMID:10733995

  10. Job-Sharing at the Greater Victoria Public Library.

    ERIC Educational Resources Information Center

    Miller, Don

    1978-01-01

    Describes the problems associated with the management of part-time library employees and some solutions afforded by a job sharing arrangement in use at the Greater Victoria Public Library. This is a voluntary work arrangement, changing formerly full-time positions into multiple part-time positions. (JVP)

  11. Persistence of neutral polymorphisms in Lake Victoria cichlid fish

    PubMed Central

    Nagl, Sandra; Tichy, Herbert; Mayer, Werner E.; Takahata, Naoyuki; Klein, Jan

    1998-01-01

    Phylogenetic trees for groups of closely related species often have different topologies, depending on the genes used. One explanation for the discordant topologies is the persistence of polymorphisms through the speciation phase, followed by differential fixation of alleles in the resulting species. The existence of transspecies polymorphisms has been documented for alleles maintained by balancing selection but not for neutral alleles. In the present study, transspecific persistence of neutral polymorphisms was tested in the endemic haplochromine species flock of Lake Victoria cichlid fish. Putative noncoding region polymorphisms were identified at four randomly selected nuclear loci and tested on a collection of 12 Lake Victoria species and their putative riverine ancestors. At all loci, the same polymorphism was found to be present in nearly all the tested species, both lacustrine and riverine. Different polymorphisms at these loci were found in cichlids of other East African lakes (Malawi and Tanganyika). The Lake Victoria polymorphisms must have therefore arisen after the flocks now inhabiting the three great lakes diverged from one another, but before the riverine ancestors of the Lake Victoria flock colonized the Lake. Calculations based on the mtDNA clock suggest that the polymorphisms have persisted for about 1.4 million years. To maintain neutral polymorphisms for such a long time, the population size must have remained large throughout the entire period. PMID:9826684

  12. A Vision for Training and Further Education in Victoria. Scenarios.

    ERIC Educational Resources Information Center

    Victoria Training Board, Melbourne (Australia).

    To revise its strategic plan for vocational education and training (VTE), the Office of Training and Further Education (OTFE) in the state of Victoria (Australia) conducted a three-stage review of strategic directions of VTE in the state. In stage 1, the internal and external environments were scanned to identify major change factors and themes.…

  13. STS-57 Earth observation of Lake Victoria, Africa

    NASA Technical Reports Server (NTRS)

    1993-01-01

    STS-57 Earth observation taken aboard Endeavour, Orbiter Vehicle (OV) 105, is of Africa's Lake Victoria, which sits in the middle of the East African Rift Valley System.Lake Victoria is a major resource in eastern Africa, especially to the countries bordering the lake -- Uganda, Kenya and Tanzania. Typical summer indicators in equatorial Africa -- puffy clouds over land mass and clear skies over the lakes -- are in the view. Lake Albert in the western section of the Rift Valley and Lake Turkana in the eastern section can be seen to the west and east of Lake Victoria, respectively. Most of the other features on the right are obscured by clouds. NASA scientists studying the STS-57 Earth photography point out that the wide perspective of this scene gives a sense of the three-dimensional profile of the whole rift system. The scientists cite the way in which the component valleys of the rift system ramp up to Lake Victoria on either side.

  14. Epilithic lichens in the Beacon sandstone formation, Victoria Land, Antarctica

    NASA Technical Reports Server (NTRS)

    Hale, M. E.; Friedmann, E. I. (Principal Investigator)

    1987-01-01

    The epilithic lichen flora on the Beacon sandstone formation in Victoria Land consists of seven species: Acarospora gwynnii Dodge & Rudolph, Buellia grisea Dodge & Baker, B. pallida Dodge & Baker, Carbonea capsulata (Dodge & Baker) Hale comb. nov., Lecanora fuscobrunnea Dodge & Baker, Lecidea cancriformis Dodge & Baker, and L. siplei Dodge & Baker. The typification of the species is given along with descriptions and distribution in Antarctica.

  15. Information Brokers in Victoria: Doing What, for Whom and How.

    ERIC Educational Resources Information Center

    Broadbent, Marianne; Kelson, Deborah

    1984-01-01

    Reports findings of study of information brokers in Victoria, Australia, which identified services offered by individual brokers and information brokerage businesses, resources used to provide those services, their clientele and pricing strategies, the attributes needed for a successful broker, and relationships between brokers and traditional…

  16. Epilithic lichens in the Beacon sandstone formation, Victoria Land, Antarctica.

    PubMed

    Hale, M E

    1987-01-01

    The epilithic lichen flora on the Beacon sandstone formation in Victoria Land consists of seven species: Acarospora gwynnii Dodge & Rudolph, Buellia grisea Dodge & Baker, B. pallida Dodge & Baker, Carbonea capsulata (Dodge & Baker) Hale comb. nov., Lecanora fuscobrunnea Dodge & Baker, Lecidea cancriformis Dodge & Baker, and L. siplei Dodge & Baker. The typification of the species is given along with descriptions and distribution in Antarctica.

  17. The Free Kindergarten Union of Victoria, 1908-80.

    ERIC Educational Resources Information Center

    Gardiner, Lyndsay

    The social history of the Free Kindergarten Union of the State of Victoria, Australia, from inception in 1908 to the year 1980 is recorded in this book. Growth of the union is described within the context of the World Wars, the Depression, and urbanization and industrialization. The story begins with volunteerism and philanthropy, and with four…

  18. Vocabulary Size Research at Victoria University of Wellington, New Zealand

    ERIC Educational Resources Information Center

    Nation, Paul; Coxhead, Averil

    2014-01-01

    The English Language Institute (now the School of Linguistics and Applied Language Studies) at Victoria University of Wellington has a long history of corpus-based vocabulary research, especially after the arrival of the second director of the institute, H. V. George, and the appointment of Helen Barnard, whom George knew in India. George's…

  19. Metal-enhanced fluorescence of single green fluorescent protein (GFP)

    SciTech Connect

    Fu Yi; Zhang Jian; Lakowicz, Joseph R.

    2008-11-28

    The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.

  20. Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants.

    PubMed

    Richards, H A; Halfhill, M D; Millwood, R J; Stewart, C N

    2003-09-01

    The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.

  1. 48 CFR 245.201-71 - GFP attachments to solicitations and awards.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 3 2011-10-01 2011-10-01 false GFP attachments to... Evaluation Procedures 245.201-71 GFP attachments to solicitations and awards. See PGI 245.201-71 for procedures for preparing GFP attachments to solicitations and awards....

  2. View of 'Bottomless Bay' on Rim of 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    As part of its investigation of 'Victoria Crater,' NASA's Mars Exploration Rover Opportunity examined a section of the scalloped rim called 'Bottomless Bay' (or 'Bahia sin Fondo'). This view shows the northeastern side of Bottomless Bay as seen from the southwest. The exposures combined into this mosaic were taken by the rover's panoramic camera through a 750-nanometer filter during the 1,019th Martian day, or sol, of Opportunity's Mars-surface mission (Dec. 5, 2006).

  3. Regional climate model performance in the Lake Victoria basin

    NASA Astrophysics Data System (ADS)

    Williams, Karina; Chamberlain, Jill; Buontempo, Carlo; Bain, Caroline

    2015-03-01

    Lake Victoria, the second largest freshwater lake in the world, plays a crucial role in the hydrology of equatorial eastern Africa. Understanding how climate change may alter rainfall and evaporation patterns is thus of vital importance for the economic development and the livelihood of the region. Regional rainfall distribution appears, up to a large extent, to be controlled by local drivers which may be not well resolved in general circulation model simulations. We investigate the performance over the Lake Victoria basin of an ensemble of UK Met Office Hadley Centre regional climate model (HadRM3P) simulations at 50 km, driven by five members of the Hadley Centre global perturbed-physics ensemble (QUMP). This is part of the validation of an ensemble of simulations that has been used to assess the impacts of climate change over the continent over the period 1950-2099. We find that the regional climate model is able to simulate a lake/land breeze over Lake Victoria, which is a significant improvement over the driving global climate model and a vital step towards reproducing precipitation characteristics in the region. The local precipitation correlates well with large-scale processes in the Pacific Ocean and Indian Ocean, which is in agreement with observations. We find that the spatial pattern of precipitation in the region and the diurnal cycle of convection is well represented although the amount of rainfall over the lake appears to be overestimated in most seasons. Reducing the observational uncertainty in precipitation over the lake through future field campaigns would enable this model bias to be better quantified. We conclude that increasing the spatial resolution of the model significantly improves its ability to simulate the current climate of the Lake Victoria basin. We suggest that, despite the higher computational costs, the inclusion of a model which allows two-way interactions between the lake and its surroundings should be seriously considered for

  4. Status of VICTORIA: NRC peer review and recent code applications

    SciTech Connect

    Bixler, N.E.; Schaperow, J.H.

    1997-12-01

    VICTORIA is a mechanistic computer code designed to analyze fission product behavior within a nuclear reactor coolant system (RCS) during a severe accident. It provides detailed predictions of the release of radioactive and nonradioactive materials from the reactor core and transport and deposition of these materials within the RCS. A summary of the results and recommendations of an independent peer review of VICTORIA by the US Nuclear Regulatory Commission (NRC) is presented, along with recent applications of the code. The latter include analyses of a temperature-induced steam generator tube rupture sequence and post-test analyses of the Phebus FPT-1 test. The next planned Phebus test, FTP-4, will focus on fission product releases from a rubble bed, especially those of the less-volatile elements, and on the speciation of the released elements. Pretest analyses using VICTORIA to estimate the magnitude and timing of releases are presented. The predicted release of uranium is a matter of particular importance because of concern about filter plugging during the test.

  5. Transcriptome pyrosequencing of the Antarctic brittle star Ophionotus victoriae.

    PubMed

    Burns, Gavin; Thorndyke, Michael C; Peck, Lloyd S; Clark, Melody S

    2013-03-01

    Brittle stars are included within a whole range of species, which contribute to knowledge in the medically important area of tissue regeneration. All brittle stars regenerate lose limbs, but the rate at which this occurs is highly variable and species-specific. One of the slowest rates of arm regeneration reported so far is that of the Antarctic Ophionotus victoriae. Additionally, O. victoriae also has an unusual delay in the onset of regeneration of about 5months. Both processes are of interest for the areas of regeneration biology and adaptation to cold environments. One method of understanding the details of regeneration events in brittle stars is to characterise the genes involved. In the largest transcriptome study of any ophiuroid to date, we describe the results of mRNA pyrosequencing from pooled samples of regenerating arms of O. victoriae. The sequencing reads resulted in 18,000 assembled contiguous sequences of which 19% were putatively annotated by blast sequence similarity searching. We focus on the identification of major gene families and pathways with potential relevance to the regenerative processes including the Wnt/β-catenin pathway, Hox genes, the SOX gene family and the TGF beta signalling pathways. These data significantly increase the amount of ophiuroid sequences publicly available and provide candidate transcripts for the further investigation of the unusual regenerative process in this Antarctic ophiuroid. PMID:23904059

  6. Transcriptome pyrosequencing of the Antarctic brittle star Ophionotus victoriae.

    PubMed

    Burns, Gavin; Thorndyke, Michael C; Peck, Lloyd S; Clark, Melody S

    2013-03-01

    Brittle stars are included within a whole range of species, which contribute to knowledge in the medically important area of tissue regeneration. All brittle stars regenerate lose limbs, but the rate at which this occurs is highly variable and species-specific. One of the slowest rates of arm regeneration reported so far is that of the Antarctic Ophionotus victoriae. Additionally, O. victoriae also has an unusual delay in the onset of regeneration of about 5months. Both processes are of interest for the areas of regeneration biology and adaptation to cold environments. One method of understanding the details of regeneration events in brittle stars is to characterise the genes involved. In the largest transcriptome study of any ophiuroid to date, we describe the results of mRNA pyrosequencing from pooled samples of regenerating arms of O. victoriae. The sequencing reads resulted in 18,000 assembled contiguous sequences of which 19% were putatively annotated by blast sequence similarity searching. We focus on the identification of major gene families and pathways with potential relevance to the regenerative processes including the Wnt/β-catenin pathway, Hox genes, the SOX gene family and the TGF beta signalling pathways. These data significantly increase the amount of ophiuroid sequences publicly available and provide candidate transcripts for the further investigation of the unusual regenerative process in this Antarctic ophiuroid.

  7. Construction of a new GFP vector and its use for Fusaruim oxysporum transformation

    PubMed Central

    Zhang, Shuo; Zhao, Baixia; Liu, Xian; Gao, Zenggui; Huang, Xinyang

    2012-01-01

    In this study, the gfp fragment as a reporter gene had integrated into the form plasmid vector pBC-hygro which contains an expressive promoter of the fungus to facilitate the transformation of Fusarium oxysporum. The resultant plasmid pBC-hygro-GFP was identified by digestion with enzymes. Binary plasmids pBC-hygro-GFP were transformed into F. oxysporum by using the PEG–CaCl2 mediated transformation technique. Results show that the recombinant plasmid pBC-hygro-GFP was constructed correctly. The gfp gene was stably maintained and did not convey any significant loss of phenotype which would affect the survival and behaviour of the tagged strains. Introduction of the gfp gene into F. oxysporum provides a simple, specific and cost-effective method of strain identification for ecological studies. Transcriptional reporter vectors were constructed for using the green fluorescent protein (GFP) reporter. PMID:23961215

  8. Nanoparticle-GFP "chemical nose" sensor for cancer cell identification.

    PubMed

    Moyano, Daniel F; Rotello, Vincent M

    2013-01-01

    Nanoparticle-based sensor arrays have been used to distinguish a wide range of bio-related molecules through pattern recognition. This "chemical nose" approach uses nanoparticles as receptors to selectively identify the analytes, while a transducer reports the binding through a readable signal (fluorescence). Here we describe a procedure that uses functionalized gold nanoparticles as receptors and green fluorescent protein (GFP) as the transducer to identify and differentiate cell state (normal, cancerous, and metastatic), an important tool in early diagnosis and treatment of tumors.

  9. Recent MELCOR and VICTORIA Fission Product Research at the NRC

    SciTech Connect

    Bixler, N.E.; Cole, R.K.; Gauntt, R.O.; Schaperow, J.H.; Young, M.F.

    1999-01-21

    The MELCOR and VICTORIA severe accident analysis codes, which were developed at Sandia National Laboratories for the U. S. Nuclear Regulatory Commission, are designed to estimate fission product releases during nuclear reactor accidents in light water reactors. MELCOR is an integrated plant-assessment code that models the key phenomena in adequate detail for risk-assessment purposes. VICTORIA is a more specialized fission- product code that provides detailed modeling of chemical reactions and aerosol processes under the high-temperature conditions encountered in the reactor coolant system during a severe reactor accident. This paper focuses on recent enhancements and assessments of the two codes in the area of fission product chemistry modeling. Recently, a model for iodine chemistry in aqueous pools in the containment building was incorporated into the MELCOR code. The model calculates dissolution of iodine into the pool and releases of organic and inorganic iodine vapors from the pool into the containment atmosphere. The main purpose of this model is to evaluate the effect of long-term revolatilization of dissolved iodine. Inputs to the model include dose rate in the pool, the amount of chloride-containing polymer, such as Hypalon, and the amount of buffering agents in the containment. Model predictions are compared against the Radioiodine Test Facility (RTF) experiments conduced by Atomic Energy of Canada Limited (AECL), specifically International Standard Problem 41. Improvements to VICTORIA's chemical reactions models were implemented as a result of recommendations from a peer review of VICTORIA that was completed last year. Specifically, an option is now included to model aerosols and deposited fission products as three condensed phases in addition to the original option of a single condensed phase. The three-condensed-phase model results in somewhat higher predicted fission product volatilities than does the single-condensed-phase model. Modeling of U02

  10. Encapsulation of GFP in Complex Coacervate Core Micelles.

    PubMed

    Nolles, Antsje; Westphal, Adrie H; de Hoop, Jacob A; Fokkink, Remco G; Kleijn, J Mieke; van Berkel, Willem J H; Borst, Jan Willem

    2015-05-11

    Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms.

  11. Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

    PubMed

    Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

    2015-01-01

    With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as

  12. Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

    PubMed Central

    Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

    2015-01-01

    With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as

  13. Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

    PubMed

    Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

    2015-01-01

    With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as

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

    SciTech Connect

    Wang Hongmin; Monteiro, Mervyn J. . E-mail: monteiro@umbi.umd.edu

    2007-08-01

    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.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Details of Layers in Victoria Crater's Cape St. Vincent

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity rover spent about 300 sols (Martian days) during 2006 and 2007 traversing the rim of Victoria Crater. Besides looking for a good place to enter the crater, the rover obtained images of rock outcrops exposed at several cliffs along the way.

    The cliff in this image from Opportunity's panoramic camera (Pancam) is informally named Cape St. Vincent. It is a promontory approximately 12 meters (39 feet) tall on the northern rim of Victoria crater, near the farthest point along the rover's traverse around the rim. Layers seen in Cape St. Vincent have proven to be among the best examples of meter scale cross-bedding observed on Mars to date. Cross-bedding is a geologic term for rock layers which are inclined relative to the horizontal and which are indicative of ancient sand dune deposits. In order to get a better look at these outcrops, Pancam 'super-resolution' imaging techniques were utilized. Super-resolution is a type of imaging mode which acquires many pictures of the same target to reconstruct a digital image at a higher resolution than is native to the camera. These super-resolution images have allowed scientists to discern that the rocks at Victoria Crater once represented a large dune field, not unlike the Sahara desert on Earth, and that this dune field migrated with an ancient wind flowing from the north to the south across the region. Other rover chemical and mineral measurements have shown that many of the ancient sand dunes studied in Meridiani Planum were modified by surface and subsurface liquid water long ago.

    This is a Mars Exploration Rover Opportunity Panoramic Camera image acquired on sol 1167 (May 7, 2007), and was constructed from a mathematical combination of 16 different blue filter (480 nm) images.

  18. Microbial community composition in soils of Northern Victoria Land, Antarctica.

    PubMed

    Niederberger, Thomas D; McDonald, Ian R; Hacker, Amy L; Soo, Rochelle M; Barrett, John E; Wall, Diana H; Cary, S Craig

    2008-07-01

    Biotic communities and ecosystem dynamics in terrestrial Antarctica are limited by an array of extreme conditions including low temperatures, moisture and organic matter availability, high salinity, and a paucity of biodiversity to facilitate key ecological processes. Recent studies have discovered that the prokaryotic communities in these extreme systems are highly diverse with patchy distributions. Investigating the physical and biological controls over the distribution and activity of microbial biodiversity in Victoria Land is essential to understanding ecological functioning in this region. Currently, little information on the distribution, structure and activity of soil communities anywhere in Victoria Land are available, and their sensitivity to potential climate change remains largely unknown. We investigated soil microbial communities from low- and high-productivity habitats in an isolated Antarctic location to determine how the soil environment impacts microbial community composition and structure. The microbial communities in Luther Vale, Northern Victoria Land were analysed using bacterial 16S rRNA gene clone libraries and were related to soil geochemical parameters and classical morphological analysis of soil metazoan invertebrate communities. A total of 323 16S rRNA gene sequences analysed from four soils spanning a productivity gradient indicated a high diversity (Shannon-Weaver values > 3) of phylotypes within the clone libraries and distinct differences in community structure between the two soil productivity habitats linked to water and nutrient availability. In particular, members of the Deinococcus/Thermus lineage were found exclusively in the drier, low-productivity soils, while Gammaproteobacteria of the genus Xanthomonas were found exclusively in high-productivity soils. However, rarefaction curves indicated that these microbial habitats remain under-sampled. Our results add to the recent literature suggesting that there is a higher

  19. Exploration of Victoria crater by the Mars rover Opportunity.

    PubMed

    Squyres, S W; Knoll, A H; Arvidson, R E; Ashley, J W; Bell, J F; Calvin, W M; Christensen, P R; Clark, B C; Cohen, B A; de Souza, P A; Edgar, L; Farrand, W H; Fleischer, I; Gellert, R; Golombek, M P; Grant, J; Grotzinger, J; Hayes, A; Herkenhoff, K E; Johnson, J R; Jolliff, B; Klingelhöfer, G; Knudson, A; Li, R; McCoy, T J; McLennan, S M; Ming, D W; Mittlefehldt, D W; Morris, R V; Rice, J W; Schröder, C; Sullivan, R J; Yen, A; Yingst, R A

    2009-05-22

    The Mars rover Opportunity has explored Victoria crater, an approximately 750-meter eroded impact crater formed in sulfate-rich sedimentary rocks. Impact-related stratigraphy is preserved in the crater walls, and meteoritic debris is present near the crater rim. The size of hematite-rich concretions decreases up-section, documenting variation in the intensity of groundwater processes. Layering in the crater walls preserves evidence of ancient wind-blown dunes. Compositional variations with depth mimic those approximately 6 kilometers to the north and demonstrate that water-induced alteration at Meridiani Planum was regional in scope.

  20. Bigger Crater Farther South of 'Victoria' on Mars

    NASA Technical Reports Server (NTRS)

    2008-01-01

    [figure removed for brevity, see original site] Annotated version

    The team operating NASA's Mars Exploration Rover Opportunity has chosen southeast as the direction for the rover's next extended journey, toward a crater more than 20 times wider than 'Victoria Crater.' Opportunity exited Victoria Crater on Aug. 28, 2008, after nearly a year investigating the interior.

    The crater to the southeast is about 22 kilometers (13.7 miles) in diameter and about 300 meters (1,000 feet) deep, exposing a much thicker stack of rock layers than those examined in Victoria Crater.

    The rover team informally calls the bigger crater 'Endeavour' and emphasizes that Opportunity may well never reach it. The rover has already operated more than 18 times longer than originally planned, and the distance to the big crater, about 12 kilometers (7 miles) matches the total distance Opportunity has driven since landing in early 2004. Driving southeastward is expected to take Opportunity to exposures of younger rock layers than is has previously seen and to provide access to rocks on the plain that were thrown long distances by impacts that excavated even deeper, more distant craters.

    The crater that Opportunity will drive toward dominates this orbital view from the Thermal Emission Imaging System (THEMIS) camera on NASA's Mars Odyssey orbiter. The much smaller Victoria Crater is the most prominent circle near the upper left corner of the image. This view is a mosaic of about 50 separate visible-light images taken by THEMIS.

    NASA's Jet Propulsion Laboratory manages the Mars Odyssey and Mars Exploration Rover missions for the NASA Science Mission Directorate, Washington, D.C. THEMIS was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the

  1. View of 'Bottomless Bay' on Rim of 'Victoria' (Altered Contrast)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    As part of its investigation of 'Victoria Crater,' NASA's Mars Exploration Rover Opportunity examined a section of the scalloped rim called 'Bottomless Bay' (or 'Bahia sin Fondo'). This view shows the northeastern side of Bottomless Bay as seen from the southwest. The exposures combined into this mosaic were taken by the rover's panoramic camera through a 750-nanometer filter during the 1,019th Martian day, or sol, of Opportunity's Mars-surface mission (Dec. 5, 2006). Contrast has been altered to improve the visibility of details in shadowed areas.

  2. Exploration of Victoria crater by the mars rover opportunity

    USGS Publications Warehouse

    Squyres, S. W.; Knoll, A.H.; Arvidson, R. E.; Ashley, James W.; Bell, J.F.; Calvin, W.M.; Christensen, P.R.; Clark, B. C.; Cohen, B. A.; De Souza, P.A.; Edgar, L.; Farrand, W. H.; Fleischer, I.; Gellert, Ralf; Golombek, M.P.; Grant, J.; Grotzinger, J.; Hayes, A.; Herkenhoff, K. E.; Johnson, J. R.; Jolliff, B.; Klingelhofer, G.; Knudson, A.; Li, R.; McCoy, T.J.; McLennan, S.M.; Ming, D. W.; Mittlefehldt, D. W.; Morris, R.V.; Rice, J. W.; Schroder, C.; Sullivan, R.J.; Yen, A.; Yingst, R.A.

    2009-01-01

    The Mars rover Opportunity has explored Victoria crater, a ???750-meter eroded impact crater formed in sulfate-rich sedimentary rocks. Impact-related stratigraphy is preserved in the crater walls, and meteoritic debris is present near the crater rim. The size of hematite-rich concretions decreases up-section, documenting variation in the intensity of groundwater processes. Layering in the crater walls preserves evidence of ancient wind-blown dunes. Compositional variations with depth mimic those ???6 kilometers to the north and demonstrate that water-induced alteration at Meridiani Planum was regional in scope.

  3. Development of Neutralization Assay Using an eGFP Chikungunya Virus.

    PubMed

    Deng, Cheng-Lin; Liu, Si-Qing; Zhou, Dong-Gen; Xu, Lin-Lin; Li, Xiao-Dan; Zhang, Pan-Tao; Li, Peng-Hui; Ye, Han-Qing; Wei, Hong-Ping; Yuan, Zhi-Ming; Qin, Cheng-Feng; Zhang, Bo

    2016-06-28

    Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

  4. Development of Neutralization Assay Using an eGFP Chikungunya Virus

    PubMed Central

    Deng, Cheng-Lin; Liu, Si-Qing; Zhou, Dong-Gen; Xu, Lin-Lin; Li, Xiao-Dan; Zhang, Pan-Tao; Li, Peng-Hui; Ye, Han-Qing; Wei, Hong-Ping; Yuan, Zhi-Ming; Qin, Cheng-Feng; Zhang, Bo

    2016-01-01

    Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV. PMID:27367716

  5. Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene.

    PubMed

    Van den Hoecke, Silvie; Verhelst, Judith; Saelens, Xavier

    2016-01-01

    Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the 'CCCGCC' motif in the GFP coding sequence. PMID:27193250

  6. Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

    PubMed Central

    Van den Hoecke, Silvie; Verhelst, Judith; Saelens, Xavier

    2016-01-01

    Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the ‘CCCGCC’ motif in the GFP coding sequence. PMID:27193250

  7. A Snapshot: Multicultural Music Teaching in Schools in Victoria, Australia, Portrayed by School Teachers

    ERIC Educational Resources Information Center

    Nethsinghe, Rohan Nishantha

    2012-01-01

    Due to the changing demographic factors and as demanded by the governmental policies and regulations, schools in Victoria, Australia, are expected to foster multicultural educational programs that address the diverse needs of students. Research has found that school teachers in Victoria struggle to provide the aspired to multicultural education…

  8. 78 FR 48318 - Approval and Promulgation of Air Quality Implementation Plans; Texas; Victoria County, 1997 8...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... AIRS on a monthly basis. The Victoria County monitoring network consists of two ambient air monitors... AGENCY 40 CFR Part 52 Approval and Promulgation of Air Quality Implementation Plans; Texas; Victoria... County, Texas, developed to ensure continued attainment of the 1997 8-hour National Ambient Air...

  9. Events Management Education through CD-ROM Simulation at Victoria University of Technology.

    ERIC Educational Resources Information Center

    Perry, Marcia; And Others

    There has been a rapid growth in the events industry in Victoria and Australia over the past five years with an increase in large scale events--resulting in substantive economic impact. The growth in events in Australia is projected to continue to beyond 2001. The Department of Management at Victoria University of Technology (VU) received a…

  10. Formula-Based Public School Funding System in Victoria: An Empirical Analysis of Equity

    ERIC Educational Resources Information Center

    Bandaranayake, Bandara

    2013-01-01

    This article explores the formula-based school funding system in the state of Victoria, Australia, where state funds are directly allocated to schools based on a range of equity measures. The impact of Victoria' funding system for education in terms of alleviating inequality and disadvantage is contentious, to say the least. It is difficult…

  11. Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera.

    PubMed

    Chachami, Georgia; Lyberopoulou, Aggeliki; Kalousi, Alkmini; Paraskeva, Efrosyni; Pantopoulos, Kostas; Simos, George

    2013-06-14

    Hepcidin, a hepatic hormone, regulates serum iron levels by controlling both intestinal iron absorption and iron release from macrophages. Although transcription of hepcidin is controlled by diverse stimuli, it remains elusive if post-transcriptional steps of its production are also regulated. To address this issue, GFP was fused to the C-terminus of hepcidin and the chimeric hepcidin-GFP protein was expressed in hepatoma Huh7 cells. Expression and secretion of hepcidin-GFP were analyzed by fluorescence microscopy or western blotting and its activity was assessed by in vitro biological assays. Transient over-expression of hepcidin-GFP resulted in production and secretion of premature forms. On the other hand, stable low-level expression led to synthesis and secretion of a properly matured hepcidin-GFP. This form was biologically active since it affected appropriately the levels of IRP2 and ferritin in human THP1 monocytes and targeted ferroportin in mouse J774 macrophages. Treatment of hepcidin-GFP expressing cells with hypoxia (0.1% O2) altered the subcellular distribution of pro-hepcidin-GFP and significantly reduced the secretion of mature hepcidin-GFP. Our hepcidin-GFP expression system allows the investigation of post-transcriptional processing of hepcidin and implicates hypoxia in its secretion control.

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

    PubMed

    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.

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

    PubMed Central

    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. Structural basis for the fast maturation of Arthropoda green fluorescent protein.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew E; Egorov, Nikolay S; Zaraisky, Andrey G; Yampolsky, Ilya V; Merzlyak, Ekaterina M; Shkoporov, Andrey N; Sander, Ian; Lukyanov, Konstantin A; Chudakov, Dmitriy M

    2006-10-01

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore. PMID:16936637

  15. Construction of a recombinant herpesvirus expressing the jellyfish green fluorescent protein.

    PubMed

    Boldogköi, Z; Erdélyi, F; Sik, A; Freund, T F; Fodor, I

    1999-01-01

    Here we report the insertion of a synthetic version of the cDNA encoding the jellyfish (Aequorea victoria) green fluorescent protein (gfph ) into the genome of pseudorabies (Aujeszky's disease) virus (PrV). A putative latency promoter (PLAT) located at the inverted repeat region of the PrV genome was chosen as the target site for the insertion. Recombinant viral DNA designated as vLAT-gfp was generated as a result of homologous recombination between the transfected viral DNA and a plasmid containing the GFP-expression cassette flanked by viral sequences homologous to the target region. Plaques containing recombinant virus were selected visually using a fluorescent microscope. We demonstrated a GFP-expression in infected neurons of rat brain which showed normal morphology at early stage of viral infection by monitoring fluorescent light emission. PMID:10398563

  16. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    SciTech Connect

    Mustafa, Huseyin . E-mail: huseyinm@hotmail.com; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

    2006-04-21

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified {sub 194}LRMEKLNI{sub 201} as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES.

  17. An examination of the selenium nutrition of sheep in Victoria.

    PubMed

    Caple, I W; Andrewartha, K A; Edwards, S J; Halpin, C G

    1980-04-01

    The selenium nutrition of sheep throughout Victoria was assessed by a survey of the blood glutathione peroxidase activity in 708 flocks. It was shown that the blood glutathione peroxidase activity in sheep had a seasonal variation with lowest levels in the spring. The enzyme activity was correlated with the blood selenium concentration. Areas where blood selenium was less than 0.03 micrograms/ml in spring were defined. Sheep with low selenium nutrition were grazing pastures in the high rainfall areas on acid soils, particularly those derived from granite. Selenium concentrations in pasture samples examined were greater than 0.02 mg/kg, and it was found that superphosphate application had no significant effect on the selenium content of pasture. However, management practices such as high stocking rates and rates of application of superphosphate to pasture were associated with low blood glutathione peroxidase activities in sheep. It was concluded that the selenium nutrition of most of the sheep flocks in Victoria is adequate, and that the deficient areas are localised. There seems little requirement for supplementation of adult sheep. As the delayed type of white muscle disease in spring lambs appears to be the main selenium-responsive disorder, direct supplementation of lambs in the low selenium areas would be the most effective method of ensuring adequate selenium nutrition.

  18. Analysis of continuous GPS measurements from southern Victoria Land, Antarctica

    USGS Publications Warehouse

    Willis, Michael J.

    2007-01-01

    Several years of continuous data have been collected at remote bedrock Global Positioning System (GPS) sites in southern Victoria Land, Antarctica. Annual to sub-annual variations are observed in the position time-series. An atmospheric pressure loading (APL) effect is calculated from pressure field anomalies supplied by the European Centre for Medium-Range Weather Forecasts (ECMWF) model loading an elastic Earth model. The predicted APL signal has a moderate correlation with the vertical position time-series at McMurdo, Ross Island (International Global Navigation Satellite System Service (IGS) station MCM4), produced using a global solution. In contrast, a local solution in which MCM4 is the fiducial site generates a vertical time series for a remote site in Victoria Land (Cape Roberts, ROB4) which exhibits a low, inverse correlation with the predicted atmospheric pressure loading signal. If, in the future, known and well modeled geophysical loads can be separated from the time-series, then local hydrological loading, of interest for glaciological and climate applications, can potentially be extracted from the GPS time-series.

  19. On the Rim of 'Victoria Crater' (Vertical Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a vertical projection with geometric seam correction.

  20. On the Rim of 'Victoria Crater' (Polar Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a polar projection with geometric seam correction.

  1. VICTORIA-92 pretest analyses of PHEBUS-FPT0

    SciTech Connect

    Bixler, N.E.; Erickson, C.M.

    1994-01-01

    FPT0 is the first of six tests that are scheduled to be conducted in an experimental reactor in Cadarache, France. The test apparatus consists of an in-pile fuel bundle, an upper plenum, a hot leg, a steam generator, a cold leg, and a small containment. Thus, the test is integral in the sense that it attempts to simulate all of the processes that would be operative in a severe nuclear accident. In FPT0, the fuel will be trace irradiated; in subsequent tests high burn-up fuel will be used. This report discusses separate pretest analyses of the FPT0 fuel bundle and primary circuit have been conducted using the USNRC`s source term code, VICTORIA-92. Predictions for release of fission product, control rod, and structural elements from the test section are compared with those given by CORSOR-M. In general, the releases predicted by VICTORIA-92 occur earlier than those predicted by CORSOR-M. The other notable difference is that U release is predicted to be on a par with that of the control rod elements; CORSOR-M predicts U release to be about 2 orders of magnitude greater.

  2. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-12-29

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

  3. Different photochemical events of a genetically encoded phenyl azide define and modulate GFP fluorescence.

    PubMed

    Reddington, Samuel C; Rizkallah, Pierre J; Watson, Peter D; Pearson, Rachel; Tippmann, Eric M; Jones, D Dafydd

    2013-06-01

    Expanding the genetic code opens new avenues to modulate protein function in real time. By genetically incorporating photoreactive phenyl azide, the fluorescent properties of green fluorescent protein (GFP) can be modulated by light. Depending on the residue in GFP programmed to incorporate the phenyl azide, different effects on function and photochemical pathways are observed.

  4. Synthesis and photochemistry of pH-sensitive GFP chromophore analogues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nobel GFP chromophore analogues containing 2-thienyl-, 5-methyl-2-furyl-, 2-pyrryl, and 6-methyl-2-pyridyl-groups were synthesized, and their fluorescence spectra were recorded across pH range of 1 to 7. The GFP chromophores prevent photoisomerizaiton in acidic media and increase their fluorescent a...

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

    PubMed Central

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

    2015-01-01

    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. PMID:26148899

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

    PubMed

    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. PMID:26263885

  7. Layers of 'Cabo Frio' in 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of 'Victoria crater' is looking southeast from 'Duck Bay' towards the dramatic promontory called 'Cabo Frio.' The small crater in the right foreground, informally known as 'Sputnik,' is about 20 meters (about 65 feet) away from the rover, the tip of the spectacular, layered, Cabo Frio promontory itself is about 200 meters (about 650 feet) away from the rover, and the exposed rock layers are about 15 meters (about 50 feet) tall. This is a red-blue stereo anaglyph generated from images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 430-nanometer filters.

  8. Layers of 'Cabo Frio' in 'Victoria Crater' (False Color)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of 'Victoria crater' is looking southeast from 'Duck Bay' towards the dramatic promontory called 'Cabo Frio.' The small crater in the right foreground, informally known as 'Sputnik,' is about 20 meters (about 65 feet) away from the rover, the tip of the spectacular, layered, Cabo Frio promontory itself is about 200 meters (about 650 feet) away from the rover, and the exposed rock layers are about 15 meters (about 50 feet) tall. This is an enhanced false color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  9. Layers of 'Cape Verde' in 'Victoria Crater' (Enhanced)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is a false color rendering (enhanced to bring out details from within the shadowed regions of the scene) of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  10. Layers of 'Cape Verde' in 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is a red-blue stereo anaglyph generated from images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 430-nanometer filters.

  11. Layers of 'Cape Verde' in 'Victoria Crater' (False Color)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is an enhanced false color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  12. Forty Meters from Entry to Victoria Crater (Vertical)

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view is presented as a vertical projection with geometric seam correction.

  13. Forty Meters from Entry to Victoria Crater (Polar)

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view is presented as a polar projection with geometric seam correction.

  14. Re-evaluation of the kinematics of Victoria Block using continuous GNSS data

    NASA Astrophysics Data System (ADS)

    Fernandes, R. M. S.; Miranda, J. M.; Delvaux, D.; Stamps, D. S.; Saria, E.

    2013-04-01

    The divergent boundary between the Somalia and Nubia plates is a complex tectonic domain where extensional processes are localized along narrow rift structures, isolating small blocks imbedded within the East African Rift. One of these tectonic units is the Victoria Block, which is the subject of this study. Here we process space-geodetic data for 37 permanent GNSS stations distributed along Nubia, Somalia and Victoria to (1) compute the motion of the three tectonic units in the ITRF2008 reference frame and (2) deduce the relative motion of Victoria with respect to its neighbouring plates. The Nubia Plate motion is computed from a set of 25 stations, the Somalia Plate motion from a set of 7 stations and the Victoria motion from a set of 5 stations. Although the number and distribution of the used stations is still not optimal, the good adjustment between observed and predicted motions confirms that Victoria acts as a rigid tectonic block. The instantaneous relative Euler poles for the Nubia-Victoria and Somalia-Victoria pairs are now evaluated as 10.66°N, 32.98E°, 0.120° Myr-1 and 8.02°S, 32.29°E, 0.159° Myr-1, respectively. The computation of the relative interplate velocities along Victoria's boundary is straightforward in most situations because the western and northeastern boundary segments correspond to well-developed rift basins, where extension is mostly normal to rift basin flanks and seismicity concentrates along narrow structures. This is particularly evident on the Western Branch between Victoria and Nubia. The southeastern limit of the Victoria Block is poorly defined, and geodetic data indicate that differential motion between Somalia and Victoria may be accommodated by a complex boundary area, which roughly encompasses the Masai Terrain. Geodetic observations of the Victoria-Somalia boundary along the Eastern Branch, particularly in the Manyara Rift, reveal highly oblique horizontal extension. In this region seismicity is sparse which suggests

  15. PCBs in fish and their cestode parasites in Lake Victoria.

    PubMed

    Oluoch-Otiego, John; Oyoo-Okoth, Elijah; Kiptoo, Kipkorir Koross Godfrey; Chemoiwa, Emily J; Ngugi, Charles C; Simiyu, Gelas; Omutange, Elijah S; Ngure, Veronica; Opiyo, Mary A

    2016-08-01

    Polychlorinated biphenyls (PCBs) are classified as persistent organic pollutants (POPs) regulated by the Stockholm Convention (2001). Although their production and use was stopped almost three decades ago, PCBs are environmental persistent, toxic, and bioaccumulate in biota. We assessed the levels of 7 PCB congeners (IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) in sediment and fish (Oreochromis niloticus, Lates niloticus, and Rastrineobola argentea) and evaluated the potential of cestode fish endoparasite (Monobothrioides sp., Proteocephalaus sp., and Ligula intestinalis) as biomonitors of PCBs in Lake Victoria, Kenya. The median concentration of Σ7PCBs in sediments and fish were 2.2-96.3 μg/kg dw and 300-3,000 μg/kg lw, respectively. At all the sampling sites, CB138, CB153, and CB180 were the dominant PCB congeners in sediment and fish samples. Compared to the muscle of the piscine host, Proteocephalaus sp. (infecting L. niloticus) biomagnified PCBs ×6-14 while Monobothrioides sp. (infecting O. niloticus) biomagnified PCBs ×4-8. Meanwhile, L. intestinalis (infecting R. argentea) biomagnified PCBs ×8-16 compared to the muscle of unparasitized fish. We demonstrate the occurrence of moderate to high levels of PCB in sediments and fish in Lake Victoria. We also provide evidence that fish parasites bioaccumulate higher levels of PCBs than their piscine hosts and therefore provide a promising biomonitor of PCBs. We urge further a long-term study to validate the use of the above cestode fish parasites as biomonitoring tools for PCBs. PMID:27456696

  16. PCBs in fish and their cestode parasites in Lake Victoria.

    PubMed

    Oluoch-Otiego, John; Oyoo-Okoth, Elijah; Kiptoo, Kipkorir Koross Godfrey; Chemoiwa, Emily J; Ngugi, Charles C; Simiyu, Gelas; Omutange, Elijah S; Ngure, Veronica; Opiyo, Mary A

    2016-08-01

    Polychlorinated biphenyls (PCBs) are classified as persistent organic pollutants (POPs) regulated by the Stockholm Convention (2001). Although their production and use was stopped almost three decades ago, PCBs are environmental persistent, toxic, and bioaccumulate in biota. We assessed the levels of 7 PCB congeners (IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) in sediment and fish (Oreochromis niloticus, Lates niloticus, and Rastrineobola argentea) and evaluated the potential of cestode fish endoparasite (Monobothrioides sp., Proteocephalaus sp., and Ligula intestinalis) as biomonitors of PCBs in Lake Victoria, Kenya. The median concentration of Σ7PCBs in sediments and fish were 2.2-96.3 μg/kg dw and 300-3,000 μg/kg lw, respectively. At all the sampling sites, CB138, CB153, and CB180 were the dominant PCB congeners in sediment and fish samples. Compared to the muscle of the piscine host, Proteocephalaus sp. (infecting L. niloticus) biomagnified PCBs ×6-14 while Monobothrioides sp. (infecting O. niloticus) biomagnified PCBs ×4-8. Meanwhile, L. intestinalis (infecting R. argentea) biomagnified PCBs ×8-16 compared to the muscle of unparasitized fish. We demonstrate the occurrence of moderate to high levels of PCB in sediments and fish in Lake Victoria. We also provide evidence that fish parasites bioaccumulate higher levels of PCBs than their piscine hosts and therefore provide a promising biomonitor of PCBs. We urge further a long-term study to validate the use of the above cestode fish parasites as biomonitoring tools for PCBs.

  17. Impact of Land Use on Soil Respiration in Southwestern Victoria

    NASA Astrophysics Data System (ADS)

    Teodosio, B.; Daly, E.; Pauwels, V. R. N.

    2015-12-01

    Land use management is one of the key contributors to the global environmental change. Considerable changes in landscapes have been experienced in Southwestern Victoria, Australia in the past two decades. Eucalyptus globulus (blue gum) plantations have expanded, resulting in possible changes in the water and carbon balances of catchments. The shift from pastures to plantations could have a significant impact on the local carbon balance with possible effects on atmospheric CO2 concentration and vegetation productivity. We present preliminary measurements from a field study comparing soil respiration in a plantation and a pasture. Adjacent catchments in Southwestern Victoria, near Gatum, were used as study areas; the prominent difference between the two catchments is the land use, with one catchment being used as a pasture for livestock grazing and the other catchment being mainly planted with blue gums. The variability of soil respiration in the pasture is governed by differences in soil moisture and substrate content due to local features of the topography and livestock grazing. Soil respiration measurements in the plantation were taken on mounds, access tracks, and open spaces. Most observations on mounds had higher soil respiration possibly due to root and mycorrhizal respiration. The measurements in open spaces had comparable values with mound measurements; this might be due to a less limited radiation. The soil respiration between trees had lower values, possibly because of radiation limitation due to the canopy cover. These preliminary measurements allow us to compare soil respiration variability across catchments with different land uses. This is important to estimate CO2 fluxes from soil to the atmosphere in large areas and will be valuable in estimating gross primary production from measurements of net ecosystem exchange.

  18. Offshore investigations on Wilkes land-Victoria land margin, Antarctica

    SciTech Connect

    Eittreim, S.L.

    1984-04-01

    In January 1984, the US Geological Survey research vessel S. P. Lee carried out investigations of the Antarctic continental margin in the Wilkes Land Victoria Land areas, using 24-channel and high-resolution seismic, sonobuoy refraction, gravity, magnetic, and bottom-sampling methods. This investigation augmented previous surveys of the Dumont d'Urville area by the French Petroleum Institute and explored new areas west and east to the boundary between the onshore Wilkes basin and the Victoria Land highlands. These surveys defined sediment thickness distribution and seismic stratigraphy in this frontier area. The tectonic style of the boundary between the East Antarctic craton and the younger crust of West Antarctica in the Ross Sea is revealed by one multichannel seismic line across this important boundary. The initial breakup of Antarctical from Australia occurred as a slowly spreading phase during the middle Cretaceous. According to Deep Sea Drilling Project results on the Tasman Rise, conditions of restricted circulation existed in the growing basin between the continents before the late Eocene. After the late Eocene, the major oceanic circulation pattern was established. Before that time, conditions were favorable for preservation of organic-carbon deposits on the sea floor. Among the questions to be addressed with this data are the following. How do apparent subsidence rates of this passive margin compare with others around the world. Does the onshore subglacial Wilkes basins to the Otway and Ceduna basins of Australia exists. What is the effect of the ice cap on the stratigraphy of this margin. Do the two major Tertiary ice advances have conspicuous seismic-stratigraphic signatures.

  19. Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs

    NASA Technical Reports Server (NTRS)

    Millwood, R. J.; Halfhill, M. D.; Harkins, D.; Russotti, R.; Stewart, C. N. Jr

    2003-01-01

    The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.

  20. Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs.

    PubMed

    Millwood, R J; Halfhill, M D; Harkins, D; Russotti, R; Stewart, C N

    2003-03-01

    The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes. PMID:12661169

  1. Creating a Global Fiducials Program (GFP) Site: Lago Cachet Dos example in Chile

    NASA Astrophysics Data System (ADS)

    Friesen, B. A.; Nimick, D. A.; Wilson, E. M.

    2012-12-01

    The USGS has been acquiring remotely sensed imagery from all over the world, collecting thousands of images at more than 200 sites, and creating Literal Image Derived Products (LIDPs) as part of the Global Fiducials Program (GFP). The GFP program enables scientists to use these images to study our rapidly changing planet at carefully selected locations around the world. New GFP Sites are created and added each year. Researchers at the University of Alaska, for instance, added areas susceptible to coastal erosion for monitoring the long-term effects of high tides and storms. This presentation will guide you through the process of creating a new GFP site for your area of interest. We will describe the components of the GFP and its archive, the Global Fiducials Library. The Global Fiducials Program—Initial Site Proposal form will be presented and we will show you how to complete each component in order to propose a new site. Lago Cachet Dos, a glacier-dammed lake in southern Chile, is currently being proposed as a new GFP site and will be used as the example for this exercise. In addition, USGS web sites will be used to illustrate how a long-term imagery record can tell a story and to demonstrate viewing and downloading the type of data that will be available online and free of charge through the GFP.

  2. Photoinduced activation of GFP-like proteins in tissues of reef corals

    NASA Astrophysics Data System (ADS)

    Salih, Anya; Wiedenmann, Joerg; Matz, Mikhail; Larkum, Anthony W.; Cox, Guy

    2006-02-01

    A variety of fluorescent and chromophoric proteins homologous to the green fluorescent protein (GFP) has been recently discovered and cloned from non-bioluminescent marine animals, such as corals, and now provide a multitude of colors for use in fluorescence imaging applications. Recently, a novel fluorescence imaging methodology has emerged that utilizes the unique photoactivatory property of several GFP-like proteins, which respond to irradiation by altering their optical properties, thereby providing a new spatio-temporal capability to the GFP-based imaging applications. During our studies of GFP-like proteins from the Great Barrier Reef corals, several novel photoactivatable (PA) GFP-like proteins have been discovered. These include fluorescence photo-amplifiers and reversible photoswitchers, similar to PA jelly-fish derived PA-GFP and Dronpa, that greatly increase their emissions following ultraviolet-A (UVA) irradiation; the red-to-green (R-to-G) converters, similar to DsRed, that rapidly change to green color following single- or 2-photon irradiation; the green-to-red (G-to-R) converters, that acquire bright red fluorescence following UV-violet irradiation, similar to Kaede-like proteins; and the kindling GFP-like proteins, that are non fluorescent, but rapidly acquire bright fluorescence after green light irradiation. We report on the various optical characteristics of these coral PA proteins that may be used to expand the scope of the available fluorescence bio-imaging technologies.

  3. VICTORIA: A mechanistic model of radionuclide behavior in the reactor coolant system under severe accident conditions

    SciTech Connect

    Heames, T.J. ); Williams, D.A.; Johns, N.A.; Chown, N.M. ); Bixler, N.E.; Grimley, A.J. ); Wheatley, C.J. )

    1990-10-01

    This document provides a description of a model of the radionuclide behavior in the reactor coolant system (RCS) of a light water reactor during a severe accident. This document serves as the user's manual for the computer code called VICTORIA, based upon the model. The VICTORIA code predicts fission product release from the fuel, chemical reactions between fission products and structural materials, vapor and aerosol behavior, and fission product decay heating. This document provides a detailed description of each part of the implementation of the model into VICTORIA, the numerical algorithms used, and the correlations and thermochemical data necessary for determining a solution. A description of the code structure, input and output, and a sample problem are provided. The VICTORIA code was developed upon a CRAY-XMP at Sandia National Laboratories in the USA and a CRAY-2 and various SUN workstations at the Winfrith Technology Centre in England. 60 refs.

  4. End‐of‐life decisions in medical practice: a survey of doctors in Victoria (Australia)

    PubMed Central

    Neil, D A; Coady, C A J; Thompson, J; Kuhse, H

    2007-01-01

    Objectives To discover the current state of opinion and practice among doctors in Victoria, Australia, regarding end‐of‐life decisions and the legalisation of voluntary euthanasia. Longitudinal comparison with similar 1987 and 1993 studies. Design and participants Cross‐sectional postal survey of doctors in Victoria. Results 53% of doctors in Victoria support the legalisation of voluntary euthanasia. Of doctors who have experienced requests from patients to hasten death, 35% have administered drugs with the intention of hastening death. There is substantial disagreement among doctors concerning the definition of euthanasia. Conclusions Disagreement among doctors concerning the meaning of the term euthanasia may contribute to misunderstanding in the debate over voluntary euthanasia. Among doctors in Victoria, support for the legalisation of voluntary euthanasia appears to have weakened slightly over the past 17 years. Opinion on this issue is sharply polarised. PMID:18055904

  5. Mitigation of environmental problems in Lake Victoria, East Africa: causal chain and policy options analyses.

    PubMed

    Odada, Eric O; Olago, Daniel O; Kulindwa, Kassim; Ntiba, Micheni; Wandiga, Shem

    2004-02-01

    Lake Victoria is an international waterbody that offers the riparian communities a large number of extremely important environmental services. Over the past three decades or so, the lake has come under increasing and considerable pressure from a variety of interlinked human activities such as overfishing, species introductions, industrial pollution, eutrophication, and sedimentation. In this paper we examine the root causes for overfishing and pollution in Lake Victoria and give possible policy options that can help remediate or mitigate the environmental degradation. PMID:15083646

  6. Mitigation of environmental problems in Lake Victoria, East Africa: causal chain and policy options analyses.

    PubMed

    Odada, Eric O; Olago, Daniel O; Kulindwa, Kassim; Ntiba, Micheni; Wandiga, Shem

    2004-02-01

    Lake Victoria is an international waterbody that offers the riparian communities a large number of extremely important environmental services. Over the past three decades or so, the lake has come under increasing and considerable pressure from a variety of interlinked human activities such as overfishing, species introductions, industrial pollution, eutrophication, and sedimentation. In this paper we examine the root causes for overfishing and pollution in Lake Victoria and give possible policy options that can help remediate or mitigate the environmental degradation.

  7. Analysis of the water balance of Lake Victoria

    NASA Astrophysics Data System (ADS)

    Nossent, J.; de Brabanter, W.; Bauwens, W.

    2009-04-01

    Lake Victoria is situated within an elevated plateau in the western part of Africa's Great Rift Valley and lies within the territory of three countries: Tanzania, Uganda and Kenya. It is Africa's largest lake and the second widest fresh water lake in the world in terms of surface area. It is also the source of the longest branch of the River Nile, the White Nile. The lake's shallowness, limited river inflow, and large surface area relative to its volume make it vulnerable to climate changes and fluctuations of the water level. This affects the surrounding countries and their people a lot, especially in terms of their food supply and economy. The aim of this study was to get more information on the causes of these fluctuations by analysing the water balance of the lake for the period 1970-1974. It was based both on historical data and measurements and new calculations, and compared with previous studies (e.g. Suttcliffe and Parks, 1999). Precipitation and evaporation over the lake surface were calculated with the Thiessen Polygons method, using measurements from stations around the lake and on the islands. The total inflow of the lake is the sum of the contributions of twelve subbasins. One of these subcatchments, the Nzoia-catchment, was modeled with SWAT (Soil and Water Assessment Tool), a physically based, semi-distributed river basin simulator, as a contribution to the development of a water balance model for Lake Victoria. To calculate the outflow at the Owen Falls Dam in Jinja (Uganda), gauge heights of the lake were used in combination with the "Agreed Curve" (the relationship between water level and flow that was set by the policy makers). As the lake is assumed to be a system with a closed mass balance, the combination of the variations in the above mentioned components resulted in changes of the lake's storage, leading to fluctuations of the water level. For the period 1970-1974 the calculated mean monthly evaporation is 133 mm, with a standard deviation

  8. Sensitivity analysis of the GNSS derived Victoria plate motion

    NASA Astrophysics Data System (ADS)

    Apolinário, João; Fernandes, Rui; Bos, Machiel

    2014-05-01

    Fernandes et al. (2013) estimated the angular velocity of the Victoria tectonic block from geodetic data (GNSS derived velocities) only.. GNSS observations are sparse in this region and it is therefore of the utmost importance to use the available data (5 sites) in the most optimal way. Unfortunately, the existing time-series were/are affected by missing data and offsets. In addition, some time-series were close to the considered minimal threshold value to compute one reliable velocity solution: 2.5-3.0 years. In this research, we focus on the sensitivity of the derived angular velocity to changes in the data (longer data-span for some stations) by extending the used data-span: Fernandes et al. (2013) used data until September 2011. We also investigate the effect of adding other stations to the solution, which is now possible since more stations became available in the region. In addition, we study if the conventional power-law plus white noise model is indeed the best stochastic model. In this respect, we apply different noise models using HECTOR (Bos et al. (2013), which can use different noise models and estimate offsets and seasonal signals simultaneously. The seasonal signal estimation is also other important parameter, since the time-series are rather short or have large data spans at some stations, which implies that the seasonal signals still can have some effect on the estimated trends as shown by Blewitt and Lavellee (2002) and Bos et al. (2010). We also quantify the magnitude of such differences in the estimation of the secular velocity and their effect in the derived angular velocity. Concerning the offsets, we investigate how they can, detected and undetected, influence the estimated plate motion. The time of offsets has been determined by visual inspection of the time-series. The influence of undetected offsets has been done by adding small synthetic random walk signals that are too small to be detected visually but might have an effect on the

  9. What do Victoria family physicians think about housecalls?

    PubMed Central

    Hammett, Tess

    2013-01-01

    Objective To determine the proportion of family physicians doing housecalls, the types of patients they think are appropriate to visit at home, whether physicians are satisfied with the number of housecalls they make, reasons family physicians list for not doing housecalls, and what they consider acceptable remuneration and travel time for housecalls. Design A 12-question paper survey was formulated specifically for this study and piloted by 6 family physicians in British Columbia. It was then mailed with a cover letter to 250 physicians' offices and faxed back anonymously. Setting Family physicians' private offices in Victoria, BC, between December 1 and 19, 2010. Participants A total of 250 randomly selected family physicians from a list of 552 physicians practising in Victoria on the College of Physicians and Surgeons of British Columbia website. Main outcome measures Proportion of physicians doing housecalls, reasons stated for not doing housecalls, and mean acceptable remuneration and travel time for a housecall. Results A total of 73 surveys (29.2%) were returned, 5 of which were not fully completed but were included for the questions that were answered. Sixty-four physicians (87.7%) did at least 1 housecall in the past year, 23 (31.5%) did housecalls at least once a month, and 12 (16.4%) did them at least once a week. Of 71 respondents, 64 physicians (90.1%) listed lack of time as a barrier to performing housecalls, 37 (52.1%) listed unsatisfactory remuneration, and 35 (49.3%) listed lengthy travel times. Most physicians indicated that appropriate remuneration for a housecall was either $142.21 (n = 30, 42.9%) or $108.41 (n = 26, 37.1%). Thirty-seven physicians (52.9%) noted that 20 minutes was an acceptable maximum 1-way travel time for a housecall, while 29 (41.4%) listed 10 minutes. Conclusion Several systemic factors, including lack of time, unsatisfactory remuneration, and large geographic catchment areas, make it difficult for urban family physicians

  10. Integrated light and scanning electron microscopy of GFP-expressing cells.

    PubMed

    Peddie, Christopher J; Liv, Nalan; Hoogenboom, Jacob P; Collinson, Lucy M

    2014-01-01

    Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes.

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

    PubMed

    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. PMID:27516189

  12. Gene trapping with GFP: the isolation of developmental mutants in the slime mold Polysphondylium.

    PubMed

    Fey, P; Cox, E C

    1997-11-01

    In order to study how a cell mass undergoes a transition from one symmetry to another in the slime mold Polysphondylium, we developed a genetic screen in which mutant phenotype and gene expression can easily be visualized in the living organism. The screen combines restriction enzyme-mediated integration (REMI) [1,2] and green fluorescent protein (GFP) [3] expression. In REMI, a restriction enzyme is electroporated along with linearized vector into cells, thus determining the site of plasmid insertion and often increasing the integration frequency. A set of transforming plasmids carrying the GFP coding sequence in three reading frames was used for transformation. The plasmids were constructed so that GFP could be expressed only under control of a host promoter. Living transformants expressing GFP spatially and temporally could be rapidly identified in a very large background of non-expressing cells and fruiting bodies. The phenotypes of representative mutants range from cells that cannot aggregate and initiate cell-cell interactions, through mutant fruiting bodies, to apparently wild-type fruiting bodies expressing GFP in all or a subpopulation of cells. The ability to screen mutant living cells and tissues for GFP expression is rapid and effective and likely to have application in many transformable systems where screening by gene and promoter trapping is essential for understanding temporal and spatial gene regulation. PMID:9382807

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

    PubMed

    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.

  14. Mumps and rubella surveillance in Victoria, 1993 to 2000.

    PubMed

    Guy, Rebecca J; Andrews, Ross M; Robinson, Priscilla M; Lambert, Stephen B

    2003-01-01

    Despite improving childhood coverage of the measles-mumps-rubella vaccine (MMR) in Victoria during the 1990s, mumps and rubella notifications in age groups eligible for vaccination persisted. This study reviewed the mumps and rubella surveillance data from 1993 to 2000 with a specific focus on method of diagnosis. There were 474 notifications of mumps over the seven-year period (annual median 61, range 40 to 77) and 3,544 notifications of rubella (annual median 297, range 66 to 1,165). The highest notifications rates for mumps were consistently among the 1-4 and 5-9 year age groups, whereas there was a marked change in the age distribution of rubella notifications during this interval. A large rubella outbreak occurred in 1995 with 1,165 notifications; the highest notification rates were males aged 15-24 years, infants under one year of age (males and females), and those aged 5-14 years (males and females), respectively. The susceptibility of 5-24 year olds reflects historical changes to the Australian Standard Vaccination Schedule. Rubella notifications returned to baseline levels in 1998 with the highest notification rates in infants aged under one year, and children aged 1-4 years. For both mumps and rubella, the majority of notifications for all age groups were clinically diagnosed, and were most common in children.

  15. Diversity of soil yeasts isolated from South Victoria Land, Antarctica

    USGS Publications Warehouse

    Connell, L.; Redman, R.; Craig, S.; Scorzetti, G.; Iszard, M.; Rodriguez, R.

    2008-01-01

    Unicellular fungi, commonly referred to as yeasts, were found to be components of the culturable soil fungal population in Taylor Valley, Mt. Discovery, Wright Valley, and two mountain peaks of South Victoria Land, Antarctica. Samples were taken from sites spanning a diversity of soil habitats that were not directly associated with vertebrate activity. A large proportion of yeasts isolated in this study were basidiomycetous species (89%), of which 43% may represent undescribed species, demonstrating that culturable yeasts remain incompletely described in these polar desert soils. Cryptococcus species represented the most often isolated genus (33%) followed by Leucosporidium (22%). Principle component analysis and multiple linear regression using stepwise selection was used to model the relation between abiotic variables (principle component 1 and principle component 2 scores) and yeast biodiversity (the number of species present at a given site). These analyses identified soil pH and electrical conductivity as significant predictors of yeast biodiversity. Species-specific PCR primers were designed to rapidly discriminate among the Dioszegia and Leucosporidium species collected in this study. ?? 2008 Springer Science+Business Media, LLC.

  16. Victorias energy efficiency and cogeneration project. Final report

    SciTech Connect

    1998-10-31

    This report describes a two-phase energy project currently contemplated for joint implementation at the Victorias Milling Company, a large sugar mill and refinery on the island of Negros in the Visayas region of the Philippines. The Energy Efficiency (EE) phase is expected to reduce of eliminate VMC`s fossil fuel consumption, which will have a direct and substantial impact on carbon emissions. Phase I is an EE project which involves the installation of equipment to reduce steam and electricity demand in the factories. Phase II, will involve retrofitting and increasing the capacity of the steam and power generation systems, and selling power to the grid. By increasing efficiency and output, the cogeneration project will allow the factory to use only bagasse sugar cane fiber waste as fuel for energy needs. The cogeneration project will also eliminate VMC`s electricity purchases and supply additional power for the island, which will offset generation capacity expansion on the island and the Visayas region.

  17. Work-related suicide in Victoria, Australia: a broad perspective.

    PubMed

    Routley, Virginia Hazel; Ozanne-Smith, Joan E

    2012-01-01

    While unintentional work-related injury is increasingly recognised as important and preventable, population studies of the full range of work related suicides have received less attention. The objective of this study is to investigate the epidemiology of work-related suicide in Victoria, July 2000-December 2007. The study draws on a database of all work-related deaths reported to the Victorian Coroner, inclusive of broadly defined work-relatedness. Inclusion criteria for work-related suicide were at least one of: suicide means was work related, work stressors were identified in police reports to the Coroners or the Coroner's finding, the suicide method involved another person's work (e.g. rail suicide, heavy vehicle) or the suicide location was a workplace. Cases still open for investigation were excluded. Of 642 work-related suicides, 55% had an association with work stressors; 32% jumped or lay in front of a train or heavy vehicle; 7% involved a work location and 6% involved work agents. Work stressor cases identified included business difficulties, recent or previous work injury, unemployment/redundancy or conflict with supervisors/colleagues (including workplace bullying). Work-related suicide is a substantial problem, for which few detailed population wide studies are available. Further research is required to understand the contribution of work stressors and effective interventions. PMID:22132703

  18. Stratigraphie relations of australites in the Port Campbell Embayment, Victoria

    USGS Publications Warehouse

    Shoemakeri, E.M.; Ralph, Uhlherr H.

    1999-01-01

    In the Port Campbell Embayment of Victoria, australites have been found in situ in channel deposits of the Hanson Plain Sand of Pliocene and Pleistocene age. The large majority of the australites, however, occur as a lag deposit at the basal contact of the Sturgess Sand of late Pleistocene and Holocene age and are spatially correlated with ferruginous sandstone clasts that are derived from the Hanson Plain Sand. Some of the tektites are imbedded in or bonded to the ferruginous sandstone clasts, but most are found as individual tektite fragments. A few percent of the tektites have nearly perfectly preserved, complete aerodynamically shaped forms. The sandstone clasts and associated tektites have been reworked from the much older underlying Hanson Plain and have been locally concentrated in the lag deposit. Some tektites also occur at higher levels in the Sturgess Sand, almost invariably in association with stone flakes, exotic stones transported by the aborigines and, locally, with middens of mollusc shells. Circumstantial evidence indicates that the aborigines transported the tektites found in the upper part of the Sturgess, particularly at Stanhope Bay. As Port Campbell australites unequivocally occur in strata much older than the late Pleistocene and Holocene Sturgess, there is no longer any conflict between the apparent stratigraphie age of the tektites and the middle Pleistocene ages obtained by various Chronometrie methods. ?? Meteoritical Society, 1999.

  19. Forty Meters from Entry to Victoria Crater (Stereo)

    NASA Technical Reports Server (NTRS)

    2007-01-01

    [figure removed for brevity, see original site] Left-eye view of a color stereo pair for PIA09972 [figure removed for brevity, see original site] Right-eye view of a color stereo pair for PIA09972

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this stereo view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view combines a stereo pair and appears three-dimensional when seen through blue-red glasses. It is presented as a cylindrical-perspective projection with geometric seam correction.

  20. Analysis of cytoskeleton dynamics and cell migration in drosophila ovaries using GFP-actin and E-cadherin-GFP fusion molecules

    NASA Astrophysics Data System (ADS)

    Verkhusha, Vladyslav V.; Tsukita, Shoichiro; Oda, Hiroki

    1999-06-01

    Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

  1. [DEVELOPMENT OF THE QUADRIVALENT LIVE ATTENUATED INFLUENZA VACCINE INCLUDING TWO INFLUENZA B LINEAGES--VICTORIA AND YAMAGATA].

    PubMed

    Desheva, Yu A; Smolonogina, T A; Doroshenko, E M; Rudenko, L G

    2016-01-01

    This work is devoted to the research of the live attenuated influenza vaccine (LAIV) comprising two reassortant B/USSR/60/69-based vaccine influenza viruses Victoria and Yamagata. The intranasal immunization of the CBA mice with both Victoria and Yamagata strains induced 100% lung protection against the subsequent infection with the wild-type influenza B viruses of any antigen lineage. The quadrivalent LAIV (qLAIV) comprising both reassortant influenza B viruses Victoria and Yamagata were safe and areactogenic in adult volunteers. Following qLAIV administration the immune response was achieved to both Victoria and Yamagata lineages. PMID:27145595

  2. [DEVELOPMENT OF THE QUADRIVALENT LIVE ATTENUATED INFLUENZA VACCINE INCLUDING TWO INFLUENZA B LINEAGES--VICTORIA AND YAMAGATA].

    PubMed

    Desheva, Yu A; Smolonogina, T A; Doroshenko, E M; Rudenko, L G

    2016-01-01

    This work is devoted to the research of the live attenuated influenza vaccine (LAIV) comprising two reassortant B/USSR/60/69-based vaccine influenza viruses Victoria and Yamagata. The intranasal immunization of the CBA mice with both Victoria and Yamagata strains induced 100% lung protection against the subsequent infection with the wild-type influenza B viruses of any antigen lineage. The quadrivalent LAIV (qLAIV) comprising both reassortant influenza B viruses Victoria and Yamagata were safe and areactogenic in adult volunteers. Following qLAIV administration the immune response was achieved to both Victoria and Yamagata lineages.

  3. Role of Coherent Low-Frequency Motion in Excited-State Proton Transfer of Green Fluorescent Protein Studied by Time-Resolved Impulsive Stimulated Raman Spectroscopy.

    PubMed

    Fujisawa, Tomotsumi; Kuramochi, Hikaru; Hosoi, Haruko; Takeuchi, Satoshi; Tahara, Tahei

    2016-03-30

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria, an essential bioimaging tool, luminesces via excited-state proton transfer (ESPT) in which the phenolic proton of the p-hydroxybenzylideneimidazolinone chromophore is transferred to Glu222 through a hydrogen-bond network. In this process, the ESPT mediated by the low-frequency motion of the chromophore has been proposed. We address this issue using femtosecond time-resolved impulsive stimulated Raman spectroscopy. After coherently exciting low-frequency modes (<300 cm(-1)) in the excited state of GFP, we examined the excited-state structural evolution and the ESPT dynamics within the dephasing time of the low-frequency vibration. A clear anharmonic vibrational coupling is found between one high-frequency mode of the chromophore (phenolic CH bend) and a low-frequency mode at ∼104 cm(-1). However, the data show that this low-frequency motion does not substantially affect the ESPT dynamics. PMID:26943852

  4. Green fluorescent protein as a tool for monitoring transgene expression in forest tree species.

    PubMed

    Tian, Lining; Levée, Valerie; Mentag, Rachid; Charest, Pierre J.; Séguin, Armand

    1999-07-01

    The gene coding for green fluorescent protein (GFP) from the jellyfish Aequorea victoria was successfully used as a vital marker for the transformation of three woody plant species, black spruce (Picea mariana (Mill.) BSP), white pine (Pinus strobus L.) and poplar (Populus spp.). The gfp gene and the gene conferring resistance to kanamycin (nptII) were introduced by microprojectile bombardment or Agrobacterium tumefaciens-mediated technology. Screening by fluorescence microscopy of the transformed plant material, under the selection of kanamycin, identified five to eight cell lines from each tree species that clearly expressed GFP. Expression of GFP was observed in somatic embryonal cells of the coniferous species and in stem sections of poplar. For all species, GFP transgene expression was stable over multiple subcultures. Stable integration of the gfp gene into plant genomes was confirmed by Southern hybridization or polymerase chain reaction (PCR) analysis. We conclude that GFP can be used as a vital marker and reporter protein in transformation experiments with gymnosperms and deciduous trees.

  5. Green fluorescence induced by EF-hand assembly in a split GFP system.

    PubMed

    Lindman, Stina; Johansson, Ida; Thulin, Eva; Linse, Sara

    2009-06-01

    The affinity between the 1-157 and 158-238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D(9k) to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1-157 of GFP are fused to residues 1-43 of calbindin, and in EF2-GFPC, residues 44-75 of calbindin are fused to residues 158-238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca(2+)-dependent. PMID:19472338

  6. GAD67-GFP+ Neurons in the Nucleus of Roller. II. Subthreshold and Firing Resonance Properties

    PubMed Central

    Berger, A. J.

    2011-01-01

    In the companion paper we show that GAD67-GFP+ (GFP+) inhibitory neurons located in the Nucleus of Roller of the mouse brain stem can be classified into two main groups (tonic and phasic) based on their firing patterns in responses to injected depolarizing current steps. In this study we examined the responses of GFP+ cells to fluctuating sinusoidal (“chirp”) current stimuli. Membrane impedance profiles in response to chirp stimulation showed that nearly all phasic cells exhibited subthreshold resonance, whereas the majority of tonic GFP+ cells were nonresonant. In general, subthreshold resonance was associated with a relatively fast passive membrane time constant and low input resistance. In response to suprathreshold chirp current stimulation at a holding potential just below spike threshold the majority of tonic GFP+ cells fired multiple action potentials per cycle at low input frequencies (<5 Hz) and either stopped firing or were not entrained by the chirp at higher input frequencies (= tonic low-pass cells). A smaller group of phasic GFP+ cells did not fire at low input frequency but were able to phase-lock 1:1 at intermediate chirp frequencies (= band-pass cells). Spike timing reliability was tested with repeated chirp stimuli and our results show that phasic cells were able to reliably fire when they phase-locked 1:1 over a relatively broad range of input frequencies. Most tonic low-pass cells showed low reliability and poor phase-locking ability. Computer modeling suggested that these different firing resonance properties among GFP+ cells are due to differences in passive and active membrane properties and spiking mechanisms. This heterogeneity of resonance properties might serve to selectively activate subgroups of interneurons. PMID:21047931

  7. GFP Facilitates Native Purification of Recombinant Perlucin Derivatives and Delays the Precipitation of Calcium Carbonate

    PubMed Central

    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 HCO3− as the first ionic interaction partner, but not necessarily for Ca2+. 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. PMID:23056388

  8. Comparative Epidemiology of Influenza B Yamagata- and Victoria-Lineage Viruses in Households.

    PubMed

    Xu, Cuiling; Chan, Kwok-Hung; Tsang, Tim K; Fang, Vicky J; Fung, Rita O P; Ip, Dennis K M; Cauchemez, Simon; Leung, Gabriel M; Peiris, J S Malik; Cowling, Benjamin J

    2015-10-15

    Influenza B viruses split into 2 distinct lineages in the early 1980s, commonly named the Victoria and Yamagata lineages. There are few data on the comparative epidemiology of Victoria- and Yamagata-lineage viruses. In 2007-2011, we enrolled 75 and 34 households containing index patients with acute respiratory illness who tested positive for Yamagata- and Victoria-lineage viruses, respectively, from outpatient clinics in Hong Kong, China. These index patients and their household contacts were followed up for 7-10 days. We examined overall risk of polymerase chain reaction-confirmed infection among household contacts and the risk of secondary infection within households using an individual-based hazard model that accounted for tertiary transmission and infections occurring outside the household. We found that for Victoria-lineage viruses, the risk of within-household infection among household contacts aged ≤15 years was significantly higher (risk ratio = 12.9, 95% credibility interval: 4.2, 43.6) than that for older household contacts, while for Yamagata-lineage viruses, the risk of within-household infection for household contacts did not differ by age. Influenza B Yamagata- and Victoria-lineage viruses have similar characteristics in terms of viral shedding and clinical illness. The mechanisms underlying these epidemiologic differences deserve further investigation. PMID:26400854

  9. Diabetic retinopathy in Victoria, Australia: the Visual Impairment Project

    PubMed Central

    McKay, R.; McCarty, C.; Taylor, H.

    2000-01-01

    AIM—To establish the prevalence, severity, and risk factors for diabetic retinopathy in a representative sample of Victorian residents aged 40 years and older.
METHODS—A population based, cluster sampling method was used to recruit 4744 participants (86% participation rate). Nine randomly selected, suburban Melbourne clusters and four randomly selected, rural Victorian clusters were used. Participants provided a detailed medical and personal history and underwent an ocular examination including funduscopy and fundus photography. Rural participants provided a blood sample, from which the glycosylated haemoglobin percentage was measured. The diagnosis of diabetic retinopathy was based on fundus photographs from participants with self reported diabetes.
RESULTS—The prevalence of diabetic retinopathy among people with self reported diabetes was 29.1%. The prevalence of untreated, vision threatening retinopathy was 2.8%. Retinopathy was positively associated with a longer reported duration of diabetes diagnosis (p<0.01) and with higher fractions of glycosylated haemoglobin (p<0.01). Retinopathy was not significantly associated with age, ethnicity, body mass index, glaucoma, myopia or intake of alcohol, tobacco, or aspirin (all p > 0.05).
CONCLUSIONS—Most people in Victoria with proliferative diabetic retinopathy or clinically significant macular oedema have received laser treatment. There remains however, a small but important group who have not received treatment and whose vision is threatened. People with diabetes should be encouraged to maintain strict glycaemic control and to undergo regular screening to delay or prevent the development of retinopathy.

 PMID:10906093

  10. Impact of cruise ship emissions in Victoria, BC, Canada

    NASA Astrophysics Data System (ADS)

    Poplawski, Karla; Setton, Eleanor; McEwen, Bryan; Hrebenyk, Dan; Graham, Mark; Keller, Peter

    2011-02-01

    Characterization of the effects of cruise ship emissions on local air quality is scarce. Our objective was to investigate community level concentrations of fine particulate matter (PM 2.5), nitrogen dioxide (NO 2) and sulphur dioxide (SO 2) associated with cruise ships in James Bay, Victoria, British Columbia (BC), Canada. Data obtained over four years (2005-2008) at the nearest air quality network site located 3.5 km from the study area, a CALPUFF modeling exercise (2007), and continuous measurements taken in the James Bay community over a three-month period during the 2009 cruise ship season were examined. Concentrations of PM 2.5 and nitrogen oxide (NO) were elevated on weekends with ships present with winds from the direction of the terminal to the monitoring station. SO 2 displayed the greatest impact from the presence of cruise ships in the area. Network data showed peaks in hourly SO 2 when ships were in port during all years. The CALPUFF modeling analysis found predicted 24-hour SO 2 levels to exceed World Health Organization (WHO) guidelines of 20 μg m -3 for approximately 3% of 24-hour periods, with a maximum 24-hour concentration in the community of 41 μg m -3; however, the CALPUFF model underestimated concentrations when predicted and measured concentrations were compared at the network site. Continuous monitoring at the location in the community predicted to experience highest SO 2 concentrations measured a maximum 24-hour concentration of 122 μg m -3 and 16% of 24-hour periods were above the WHO standard. The 10-minute concentrations of SO 2 reached up to 599 μg m -3 and exceeded the WHO 10-minute SO 2 guideline (500 μg m -3) for 0.03% of 10-minute periods. No exceedences of BC Provincial or Canadian guidelines or standards were observed.

  11. E. coli RS2GFP Retention Mechanisms in Laboratory-Scale Fractured Rocks: A Statistical Model

    NASA Astrophysics Data System (ADS)

    Rodrigues, S. N.; Qu, J.; Dickson, S. E.

    2011-12-01

    With billions of gallons of groundwater being withdrawn every day in the US and Canada, it is imperative to understand the mechanisms which jeopardize this resource and the health of those who rely on it. Porous media aquifers have typically been considered to provide significant filtration of particulate matter (e.g. microorganisms), while the fractures in fractured rock aquifers and aquitards are considered to act as contaminant highways allowing a large fraction of pathogens to travel deep into an aquifer relatively quickly. Recent research results indicate that fractured rocks filter out more particulates than typically believed. The goal of the research presented here is to quantify the number of E. coli RS2GFP retained in a single, saturated, laboratory-scale fracture, and to relate the retention of E. coli RS2GFP to the aperture field characteristics and groundwater flow rate. To achieve this goal, physical experiments were conducted at the laboratory-scale to quantify the retention of E. coli RS2GFP through several single, saturated, dolomitic limestone fractures under a range of flow rates. These fractures were also cast with a transparent epoxy in order to visualize the transport mechanisms in the various different aperture fields. The E. coli RS2GFP is tagged with a green-fluorescent protein (GFP) that is used to obtain visualization data when excited by ultraviolet light. A series of experiments was conducted, each of which involved the release of a known number of E. coli RS2GFP at the upstream end of the fracture and measuring the effluent concentration profile. These experiments were conducted using both the natural rock and transparent cast of several different aperture fields, under a range of flow rates. The effects of different aperture field characteristics and flow rates on the retention of E. coli RS2GFP will be determined by conducting a statistical analysis of the retention data under different experimental conditions. The images captured

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

    NASA Technical Reports Server (NTRS)

    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.

  13. A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

    PubMed

    Leonetti, Manuel D; Sekine, Sayaka; Kamiyama, Daichi; Weissman, Jonathan S; Huang, Bo

    2016-06-21

    A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. PMID:27274053

  14. A simple system for converting lacZ to gfp reporter fusions in diverse bacteria.

    PubMed

    Goulian, Mark; van der Woude, Marjan

    2006-05-10

    We describe new plasmids that facilitate the rapid conversion of lacZ fusions to gfp transcriptional fusions in bacteria. The exchange is based on a double recombination between lacZ sequences in a suicide vector and the recipient chromosome. The suicide vector is a mobilizable, conditionally replicative plasmid that contains the gene for gfp with flanking lacZ homology and is derived from a broad host range plasmid that has been successfully used in a wide range of bacterial species. The technique was used to convert lacZ reporter fusions to gfp fusions in Escherichia coli, Bordetella bronchiseptica and Agrobacterium tumefaciens. Green fluorescent protein expression in the new recombinants reflected the beta galactosidase expression in the parent strains. GFP is particularly useful for rapid quantification of gene expression in real time and in single cells. As a demonstration of an application of this system, we studied the induction of virE transcription by the VirA/VirG two-component system in A. tumefaciens in response to various levels of phenolic inducer. Analysis of GFP fluorescence in single cells revealed that at intermediate levels of inducer the population of cells was remarkably heterogeneous. The tools described here will be useful for general studies of transcriptional regulation as well as for applications that require spatial and temporal identification of gene expression, such as in the study of biofilms, and interactions between bacteria and their environment. PMID:16500039

  15. DsRed as a Potential FRET Partner with CFP and GFP

    PubMed Central

    Erickson, Michael G.; Moon, Daniel L.; Yue, David T.

    2003-01-01

    Fluorescence resonance energy transfer (FRET) between mutant green fluorescent proteins (GFP) provides powerful means to monitor in vivo protein-protein proximity and intracellular messengers. However, the leading FRET pair of this class (CFP/YFP) entails suboptimal donor excitation by Argon lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. By contrast, DsRed, along with other members of a growing family of red-shifted sea coral fluorophores, features spectra that could obviate such limitations, using DsRed as FRET acceptor, and GFP or CFP as donor. Nonetheless, DsRed suffers from slow chromophore maturation, which confounds quantitative FRET. Here, we develop strategies minimizing the resulting complexity: 1), Pulsed activation of inducible promoters, driving expression of DsRed-tagged molecules, yields a uniform bolus of mature fluorophore; 2), The 33-FRET detection algorithm, adapted for CFP/DsRed and GFP/DsRed, proves insensitive to distortion by slow maturation. We thus show that DsRed supports strong FRET in CFP-DsRed or GFP-DsRed concatemers. These results reveal the promise of sea coral fluorophores like DsRed as FRET partners with GFP or CFP. PMID:12829514

  16. In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice.

    PubMed

    Takaishi, Shigeo; Shibata, Wataru; Tomita, Hiroyuki; Jin, Guangchun; Yang, Xiangdong; Ericksen, Russell; Dubeykovskaya, Zinaida; Asfaha, Samuel; Quante, Michael; Betz, Kelly S; Shulkes, Arthur; Wang, Timothy C

    2011-02-01

    Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

  17. Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

    PubMed

    Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

    2011-08-15

    Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

  18. Understanding virtual water flows: A multiregion input-output case study of Victoria

    NASA Astrophysics Data System (ADS)

    Lenzen, Manfred

    2009-09-01

    This article explains and interprets virtual water flows from the well-established perspective of input-output analysis. Using a case study of the Australian state of Victoria, it demonstrates that input-output analysis can enumerate virtual water flows without systematic and unknown truncation errors, an issue which has been largely absent from the virtual water literature. Whereas a simplified flow analysis from a producer perspective would portray Victoria as a net virtual water importer, enumerating the water embodiments across the full supply chain using input-output analysis shows Victoria as a significant net virtual water exporter. This study has succeeded in informing government policy in Australia, which is an encouraging sign that input-output analysis will be able to contribute much value to other national and international applications.

  19. Reactivated tectonic boundaries and implications for the reconstruction of southeastern Australia and northern Victoria Land, Antarctica

    SciTech Connect

    Foster, D.A.; Gleadow, A.J.W. )

    1992-03-01

    Rifted continental margins are strongly influenced by the location of zones of preexisting crustal weakness. Apatite fission track thermochronological data indicate that the Paleozoic lithospheric boundary between the Kanmantoo and Lachlan fold belts in western Victoria, Australia, was reactivated during Mesozoic rifting as a transfer fault. To the east of this boundary, 1-2 km of material was denuded during extension, while essentially no denudation took place during rifting to the west. The correlated tectonic boundary within the Bowers terrane of northern Victoria Land, Antarctica, was also reactivated upon rifting. During fragmentation of Gondwana, movement on this zone perpendicular to the direction of rifting probably determined the location of the Tasman Fracture Zone. When Australia and Antarctica are reconstructed along the trace of the Tasman Fracture Zone, the tectonic boundary in Victoria falls into precise alignment with the Bowers terrane.

  20. A Cross-National Comparison of School Drug Policies in Washington State, United States, and Victoria, Australia.

    ERIC Educational Resources Information Center

    Beyers, Jennifer M.; Evans-Whipp, Tracy; Mathers, Megan; Toumbourou, John W.; Catalano, Richard F.

    2005-01-01

    Using mail survey data collected from primary and secondary school administrators in Washington State, United States, and in Victoria, Australia, this study compared aspects of the school drug policy, environment in the 2 states. Documented substance-use policies were prevalent in Washington and Victoria but less prevalent in primary schools,…

  1. Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo.

    PubMed

    To, Tsz-Leung; Schepis, Antonino; Ruiz-González, Rubén; Zhang, Qiang; Yu, Dan; Dong, Zhiqiang; Coughlin, Shaun R; Shu, Xiaokun

    2016-07-21

    Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

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

    SciTech Connect

    Fujimura, Juri; E-mail: juri-f@nms.ac.jp; Ogawa, Rei; Mizuno, Hiroshi; Fukunaga, Yoshitaka; Suzuki, Hidenori

    2005-07-22

    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.

  3. A norovirus intervariant GII.4 recombinant in Victoria, Australia, June 2016: the next epidemic variant?

    PubMed Central

    Bruggink, Leesa; Catton, Michael; Marshall, John

    2016-01-01

    A norovirus recombinant GII.P4_NewOrleans_2009/GII.4_Sydney_2012 was first detected in Victoria, Australia, in August 2015 at low frequency, and then re-emerged in June 2016, having undergone genetic changes. Analysis of 14 years’ surveillance data from Victoria suggests a typical delay of two to seven months between first detection of a new variant and occurrence of a subsequent epidemic linked to that variant. We consider that the current recombinant strain has the potential to become a pandemic variant. PMID:27719750

  4. Stratigraphic architecture of bedrock reference section, Victoria Crater, Meridiani Planum, Mars

    USGS Publications Warehouse

    Edgar, Lauren A.; Grotzinger, John P.; Hayes, Alex G.; Rubin, David M.; Squyres, Steve W.; Bell, James F.; Herkenhoff, Ken E.

    2012-01-01

    The Mars Exploration Rover Opportunity has investigated bedrock outcrops exposed in several craters at Meridiani Planum, Mars, in an effort to better understand the role of surface processes in its geologic history. Opportunity has recently completed its observations of Victoria crater, which is 750 m in diameter and exposes cliffs up to ~15 m high. The plains surrounding Victoria crater are ~10 m higher in elevation than those surrounding the previously explored Endurance crater, indicating that the Victoria crater exposes a stratigraphically higher section than does the Endurance crater; however, Victoria strata overlap in elevation with the rocks exposed at the Erebus crater. Victoria crater has a well-developed geomorphic pattern of promontories and embayments that define the crater wall and that reveal thick bedsets (3–7m) of large-scale cross-bedding, interpreted as fossil eolian dunes. Opportunity was able to drive into the crater at Duck Bay, located on the western margin of Victoria crater. Data from the Microscopic Imager and Panoramic Camera reveal details about the structures, textures, and depositional and diagenetic events that influenced the Victoria bedrock. A lithostratigraphic subdivision of bedrock units was enabled by the presence of a light-toned band that lines much of the upper rim of the crater. In ascending order, three stratigraphic units are named Lyell, Smith, and Steno; Smith is the light-toned band. In the Reference Section exposed along the ingress path at Duck Bay, Smith is interpreted to represent a zone of diagenetic recrystallization; however, its upper contact also coincides with a primary erosional surface. Elsewhere in the crater the diagenetic band crosscuts the physical stratigraphy. Correlation with strata present at nearby promontory Cape Verde indicates that there is an erosional surface at the base of the cliff face that corresponds to the erosional contact below Steno. The erosional contact at the base of Cape Verde

  5. Monitoring the water balance of Lake Victoria, East Africa, from space

    NASA Astrophysics Data System (ADS)

    Swenson, Sean; Wahr, John

    2009-05-01

    SummaryUsing satellite gravimetric and altimetric data, we examine trends in water storage and lake levels of multiple lakes in the Great Rift Valley region of East Africa for the years 2003-2008. GRACE total water storage estimates reveal that water storage declined in much of East Africa, by as much as 60 {mm}/{year}, while altimetric data show that lake levels in some large lakes dropped by as much as 1-2 m. The largest declines occurred in Lake Victoria, the Earth's second largest freshwater body. Because the discharge from the outlet of Lake Victoria is used to generate hydroelectric power, the role of human management in the lake's decline has been questioned. By comparing catchment water storage trends to lake level trends, we confirm that climatic forcing explains only about 50decline. This analysis provides an independent means of assessing the relative impacts of climate and human management on the water balance of Lake Victoria that does not depend on observations of dam discharge, which may not be publically available. In the second part of the study, the individual components of the lake water balance are estimated. Satellite estimates of changes in lake level, precipitation, and evaporation are used with observed lake discharge to develop a parameterization for estimating subsurface inflows due to changes in groundwater storage estimated from satellite gravimetry. At seasonal timescales, this approach provides closure to Lake Victoria's water balance to within 17 {mm}/{month}. The third part of this study uses the water balance of a downstream water body, Lake Kyoga, to estimate the outflow from Lake Victoria remotely. Because Lake Kyoga is roughly 20 times smaller in area than Lake Victoria, its water balance is strongly influenced by inflow from Lake Victoria. Lake Kyoga has been shown to act as a linear reservoir, where its outflow is proportional to the height of the lake. This model can be used with satellite altimetric lake levels to estimate a

  6. Use of mouthguards by basketball players in Victoria, Australia.

    PubMed

    Cornwell, Helen; Messer, Louise Brearley; Speed, Harriet

    2003-08-01

    Basketball is a popular sport in Australia. Although orofacial injuries are common, mouthguard (MG) wear in basketball appears to be low. The purposes of this study were: to measure mouthguard wear by basketball players before and after a promotional intervention; to assess players' knowledge of the value of mouthguards for prevention of injury; and to describe their experience of orofacial injury. Two questionnaires (baseline and follow-up) were administered to a convenience sample of 496 basketball players in Victoria, Australia. Players recruited were youths (12-15-year olds, n = 208) and adults (18 years and over, n = 288), from all basketball levels (social to elite). Completion of the baseline questionnaire was followed immediately by an intervention comprising written and verbal information, a mouthguard blank and instructions on mouthguard construction. The follow-up questionnaire was mailed to all respondents 10-12 weeks later; 135 youths (65%) and 157 adults (54%) completed this. Mouthguard wear at baseline was low but was more frequent at games (62%) than at training (25%). Despite 90% of players acknowledging the protective value of a mouthguard, wear by youths did not increase following the intervention, and wear by adults increased by only 14% for training and 10% at games. Previous orofacial injury was recorded at baseline by 23% of players, but few had requested compensation from Basketball Australia (youths, 17%; adults, 30%). Two predictor variables were statistically identified as related to mouthguard wear: previous orofacial injury and age group. Mouthguard wear was significantly more frequent amongst players with previous injury; such players were 2.76 times more likely to be wearers than those without previous injury. Youths were 2.31 times more likely to wear mouthguards than adults. Only 34 players (12% of respondents at follow-up) had a mouthguard constructed from the blank provided. Although youth and adult groups differed, the overall

  7. Transformation of tobacco plants by Yali PPO-GFP fusion gene and observation of subcellular localization.

    PubMed

    Qi, Jing; Li, Gui-Qin; Dong, Zhen; Zhou, Wei

    2016-01-01

    To explore the subcellular localization of Polyphenol oxidase (PPO) from Pyrus bretschneideri, the 1779 bp cDNA of PPO gene excluding the termination codon TAA was cloned and fused with GFP to construct a binary vector pBI121-PPO-GFP. Then, the binary vector was transformed into Nicotiana tabacum by the tumefanciens-mediated method. Using confocal laser scanning microscopy, green fluorescent signals were localized in chloroplasts of the transformed Nicotiana tabacum cell, suggesting that the Polyphenol oxidase from Pyrus bretschneideri was a chloroplast protein. PMID:27158362

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

    PubMed Central

    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

    2015-01-01

    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 Cγ—Cδ 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. PMID:26249350

  9. Reduction of Photo Bleaching and Long Term Archiving of Chemically Cleared GFP-Expressing Mouse Brains

    PubMed Central

    Becker, Klaus; Hahn, Christian Markus; Saghafi, Saiedeh; Jährling, Nina; Wanis, Martina; Dodt, Hans-Ulrich

    2014-01-01

    Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years. PMID:25463047

  10. Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

    PubMed Central

    Schauer, Christian; Leinders-Zufall, Trese

    2012-01-01

    Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites. New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic β-cells and GFP

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

    PubMed

    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.

  12. Attachment of anti-GFP antibodies to microspheres for optical trapping experiments.

    PubMed

    Spudich, James A; Rice, Sarah E; Rock, Ronald S; Purcell, Thomas J; Warrick, Hans M

    2011-11-01

    In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

  13. Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

    PubMed

    Marr, Lisa; Lülf, Anna-Theresa; Freudenstein, Astrid; Sutter, Gerd; Volz, Asisa

    2016-04-01

    Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens. PMID:26864442

  14. Structural Analysis of the Victoria Quadrangle (H2) of Mercury based on NASA MESSENGER Data

    NASA Astrophysics Data System (ADS)

    Galluzzi, Valentina

    2015-04-01

    Objective of this thesis is the mapping and structural analysis of the H2 quadrangle, “Victoria”, and a reconnaissance study of the geometry and kinematics of lobate scarps on Mercury. To this end, I produced a 1:3,000,000 geologic map of the area using the images provided by the NASA spacecraft MESSENGER, which has been orbiting the planet since March, 2011. The geologic map shows the distribution of smooth plains, intermediate plains, intercrater plains units and a classification of crater materials based on an empirical distinction among three stages of degradation. Structural mapping shows that the H2 quadrangle is dominated by N-S faults (here grouped into the Victoria system) to the east and NE-SW faults (Larrocha system) to the west, with the secondary existence of NW-SE-trending faults (Carnegie system) in the north-western area of the quadrangle. A systematic analysis of these systems has led to the following results. 1) The Victoria system is characterized by a main array of faults located along Victoria Rupes - Endeavour Rupes - Antoniadi Dorsum. The segmentation of this array into three different sectors changes from north to south and is spatially linked to the presence of three volcanic vents located at the boundaries between each sector and at the northern end of the Victoria Rupes sector, suggesting that volcanism and faulting are interrelated. 2) The main array of Carnegie system is kinematically linked and antithetical to the Victoria system. Both systems have arguably controlled the growth of a longitudinal, fault-free, crustal and gravimetric bulge in the central area of the Victoria quadrangle, which is interpreted as a regional contractional pop-up. 3) The Larrocha system is interrupted against the central bulge and thus is probably older than the Victoria and Carnegie systems. Buffered crater counting performed on the Victoria system confirms the young relative age of its fault segments with respect to the map units. The faults of the

  15. Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish

    PubMed Central

    Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-ichi; Kawahara, Atsuo

    2016-01-01

    The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes. PMID:27725766

  16. Connections '99. Proceedings of a Faculty Conference (5th, Victoria, British Columbia, Canada, May 1999).

    ERIC Educational Resources Information Center

    Gibbons, Sandra L., Ed.; Liedtke, Werner W., Ed.

    This proceedings contains 13 papers from the 1999 annual conference of the Faculty of Education, University of Victoria (British Columbia). The papers are: (1) "Sacred and the Profane in Advertising Art" (Bill Zuk, Robert Dalton); (2) "Finding the Fund$ in Fun Run: Evaluating the Effectiveness and Efficiency of Physical Activity Events as…

  17. Diseases diagnosed in broiler chicken flocks in Victoria, Australia, 1977 to 1984.

    PubMed

    Reece, R L; Beddome, V D; Barr, D A

    1985-03-23

    During the period July 1977 to June 1984, specimens from 1402 broiler chicken flocks were submitted to the Veterinary Research Institute, Victoria, Australia for investigation of morbidity, mortality and, or, poor performance. A total of 19,450 post mortem examinations, as well as numerous ancillary tests, were performed. The findings on these flocks are presented and the consequences of some particular diseases noted.

  18. Connections '96. Proceedings of a Faculty Conference (2nd, Victoria, British Columbia, Canada, May 1996).

    ERIC Educational Resources Information Center

    Dayton-Sakari, Mary, Ed.; Miller, Carole S., Ed.; Liedtke, Werner, Ed.

    This proceedings contains 19 papers presented at the second annual faculty conference at the University of Victoria (British Columbia, Canada). Papers cover a wide variety of disciplines, including preschool education, classroom communication, mathematics instruction, theater, attention deficit disorders, distance learning by rural home schoolers,…

  19. Increase in Meningococcal Serogroup W Disease, Victoria, Australia, 2013–2015

    PubMed Central

    Stevens, Kerrie; Sohail, Asma; Franklin, Lucinda J.; Bond, Katherine A.; Brahmi, Aicha; Romanes, Finn; Ong, Katherine S.

    2016-01-01

    In Victoria, Australia, invasive meningococcal disease caused by Neisseria meningitidis serogroup W increased from 4% of all cases in 2013 to 30% in 2015. This increase resulted largely from strains similar to those in the serogroup W sequence type 11 clonal complex, previously described in the United Kingdom and South America. PMID:27648521

  20. Employment Shifts in the Technical and Further Education Workforce in Victoria.

    ERIC Educational Resources Information Center

    Shah, Chandra

    2003-01-01

    Analyzes changes in Technical and Further Education (TAFE) work force in Victoria, Australia, that occurred during the period 1993-98. Main changes include increased participation of women, significant growth in part-time employment, decline in full-time (mainly male) employment, and an increased use of seasonal teachers. (Includes 10 figures and…

  1. 77 FR 61645 - Exelon Generation Company, LLC, Victoria County Station Site; Notice of Withdrawal of Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-10

    ... availability of this application was published in the Federal Register on April 28, 2010 (75 FR 22434). On June 14, 2010 (75 FR 33653), a subsequent notice was published in the Federal Register announcing the...) submitted an application for an Early Site Permit (ESP) for the Victoria County Station (VCS) site...

  2. From Aspiration to Destination: Understanding the Decisions of University Applicants in Regional Victoria

    ERIC Educational Resources Information Center

    Harvey, Andrew; Burnheim, Catherine; Joschko, Lucie; Luckman, Michael

    2012-01-01

    This paper examines the choices and destinations of prospective university students from three regional areas in Victoria. The study is based on information collected for tertiary applicants in the Gippsland, Bendigo and Mildura areas, all of which host a local university campus. Using application and enrollment data, we examine the choices that…

  3. Public Library Development in Victoria; Current Problems. A Seminar in Four Sessions.

    ERIC Educational Resources Information Center

    Williams, R. P., Ed.; And Others

    The eight papers included in this anthology were originally presented during the 1968 quarterly meeting of the Public Libraries Section, Victorian Division of the Library Association of Australia. The papers are: (1) Some Current Problems in Public Library Development in Victoria, (2) The Role of Government and Public Libraries, (3) Regional…

  4. Do invasive bullfrogs in Victoria, British Columbia, Canada, show evidence of parasite release?

    PubMed

    Dare, O K; Forbes, M R

    2013-06-01

    Few studies have examined vertebrate models of invasive species to explore parasite release as a proposed mechanism through which host species might become invasive. In this study, we examined evidence for parasite release in invasive American bullfrogs (Rana catesbeiana/Lithobates catesbeianus) from five sites in Victoria, British Columbia, Canada. We examined helminth species richness, as well as the prevalence, intensity and abundance of lung and kidney fluke infections. These flukes are expected to impose costs on host survival, growth and reproductive output. We compared measures of these parasite taxa with bullfrogs from Ontario and New Brunswick where they are endemic. Helminth species richness in bullfrogs from the Victoria sites was lower than in Ontario bullfrogs, but comparable to reported indices for other endemic populations. The prevalence of lung flukes (Haematoloechus spp.) in bullfrogs from Victoria was twice as high as was observed in the Ontario bullfrogs, and higher than has been reported from other endemic locations. In four of the five study sites in Victoria, numbers of Echinostoma spp. kidney cysts were lower than observed in endemic populations; however, the fifth site had uncharacteristically high numbers of cysts. In this study, there did not appear to be clear evidence to support parasite release using either parasite species numbers, or infection by specific parasite taxa. Instead, the invasive bullfrogs demonstrated high parasite species richness and high levels of infection for parasites known to be harmful to their hosts. PMID:22716948

  5. Connections '98. Proceedings of a Faculty Conference (4th, Victoria, British Columbia, Canada, May 1998).

    ERIC Educational Resources Information Center

    Gibbons, Sandra L., Ed.; Anderson, John O., Ed.

    This proceedings contains 13 papers from the 1998 annual Faculty of Education conference at the University of Victoria, British Columbia (Canada). The papers are: (1) "Struggling with Re-Presentation, Voice, and Self in Narrative Research" (Marla Arvay); (2) "Women's Soccer in Canada: A Slow Road to Equity" (Meredith Bogle, Bruce Howe); (3)…

  6. Employment Shifts in the TAFE Workforce in Victoria, 1993-98. Working Paper.

    ERIC Educational Resources Information Center

    Shah, Chandra

    Data on the work force in Technical and Further Education (TAFE) institutes in Victoria, Australia, for 1993-1998 reveal a number of structural changes. First, the number of women staff increased from 46% to 53%, although men still constitute 54% of the teaching staff. As full-time staff employment dropped an average of 1.1% annually, part-time…

  7. Chlamydia pecorum Infection in Free-ranging Koalas ( Phascolarctos cinereus ) on French Island, Victoria, Australia.

    PubMed

    Legione, Alistair R; Amery-Gale, Jemima; Lynch, Michael; Haynes, Leesa; Gilkerson, James R; Sansom, Fiona M; Devlin, Joanne M

    2016-04-28

    We detected Chlamydia pecorum in two koalas ( Phascolarctos cinereus ) from a closed island population in Victoria, Australia, previously free of Chlamydia infection. The ompA and multilocus sequence type were most closely related to published isolates of livestock rather than koala origin, suggesting potential cross-species transmission of C. pecorum . PMID:26981690

  8. Vulnerability to Bushfires in Rural Australia: A Case Study from East Gippsland, Victoria

    ERIC Educational Resources Information Center

    Whittaker, Joshua; Handmer, John; Mercer, David

    2012-01-01

    This paper investigates the nature and causes of vulnerability to bushfires in the Wulgulmerang district of East Gippsland, Victoria, in south-eastern Australia. In 2003 bushfires devastated the small population of this isolated farming district, destroying homes, agricultural assets and public infrastructure. The fires also adversely affected the…

  9. The Workers' Educational Association of Victoria and the University of Melbourne: A Clash of Purpose?

    ERIC Educational Resources Information Center

    Dadswell, Gordon

    2005-01-01

    The paper challenges an argument made by Alf Wesson in 1972. His argument was that the failure of the University of Melbourne Extension Board to work effectively with the Worker's Educational Association of Victoria was almost exclusively as a result of the poor management skills and personality of the Director of University Extension, Professor…

  10. A Vision for Training and Further Education in Victoria. Vision Statement.

    ERIC Educational Resources Information Center

    Victoria Training Board, Melbourne (Australia).

    The state of Victoria (Australia) needs a well-educated, adaptable work force to face the numerous challenges stemming from the following: shifts in employment toward "knowledge workers"; the impact of competition, technological change, and microeconomic reform; aging of the population; disparate views regarding the purpose of education; and the…

  11. 40 CFR 81.136 - Corpus Christi-Victoria Intrastate Air Quality Control Region.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 17 2010-07-01 2010-07-01 false Corpus Christi-Victoria Intrastate Air Quality Control Region. 81.136 Section 81.136 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... County, Jackson County, Jim Wells County, Kenedy County, Kleberg County, Lavaca County, Live Oak...

  12. Predator removal: effect on fisheries yields in lake victoria (East Africa).

    PubMed

    Marten, G G

    1979-02-16

    Lake Victoria's artisanal fishery has an overfishing problem. A possible solution is suggested by records showing that fish catches are best where predator populations have been reduced by fishing. It may be possible to remedy overfishing by increasing fishing effort, provided the additional effort is directed toward predators. PMID:17813374

  13. Connections '97. Proceedings of a Faculty Conference (3rd, Victoria, British Columbia, Canada, May 1997).

    ERIC Educational Resources Information Center

    Liedtke, Werner, Ed.

    This proceedings contains 17 papers presented at the third annual faculty conference at the University of Victoria (British Columbia, Canada). Papers cover a wide variety of disciplines and topics, including student teaching, athletics, researcher-teacher collaboration, hands-on science instruction, violence prevention, youth violence, counseling,…

  14. Equity Indicators: Measures of Socio-Economic Status at Victoria University.

    ERIC Educational Resources Information Center

    Sinclair, Genevieve; Doughney, James; Palermo, Josephine

    After a review of relevant literature on socioeconomic status (SES) and the ways in which is used for higher education institutional research and policy, a detailed data analysis of Victoria University (VU), Australia student data was undertaken. Between 10,000 and 15,000 domestic student addresses were geocoded to Australian Bureau of Statistics…

  15. People Management Practices in the Public Health Sector: Developments from Victoria, Australia

    ERIC Educational Resources Information Center

    Stanton, Pauline; Bartram, Timothy; Harbridge, Raymond

    2004-01-01

    This study investigates the impact on human resource management (HRM) practices in the public health sector in Victoria, Australia of two different government policy environments. First, it explores the Liberal Coalition Government's decentralisation of public health sector management, from 1992-1999 and second, the Labor Government's…

  16. 78 FR 76403 - Requested Administrative Waiver of the Coastwise Trade Laws: Vessel VICTORIA; Invitation for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-17

    ... Maritime Administration Requested Administrative Waiver of the Coastwise Trade Laws: Vessel VICTORIA... Administration (MARAD), is authorized to grant waivers of the U.S.-build requirement of the coastwise laws under... association, business, labor union, etc.). You may review DOT's complete Privacy Act Statement in the...

  17. Visy Cares Hub and Victoria University: Making the Door of a University Open to the Community

    ERIC Educational Resources Information Center

    Broadbent, Robyn

    2011-01-01

    In 1999, a group of men embarked on a remarkable project that resulted in building a two million dollar youth centre in one of Melbourne's most disadvantaged communities. From the outset, Victoria University (VU) was a keen partner in the project. This project had key synergies with the current experiences of the University--a dual sector higher…

  18. Predator removal: effect on fisheries yields in lake victoria (East Africa).

    PubMed

    Marten, G G

    1979-02-16

    Lake Victoria's artisanal fishery has an overfishing problem. A possible solution is suggested by records showing that fish catches are best where predator populations have been reduced by fishing. It may be possible to remedy overfishing by increasing fishing effort, provided the additional effort is directed toward predators.

  19. The Killing of the Workers' Educational Association of Victoria: A Myth Challenged

    ERIC Educational Resources Information Center

    Dadswell, Gordon

    2004-01-01

    On 21 March 1941, the Council of the Workers' Educational Association of Victoria, Australia, (the Association) voted the organisation out of existence. The demise was in no way contemplated, and there was no practical reason why the Council acted in the way it did. This paper is the story of the destruction of a successful adult education…

  20. Educational Enrolment of Students with a Disability in New South Wales and Victoria.

    ERIC Educational Resources Information Center

    Dempsey, Ian; Foreman, Phil; Jenkinson, Josephine

    2002-01-01

    This article discusses the impact of legislative and special education policy on the educational enrollment of students with a disability across Australia. Enrollment trends in New South Wales and Victoria are examined and discussed within the context of their respective special education policies, disability discrimination legislation, and…

  1. Perception of Aquaculture Education to Support Further Growth of Aquaculture Industry in Victoria, Australia

    ERIC Educational Resources Information Center

    Awal, Sadiqul; Christie, Andrew; Watson, Matthew; Hannadige, Asanka G. T.

    2012-01-01

    Purpose: The central aim of this study was to determine the perception of aquaculture educational provisions in the state of Victoria, and whether they are sufficient to ultimately support further growth of the industry. Design/methodology/approach: Questionnaires were formulated and distributed to participants in a variety of ways, including via…

  2. History and timing of human impact on Lake Victoria, East Africa.

    PubMed

    Verschuren, Dirk; Johnson, Thomas C; Kling, Hedy J; Edgington, David N; Leavitt, Peter R; Brown, Erik T; Talbot, Michael R; Hecky, Robert E

    2002-02-01

    Lake Victoria, the largest tropical lake in the world, suffers from severe eutrophication and the probable extinction of up to half of its 500+ species of endemic cichlid fishes. The continuing degradation of Lake Victoria's ecological functions has serious long-term consequences for the ecosystem services it provides, and may threaten social welfare in the countries bordering its shores. Evaluation of recent ecological changes in the context of aquatic food-web alterations, catchment disturbance and natural ecosystem variability has been hampered by the scarcity of historical monitoring data. Here, we present high-resolution palaeolimnological data, which show that increases in phytoplankton production developed from the 1930s onwards, which parallels human-population growth and agricultural activity in the Lake Victoria drainage basin. Dominance of bloom-forming cyanobacteria since the late 1980s coincided with a relative decline in diatom growth, which can be attributed to the seasonal depletion of dissolved silica resulting from 50 years of enhanced diatom growth and burial. Eutrophication-induced loss of deep-water oxygen started in the early 1960s, and may have contributed to the 1980s collapse of indigenous fish stocks by eliminating suitable habitat for certain deep-water cichlids. Conservation of Lake Victoria as a functioning ecosystem is contingent upon large-scale implementation of improved land-use practices.

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

    PubMed Central

    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

  4. Resolution of multiple GFP color variants and dyes using two-photon microscopy and imaging spectroscopy

    NASA Astrophysics Data System (ADS)

    Lansford, Rusty; Bearman, Gregory H.; Fraser, Scott E.

    2001-07-01

    The imaging of living cells and tissues using laser-scanning microscopy is offering dramatic insights into the spatial and temporal controls of biological processes. The availability of genetically encoded labels such as green fluorescent protein (GFP) offers unique opportunities by which to trace cell movements, cell signaling or gene expression dynamically in developing embryos. Two-photon laser scanning microscopy (TPLSM) is ideally suited to imaging cells in vivo due to its deeper tissue penetration and reduced phototoxicity; however, in TPLSM the excitation and emission spectra of GFP and its color variants [e.g., CyanFP (CFP); yellowFP (YFP)] are insufficiently distinct to be uniquely imaged by conventional means. To surmount such difficulties, we have combined the technologies of TPLSM and imaging spectroscopy to unambiguously identify CFP, GFP, YFP, and redFP (RFP) as well as conventional dyes, and have tested the approach in cell lines. In our approach, a liquid crystal tunable filter was used to collect the emission spectrum of each pixel within the TPLSM image. Based on the fluorescent emission spectra, supervised classification and linear unmixing analysis algorithms were used to identify the nature and relative amounts of the fluorescent proteins expressed in the cells. In a most extreme case, we have used the approach to separate GFP and fluorescein, separated by only 7 nm, and appear somewhat indistinguishable by conventional techniques. This approach offers the needed ability to concurrently image multiple colored, spectrally overlapping marker proteins within living cells.

  5. Non-invasive imaging of transgenic GFP expression in neonatal mouse brain

    NASA Astrophysics Data System (ADS)

    Ho, Gideon; Zhang, Chunyan; Zhuo, Lang

    2007-02-01

    Glial fibrillary acidic protein (GFAP) is a traditional biomarker for astrocytes of the central nervous system. In this study, non-invasive in vivo imaging of GFAP-GFP (green fluorescent protein) expression in the brain of neonatal transgenic mice is used as a novel method to investigate the relationship between the expression of the transgene at 0, 2, 4, 6 and 8 hr post-treatment in mice subjected to a single administration of 12 mg/kg of neurotoxin 1-methyl-4(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH 3-MPTP). The GFP elevation was found to peak at 6 hr and lasted to at least 8 hr after the toxin treatment. Histological examination of fixed brain sections using immunohistochemistry (IHC) shows an increase in GFP and GFAP signal from the substantia nigra pars compacta (SNpc) and the hippocampus. The results have provided quantitative fluorescence and qualitative histological evidence for the activation of the GFAP-GFP transgene in astrocytes following neurotoxin 2'-CH 3-MPTP administration, suggesting that the model described here could be used to study neuronal degeneration such as Parkinson's disease and in general, developmental neurotoxicity in live animals.

  6. In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.

    PubMed

    Lark, Arianna R; Kitamoto, Toshihiro; Martin, Jean-René

    2016-01-08

    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

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

    PubMed Central

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP.

    PubMed

    Hyun, Seong-In; Maruri-Avidal, Liliana; Moss, Bernard

    2015-07-01

    The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER-associated proteins. A 215-amino acid fragment of GFP (S1-10) was expressed in the cytoplasm as a free protein or fused to the N-terminus of calnexin and in the ER as an intraluminal protein or fused to the C-terminus of calnexin. A 16-amino acid fragment of GFP (S11) was fused to the N- or C-terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N- and C-termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N- and C-termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.

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

    PubMed

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

    2016-01-01

    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. PMID:27173288

  11. Colonization of spinach (Spinacia oleracea L.) by GFP-tagged verticillium dahliae.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soilborne fungus, Verticillium dahliae, causes wilt in a wide range of hosts, including spinach (Spinacia oleracea L.). The interaction between a green fluorescent protein (GFP)-tagged V. dahliae strain and spinach was studied by confocal laser scanning microscopy. The roots of spinach seedlings...

  12. An infectious West Nile virus that expresses a GFP reporter gene.

    PubMed

    Pierson, Theodore C; Diamond, Michael S; Ahmed, Asim A; Valentine, Laura E; Davis, Carl W; Samuel, Melanie A; Hanna, Sheri L; Puffer, Bridget A; Doms, Robert W

    2005-03-30

    West Nile virus is a mosquito-borne, neurotropic flavivirus that causes encephalitis in humans and animals. Since being introduced into the Western hemisphere in 1999, WNV has spread rapidly across North America, identifying this virus as an important emerging pathogen. In this study, we developed a DNA-launched infectious molecular clone of WNV that encodes a GFP reporter gene. Transfection of cells with the plasmid encoding this recombinant virus (pWNII-GFP) resulted in the production of infectious WNV capable of expressing GFP at high levels shortly after infection of a variety of cell types, including primary neurons and dendritic cells. Infection of cells with WNII-GFP virus was productive, and could be inhibited with both monoclonal antibodies and interferon-beta, highlighting the potential of this system in the development and characterization of novel inhibitors and therapeutics for WNV infection. As expected, insertion of the reporter gene into the viral genome was associated with a reduced rate of viral replication, providing the selective pressure for the development of variants that no longer encoded the full-length reporter gene cassette. We anticipate this DNA-based, infectious WNV reporter virus will allow novel approaches for the study of WNV infection and its inhibition both in vitro and in vivo.

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

    PubMed

    Chu, Lili; 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

  14. GFP-LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy.

    PubMed

    Korkhov, Vladimir M

    2009-05-01

    Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP-LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP-LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP-Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin-positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP-LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP-LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP-LC3, a fusion protein widely used as a marker for autophagic vesicles and pre-autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes.

  15. Sorting of GFP Tagged NtSyr1, an ABA Related Syntaxin

    PubMed Central

    Gigante, Massimiliano; De Domenico, Stefania; Piro, Gabriella; Dalessandro, Giuseppe

    2006-01-01

    Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers. NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane. In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane. PMID:19521480

  16. Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.

    PubMed

    Bhaskar, Ashima; Chawla, Manbeena; Mehta, Mansi; Parikh, Pankti; Chandra, Pallavi; Bhave, Devayani; Kumar, Dhiraj; Carroll, Kate S; Singh, Amit

    2014-01-01

    Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (EMSH ) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial EMSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of EMSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in EMSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in EMSH , suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied EMSH during infection and show that subpopulation with higher EMSH are susceptible to clinically relevant antibiotics, whereas lower EMSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence. PMID:24497832

  17. Regional nitrogen budget of the Lake Victoria Basin, East Africa: syntheses, uncertainties and perspectives

    NASA Astrophysics Data System (ADS)

    Zhou, Minghua; Brandt, Patric; Pelster, David; Rufino, Mariana C.; Robinson, Timothy; Butterbach-Bahl, Klaus

    2014-10-01

    Using the net anthropogenic nitrogen input (NANI) approach we estimated the N budget for the Lake Victoria Basin in East Africa. The NANI of the basin ranged from 887 to 3008 kg N km-2 yr-1 (mean: 1827 kg N km-2 yr-1) for the period 1995-2000. The net nitrogen release at basin level is due primarily to livestock and human consumption of feed and foods, contributing between 69% and 85%. Atmospheric oxidized N deposition contributed approximately 14% to the NANI of the Lake Victoria Basin, while either synthetic N fertilizer imports or biological N fixations only contributed less than 6% to the regional NANI. Due to the low N imports of feed and food products (<20 kg N km-2 yr-1), nitrogen release to the watershed must be derived from the mining of soil N stocks. The fraction of riverine N export to Lake Victoria accounted for 16%, which is much lower than for watersheds located in Europe and USA (25%). A significant reduction of the uncertainty of our N budget estimate for Lake Victoria Basin would be possible if better data on livestock systems and riverine N export were available. Our study indicates that at present soil N mining is the main source of nitrogen in the Lake Victoria Basin. Thus, sustainable N management requires increasing agricultural N inputs to guarantee food security and rehabilitation and protection of soils to minimize environmental costs. Moreover, to reduce N pollution of the lake, improving management of human and animal wastes needs to be carefully considered in future.

  18. Framework for Integrated Water, Energy, and Environmental Resources Assessment, Planning, and Management: Lake Victoria Case Study

    NASA Astrophysics Data System (ADS)

    Georgakakos, A.; Yao, H.; Tidwell, A.

    2006-12-01

    This article describes a recent assessment study for Lake Victoria in East Africa. The study includes three interlinked components. The first pertains to Lake Victoria regulation and includes climate and hydrologic forecasting (seasonal, inter-annual, and centennial), and outflow regulation (water resources planning). The second pertains to energy system planning, and the third to environmental and socio-economic impact assessments. These components converge at the operational level where water, energy, and environmental management are harmonized through the use of a decision support system, the Lake Victoria Decision Support Tool. Some of the broad study findings are summarized below: (1)Lake Victoria is entering a new era in which sustainable water resources management is tightly linked to and can only be achieved by proactive energy planning. To meet this new challenge, and maximize the benefit of the decision support system, water and energy sector decisions must be institutionally coordinated. (2)Climate and hydrologic forecasts of sufficient skill are critical for effective water resources and energy planning and management. More specifically, extensive assessments with several GCMs and climate scenarios indicate that Lake Victoria will most likely be adversely impacted by climate change with potentially serious local and regional consequences. Furthermore, seasonal and inter-annual forecasts are very important in meeting medium term water and energy demands and minimizing the costs of thermal energy generation. (3)Wetland ecological and socio-economic benefits are comparable to power sector benefits, underscoring the need for more comprehensive evaluations of non-power water uses and integrative water, energy, and environmental planning and management. (4)Integrated forecast-decision systems are excellent means to bring to bear and make practically available advances in various water-related disciplines for the benefit of managers and policy makers.

  19. Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

    PubMed Central

    Kay, Jennifer E.; Na, Li; Rowland, Elizabeth A.; Winther, Kelly E.; Chow, Danielle N.; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S.; Maklakova, Vilena I.; Singh, Vijay R.; Wadduwage, Dushan N.; Rajapakse, Jagath; So, Peter T. C.; Collier, Lara S.; Engelward, Bevin P.

    2014-01-01

    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. PMID:24901438

  20. Preferential and Bidirectional Labeling of the Rubrospinal Tract with Adenovirus-GFP for Monitoring Normal and Injured Axons

    PubMed Central

    Wang, Xiaofei; Smith, George M.

    2011-01-01

    Abstract The rodent rubrospinal tract (RST) has been studied extensively to investigate regeneration and remodeling of central nervous system (CNS) axons. Currently no retrograde tracers can specifically label rubrospinal axons and neurons (RSNs). The RST can be anterogradely labeled by injecting tracers into the red nucleus (RN), but accurately locating the RN is a technical challenge. Here we developed a recombinant adenovirus carrying a green fluorescent protein reporter gene (Adv-GFP) which can preferentially, intensely, and bi-directionally label the RST. When Adv-GFP was injected into the second lumbar spinal cord, the GFP was specifically transported throughout the entire RST, with peak labeling seen at 2 weeks post-injection. When Adv-GFP was injected directly into the RN, GFP was anterogradely transported throughout the RST. Following spinal cord injury (SCI), injection of Adv-GFP resulted in visualization of GFP in transected, spared, or sprouted RST axons bi-directionally. Thus Adv-GFP could be used as a novel tool for monitoring and evaluating strategies designed to maximize RST axonal regeneration and remodeling following SCI. PMID:21299337

  1. Generation of recombinant rabies Virus CVS-11 expressing eGFP applied to the rapid virus neutralization test.

    PubMed

    Xue, Xianghong; Zheng, Xuexing; Liang, Hongru; Feng, Na; Zhao, Yongkun; Gao, Yuwei; Wang, Hualei; Yang, Songtao; Xia, Xianzhu

    2014-04-01

    The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

  2. A GFP-based motif-trap reveals a novel mechanism of targeting for the Toxoplasma ROP4 protein.

    PubMed

    Bradley, Peter J; Li, Nancy; Boothroyd, John C

    2004-09-01

    The protozoan parasite Toxoplasma gondii is a highly specialized eukaryote that contains a remarkable number of intracellular compartments, some unique to Apicomplexans and others typical of eukaryotes in general. We have established a green fluorescent protein (GFP)-based motif-trap to identify proteins targeted to different intracellular locations and subsequently the signals responsible for this sorting. The motif-trap involves the transfection and integration of a linearized GFP construct which lacks a promoter and an initiator methionine codon. FACS is used to isolate parasites in which GFP fuses in-frame into a coding region followed by screening by fluorescence microscopy for those containing GFP targeted to specific intracellular compartments. GFP trapping was successful using vectors designed for integration into regions encoding exons and vectors that were engineered with a splice acceptor site for integration into regions encoding introns. This strategy differs from most protein traps in that the resulting fusions are expressed from the endogenous promoter and starting methionine. Thus, problems from inappropriate expression levels or the creation of fortuitous targeting signals seen in library-based traps are diminished. Using this approach, we have trapped GFP localized to a number of intracellular compartments including the nucleus, nucleolus, endoplasmic reticulum, cytosol, parasite surface and rhoptries of Toxoplasma. Further analysis of a parasite clone containing GFP targeted to the rhoptries shows GFP fused to the gene encoding the rhoptry protein ROP4 and has elucidated an additional mechanism for targeting of this protein.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

    PubMed Central

    Pletnev, Vladimir Z.; Pletneva, Nadya V.; Lukyanov, Konstantin A.; Souslova, Ekaterina A.; Fradkov, Arkady F.; Chudakov, Dmitry M.; Chepurnykh, Tatyana; Yampolsky, Ilia V.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-01-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 Cβ atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis. PMID:23999308

  5. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

    PubMed

    Pletnev, Vladimir Z; Pletneva, Nadya V; Lukyanov, Konstantin A; Souslova, Ekaterina A; Fradkov, Arkady F; Chudakov, Dmitry M; Chepurnykh, Tatyana; Yampolsky, Ilia V; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-09-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(β) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.

  6. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

    PubMed

    Pletnev, Vladimir Z; Pletneva, Nadya V; Lukyanov, Konstantin A; Souslova, Ekaterina A; Fradkov, Arkady F; Chudakov, Dmitry M; Chepurnykh, Tatyana; Yampolsky, Ilia V; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-09-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(β) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis. PMID:23999308

  7. Soil surface lowering due to soil erosion in villages near Lake Victoria, Uganda

    NASA Astrophysics Data System (ADS)

    de Meyer, A.; Deckers, J.; Poesen, J.; Isabirye, M.

    2009-04-01

    In the effort to pinpoint the sources of sediment pollution in Lake Victoria, the contribution of sedi-ment from compounds, landing sites, main roads and footpaths is determined in the catchment of Na-bera Bay and Kafunda Bay at the northern shore of Lake Victoria in southern Uganda. The amount of soil loss in compounds and landing sites is determined by the reconstruction of the original and current soil surface according to botanical and man-made datable objects. The soil erosion rate is then deter-mined by dividing the eroded soil volume (corrected for compaction) by the age of the oldest datable object. In the study area, the average soil erosion rate in compounds amounts to 107 Mg ha-1 year-1 (per unit compound) and in landing sites to 207 Mg ha-1 year-1 (per unit landing site). Although com-pounds and landing sites occupy a small area of the study area (1.1 %), they are a major source of sediment to Lake Victoria (63 %). The soil loss on footpaths and main roads is calculated by multip-lying the total length of footpaths and main roads with the average width and depth (measured towards a reference surface). After the correction for compaction is carried out, the soil erosion rate on foot-paths amounts to 34 Mg ha-1 year-1 and on main roads to 35 Mg ha-1 year-1. Also footpaths and main roads occupy a small area of the study area (1.1 %), but contribute disproportionately to the total soil loss in the catchment (22 %). In this research, the information about the village/compound given by the villager/owner is indispensable. In accordance to an adaptation of the model of McHugh et al. (2002), 32 % of the sediment that is generated in the catchment, is deposited in Lake Victoria (i.e. 2 209 Mg year-1 or 0.7 Mg ha-1 year-1). The main buffer in the study area is papyrus at the shore of Lake Victoria. Also sugarcane can be a major buffer. However, the sugarcane-area is intersected by com-pounds, landing sites, footpaths and main roads that generate large amounts of

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

    SciTech Connect

    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.

  9. Formation of Schiff-base for photoreaction mechanism of red shift of GFP spectra.

    PubMed

    Koseki, Jun; Kita, Yukiumi; Tachikawa, Masanori

    2010-04-01

    We have proposed the formation of Schiff-base between R96 and chromophore (CRO) to elucidate the reaction mechanism for the irreversible red shift of green fluorescent protein (GFP) spectra under the absence of oxygen. The difference between absorption energies of reactant and product for our GFP models with CIS(D)/6-31G* level is 0.21eV, which is in reasonable agreement with the corresponding experimental value of 0.25eV. We have suggested the irreversible photoreaction mechanism, where the CRO excited from ground (S(0)) state to first excited singlet (S(1)) state immediately turns to the first excited triplet (T(1)) state, and the nucleophilic addition reaction occurs on the T(1) state.

  10. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  11. Complete Proton Transfer Cycle in GFP and Its T203V and S205V Mutants.

    PubMed

    Laptenok, Sergey P; Lukacs, Andras; Gil, Agnieszka; Brust, Richard; Sazanovich, Igor V; Greetham, Gregory M; Tonge, Peter J; Meech, Stephen R

    2015-08-01

    Proton transfer is critical in many important biochemical reactions. The unique three-step excited-state proton transfer in avGFP allows observations of protein proton transport in real-time. In this work we exploit femtosecond to microsecond transient IR spectroscopy to record, in D2 O, the complete proton transfer photocycle of avGFP, and two mutants (T203V and S205V) which modify the structure of the proton wire. Striking differences and similarities are observed among the three mutants yielding novel information on proton transfer mechanism, rates, isotope effects, H-bond strength and proton wire stability. These data provide a detailed picture of the dynamics of long-range proton transfer in a protein against which calculations may be compared. PMID:26087935

  12. Complete Proton Transfer Cycle in GFP and Its T203V and S205V Mutants**

    PubMed Central

    Laptenok, Sergey P; Lukacs, Andras; Gil, Agnieszka; Brust, Richard; Sazanovich, Igor V; Greetham, Gregory M; Tonge, Peter J; Meech, Stephen R

    2015-01-01

    Proton transfer is critical in many important biochemical reactions. The unique three-step excited-state proton transfer in avGFP allows observations of protein proton transport in real-time. In this work we exploit femtosecond to microsecond transient IR spectroscopy to record, in D2O, the complete proton transfer photocycle of avGFP, and two mutants (T203V and S205V) which modify the structure of the proton wire. Striking differences and similarities are observed among the three mutants yielding novel information on proton transfer mechanism, rates, isotope effects, H-bond strength and proton wire stability. These data provide a detailed picture of the dynamics of long-range proton transfer in a protein against which calculations may be compared. PMID:26087935

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

    NASA Astrophysics Data System (ADS)

    Awazu, Kunio; Kinpara, Takeshi; Tamiya, Eiichi

    2002-05-01

    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 2. FEL irradiation at a wavelength of 5.75 and 6.1 μm, 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 μm. The maximum transfer efficiency was about 0.5%.

  14. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore

    SciTech Connect

    Huang, Hao; Suslov, Nikolai B.; Li, Nan-Sheng; Shelke, Sandip A.; Evans, Molly E.; Koldobskaya, Yelena; Rice, Phoebe A.; Piccirilli, Joseph A.

    2014-08-21

    Spinach is an in vitro–selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

  15. Evaluation of a GFP reporter gene construct for environmental arsenic detection.

    PubMed

    Roberto, Francisco F; Barnes, Joni M; Bruhn, Debby F

    2002-08-16

    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 beta-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.

  16. Application of fluorescence spectroscopy and multispectral imaging for non-invasive estimation of GFP transfection efficiency

    NASA Astrophysics Data System (ADS)

    Tamošiūnas, M.; Jakovels, D.; Lihačovs, A.; Kilikevičius, A.; Baltušnikas, J.; Kadikis, R.; Šatkauskas, S.

    2014-10-01

    Electroporation and ultrasound induced sonoporation has been showed to induce plasmid DNA transfection to the mice tibialis cranialis muscle. It offers new prospects for gene therapy and cancer treatment. However, numerous experimental data are still needed to deliver the plausible explanation of the mechanisms governing DNA electro- or sono-transfection, as well as to provide the updates on transfection protocols for transfection efficiency increase. In this study we aimed to apply non-invasive optical diagnostic methods for the real time evaluation of GFP transfection levels at the reduced costs for experimental apparatus and animal consumption. Our experimental set-up allowed monitoring of GFP levels in live mice tibialis cranialis muscle and provided the parameters for DNA transfection efficiency determination.

  17. A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene.

    PubMed

    Gadea, Gilles; Bos, Sandra; Krejbich-Trotot, Pascale; Clain, Elodie; Viranaicken, Wildriss; El-Kalamouni, Chaker; Mavingui, Patrick; Desprès, Philippe

    2016-10-01

    Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766(NIID), the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.

  18. A G-Quadruplex-Containing RNA Activates Fluorescence in a GFP-Like Fluorophore

    PubMed Central

    Huang, Hao; Suslov, Nikolai B.; Li, Nan-Sheng; Shelke, Sandip A.; Evans, Molly E.; Koldobskaya, Yelena; Rice, Phoebe A.; Piccirilli, Joseph A.

    2014-01-01

    Spinach is an in vitro selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence.Spinach is thus an RNA analog of GFP, and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2 and 2.4 Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially pre-formed binding site for the fluorophore.The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers. PMID:24952597

  19. Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria.

    PubMed

    Qazi, S.N.A.; Rees, C.E.D.; Mellits, K.H.; Hill, P.J.

    2001-02-01

    The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the Pxyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the Pxyn promoter was replaced with the PxylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population. High-level expression of these reporter constructs in L. monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines. The gfp3+ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.

  20. Multipixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation

    NASA Astrophysics Data System (ADS)

    Greenbaum, Lior; Rothmann, Chana; Hanania, Judith; Malik, Zvi

    2000-12-01

    Spectrally resolved imaging of Green fluorescent protein (GFP) expressed in living COS-7 kidney cells distinguished the subcellular localization and demarcated the processes of protein folding and chromophore formation. COS-7 kidney cells were transfected by a plasmid pEGFP-N1 plasmid followed by incubation for 15 hours for gen expression. At different intervals the cells were examined by fluorescence microscopy and analyzed by a spectral imaging system. After 7 hours, GFP was detected in the cytoplasm, concentrated in a localized form. Spectra of the initial GFP evinced several components that belong both tot he typical fluorescent signal as well as to unspecific fingerprints. At 10 and 15 hours, GFP was seen spread in the cytoplasm as well as in the nucleus, and the specific spectra of the GFP were dominant at the later time. The typical GFP spectral fingerprint is the result of protein folding and chromophore formation following internal oxidation reactions. This folding and chromophore formation process, up to final conformation, was detected by spectral imaging as localized in the nucleus rather than in the cytosol. Thus, the method of fluorescence microscopy combined with multiplex spectral imaging demonstrates distinct biochemical pathways leading to GFP conformation.

  1. Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed.

    PubMed

    Rajasekaran, Kanniah; Cary, Jeffrey W; Cotty, Peter J; Cleveland, Thomas E

    2008-02-01

    Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination.

  2. Photoconversion of matrix targeted GFP enables analysis of continuity and intermixing of the mitochondrial lumen.

    PubMed

    Jakobs, Stefan; Schauss, Astrid C; Hell, Stefan W

    2003-11-01

    We establish photoconversion of green fluorescent protein (GFP) as an optical 'highlighter' to investigate the continuity of the mitochondrial matrix in living budding yeast (Saccharomyces cerevisiae). Photoconversion of GFP resulting in a marked shift of the absorption and emission spectra to longer wavelengths is elicited, under low oxygen conditions, by irradiation with blue light. Photoconversion induced a several 100-fold increase in red fluorescence of matrix targeted GFP without affecting cell viability. The color changing facilitates simple and effective regional optical marking in a conventional fluorescence microscope. We found the mitochondrial compartment of S. cerevisiae to generally consist of one luminally continuous large part and occasionally some additional smaller fragments. Separated fragments fuse within a few minutes to the large part, resulting in a rapid intermixing of the entire mitochondrial matrix compartment. In Deltafis1 and Deltadnm1 mutants restricted in outer membrane fission, the mitochondria are still luminally continuous, suggesting a tight coupling of inner and outer membrane fissions. Matrix constrictions frequently occurring in wild type cells as well as in Deltafis1 and Deltadnm1 mutants do not interfere with luminal continuity.

  3. Strain-specific regulation of striatal phenotype in Drd2-eGFP BAC transgenic mice.

    PubMed

    Chan, C Savio; Peterson, Jayms D; Gertler, Tracy S; Glajch, Kelly E; Quintana, Ruth E; Cui, Qiaoling; Sebel, Luke E; Plotkin, Joshua L; Shen, Weixing; Heiman, Myriam; Heintz, Nathaniel; Greengard, Paul; Surmeier, D James

    2012-07-01

    Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists, providing a powerful means of dissecting complex neural circuits in the brain. Recently, it was reported that one popular line of these mice--mice possessing a BAC transgene with a D(2) dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter--had abnormal striatal gene expression, physiology, and motor behavior. Unlike most of the work using BAC mice, this interesting study relied upon mice backcrossed on the outbred Swiss Webster (SW) strain that were homozygous for the Drd2-eGFP BAC transgene. The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported, SW mice were very sensitive to transgene expression. However, in more commonly used inbred strains of mice (C57BL/6, FVB/N) that were hemizygous for the transgene, the Drd2-eGFP BAC transgene did not alter striatal gene expression, physiology, or motor behavior. Thus, the use of inbred strains of mice that are hemizygous for the Drd2 BAC transgene provides a reliable tool for studying basal ganglia function.

  4. A Novel Binary T-Vector with the GFP Reporter Gene for Promoter Characterization

    PubMed Central

    Jiang, Shu-Ye; Vanitha, Jeevanandam; Bai, Yanan; Ramachandran, Srinivasan

    2014-01-01

    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. PMID:25197968

  5. Detection of Autophagy in Caenorhabditis elegans Using GFP::LGG-1 as an Autophagy Marker.

    PubMed

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-01-04

    In yeast and mammalian cells, the autophagy protein Atg8/LC3 (microtubule-associated proteins 1A/1B light chain 3B encoded by MAP1LC3B) has been the marker of choice to detect double-membraned autophagosomes that are produced during the process of autophagy. A lipid-conjugated form of Atg8/LC3B is localized to the inner and outer membrane of the early-forming structure known as the phagophore. During maturation of autophagosomes, Atg8/LC3 bound to the inner autophagosome membrane remains in situ as the autophagosomes fuse with lysosomes. The nematode Caenorhabditis elegans is thought to conduct a similar process, meaning that tagging the nematode ortholog of Atg8/LC3-known as LGG-1-with a fluorophore has become a widely accepted method to visualize autophagosomes. Under normal growth conditions, GFP-modified LGG-1 displays a diffuse expression pattern throughout a variety of tissues, whereas, when under conditions that induce autophagy, the GFP::LGG-1 tag labels positive punctate structures, and its overall level of expression increases. Here, we present a protocol for using fluorescent reporters of LGG-1 coupled to GFP to monitor autophagosomes in vivo. We also discuss the use of alternative fluorescent markers and the possible utility of the LGG-1 paralog LGG-2.

  6. Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

    PubMed

    Masullo, Tiziana; Armata, Nerina; Pendolino, Flavio; Colombo, Paolo; Lo Celso, Fabrizio; Mazzola, Salvatore; Cuttitta, Angela

    2016-02-01

    The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery. PMID:26490379

  7. Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

    PubMed

    Masullo, Tiziana; Armata, Nerina; Pendolino, Flavio; Colombo, Paolo; Lo Celso, Fabrizio; Mazzola, Salvatore; Cuttitta, Angela

    2016-02-01

    The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.

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

    PubMed

    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. PMID:25423905

  9. Selection of IgE-binding aptameric green fluorescent protein (Ap-GFP) by the ribosome display (RD) platform

    SciTech Connect

    Chen, S.-S. Yang Yongmin; Barankiewicz, Teresa J.

    2008-09-26

    GFP-C{kappa} fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 {mu}g of the dual site aptameric GFP library exhibiting a diversity of 7.5 x 10{sup 11} was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.

  10. Episodic rifting of phanerozoic rocks in the victoria land basin, Western ross sea, antarctica.

    PubMed

    Cooper, A K; Davey, F J

    1985-09-13

    Multichannel seismic-reflection data show that the Victoria Land-basin, unlike other sedimentary basins in the Ross Sea, includes a rift-depression 15 to 25 kilometers wide that parallels the Transantarctic Mountains and contains up to 12 kilometers of possible Paleozoic to Holocene age sedimentary rocks. An unconformity separates the previously identified Cenozoic sedimentary section from the underlying strata of possible Mesozoic and Paleozoic age. Late Cenozoic volcanic rocks intrude into the entire section along the eastern flank of the basin. The Victoria Land basin is probably part of a more extensive rift system that has been active episodically since Paleozoic time. Inferred rifting and basin subsidence during Mesozoic and Cenozoic time may be associated with regional crustal extension and uplift of the nearby Transantarctic Mountains.

  11. VLUIS, a land use data product for Victoria, Australia, covering 2006 to 2013

    PubMed Central

    Morse-McNabb, Elizabeth; Sheffield, Kathryn; Clark, Rob; Lewis, Hayden; Robson, Susan; Cherry, Don; Williams, Steve

    2015-01-01

    Land Use Information is a key dataset required to enable an understanding of the changing nature of our landscapes and the associated influences on natural resources and regional communities. The Victorian Land Use Information System (VLUIS) data product has been created within the State Government of Victoria to support land use assessments. The project began in 2007 using stakeholder engagement to establish product requirements such as format, classification, frequency and spatial resolution. Its genesis is significantly different to traditional methods, incorporating data from a range of jurisdictions to develop land use information designed for regular on-going creation and consistency. Covering the entire landmass of Victoria, the dataset separately describes land tenure, land use and land cover. These variables are co-registered to a common spatial base (cadastral parcels) across the state for the period 2006 to 2013; biennially for land tenure and land use, and annually for land cover. Data is produced as a spatial GIS feature class. PMID:26602150

  12. The Cambrian Ross Orogeny in northern Victoria Land (Antarctica) and New Zealand: A synthesis

    USGS Publications Warehouse

    Federico, L.; Capponi, G.; Crispini, L.; Bradshaw, J.D.

    2007-01-01

    In the Cambrian, the paleo-Pacific margin of the Gondwana supercontinent included East Antarctica, Australia, Tasmania and New Zealand and was affected by themajor Ross-Delamerian Orogeny. In Antarctica, evidence suggests that this resulted from oblique subduction and that in northern Victoria Land it was accompanied by the opening and subsequent closure of a back-arc basin. Comparison of the type and timing of sedimentary, magmatic and metamorphic events in areas noted above shows strong similarities between northern Victoria Land and New Zealand. In both regions Middle Cambrian volcanites are interpreted as arc/back-arc assemblages produced by west-directed subduction; sediments interbedded with the volcanites show provenance both from the arc and from the Gondwana margin and therefore place the basin close to the continent. Back-arc closure in the Late Cambrian was likely accomplished through a second subduction system

  13. Distribution of Cr, Pb, Cd, Zn, Fe and Mn in Lake Victoria sediments, East Africa

    SciTech Connect

    Onyari, J.M.; Wandiga, S.O. )

    1989-06-01

    The presence of many metals at trace or ultra-trace levels in the human environment has received increased global attention. Sediments as a sink for pollutants are widely recognized pollution sources and diagenesis and biochemical transformations within the sediment may mobilize pollutants posing a threat to a wider biological community. The natural (background) concentrations of heavy metals in lake sediments can be estimated either by analysis of surface sediments in non-polluted regions or by analysis of core samples antedating modern pollution. The distribution pattern of heavy metals in tropical freshwater systems has been little studied. The authors found increased concentrations of lead and other trace metals in Lake Victoria. Thus this study was initiated in order to further investigate the distribution patterns of lead and other metals in Lake Victoria.

  14. VLUIS, a land use data product for Victoria, Australia, covering 2006 to 2013.

    PubMed

    Morse-McNabb, Elizabeth; Sheffield, Kathryn; Clark, Rob; Lewis, Hayden; Robson, Susan; Cherry, Don; Williams, Steve

    2015-01-01

    Land Use Information is a key dataset required to enable an understanding of the changing nature of our landscapes and the associated influences on natural resources and regional communities. The Victorian Land Use Information System (VLUIS) data product has been created within the State Government of Victoria to support land use assessments. The project began in 2007 using stakeholder engagement to establish product requirements such as format, classification, frequency and spatial resolution. Its genesis is significantly different to traditional methods, incorporating data from a range of jurisdictions to develop land use information designed for regular on-going creation and consistency. Covering the entire landmass of Victoria, the dataset separately describes land tenure, land use and land cover. These variables are co-registered to a common spatial base (cadastral parcels) across the state for the period 2006 to 2013; biennially for land tenure and land use, and annually for land cover. Data is produced as a spatial GIS feature class. PMID:26602150

  15. Adenocarcinoma of the nose and paranasal sinuses in woodworkers in the state of Victoria, Australia.

    PubMed

    Ironside, P; Matthews, J

    1975-09-01

    The case index of the Cancer Institute of Victoria (Australia) contained 19 cases of adenocarcinoma of the nose and paranasal sinuses. Eighteen of the cases were in men and 1 in a women. Routine questioning of these patients revealed an occupation involving woodworking in 7 cases, whereas among 80 cases of other malignant tumors of the nose and sinuses there were only 4 who had been woodworkers. Among the patients with adenocarcinoma of the nose and sinuses, there was a significantly higher proportion of woodworkers than in the general population. The findings are consistent with European reports associating nasal adenocarcinoma with wood dust, but whereas the workers at risk in Europe are mainly in the furniture industry, some of the workers affected in Victoria have been sawmillers or carpenters. The specific salivary patterns of tumors of mucous glands are not associated with woodworking.

  16. Mortality-temperature thresholds for ten major population centres in rural Victoria, Australia.

    PubMed

    Loughnan, Margaret; Nicholls, Neville; Tapper, Nigel

    2010-11-01

    Mortality-temperature relationships in small regional towns in Victoria, Australia, were used to ascertain whether the effects of high ambient temperatures documented in the literature for major population centres in Europe and America are also noted in small rural communities in Australia. The establishment of threshold temperatures in all major rural regions of Victoria indicate that hot weather results in an increase in mortality in persons aged 65 years and older. This adds considerable strength to the argument that human populations are vulnerable to heat events regardless of location. Heat alerts can be issued through local health and welfare agencies, to increase awareness of 'hot' weather as a health hazard for elderly people by providing education campaigns involving local authorities based on these simple thresholds. PMID:20797898

  17. A remembrance of Victoria and the Canadian Army Medical Corps in the Great War.

    PubMed

    Carter, Preston L

    2008-05-01

    The North Pacific Surgical Association first met in Victoria in December, 1917, in the midst of World War I, or as it was known then, the Great War. On all sides, the toll in human life was staggering. Canada alone lost more than 60,000 men in the war. Our Association now returns to Victoria as the very last survivors of that generation pass into history. We honor the great sacrifice of the Canadian Army, recall the horrific conditions they endured, and honor the doctors and nurses who attended the countless wounded through the experiences of a Canadian surgeon from Calgary, Dr. Harold McGill, who served for 3 years in the thick of action on the Western Front.

  18. Newborn screening in Victoria: a case study of tissue banking regulation.

    PubMed

    Lawson, Charles

    2008-12-01

    The regulation of human tissue collections is increasingly important in maintaining public trust (and legitimacy) for critical practices and resources directed to public health programs and research. This article examines the governance arrangements applying to VCGS Ltd (under its various incarnations as "Genetic Health Services Victoria", "VCGS Pathology", and so on) and the existing collection of population-wide blood samples maintained on newborn screening cards (or Guthrie cards) in Victoria. The analyses reveal a complex web of regulations (and possibly even no regulation) and the limited role of significant statutory schemes that are generally assumed to apply to human tissue collections and the data and information derived from those materials. The article argues that, without a clear regulatory framework (and in particular meaningful consent), there is likely to be a decline in public trust (and legitimacy) with a consequent decreased participation in what is a public health program with immediate and quantifiable benefits and a valuable research resource for the future.

  19. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  20. Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

    NASA Technical Reports Server (NTRS)

    Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

    1998-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

  1. Transient-state kinetic analysis of complex formation between photoprotein clytin and GFP from jellyfish Clytia gregaria.

    PubMed

    Eremeeva, Elena V; van Berkel, Willem J H; Vysotski, Eugene S

    2016-02-01

    Luminous organisms use different protein-mediated strategies to modulate light emission color. Here, we report the transient-state kinetic studies of the interaction between photoprotein clytin from Clytia gregaria and its antenna protein, cgreGFP. We propose that cgreGFP forms a transient complex with Ca(2+)-bound clytin before the excited singlet state of the coelenteramide product is formed. From the spectral distribution and donor-acceptor separation distance, we infer that clytin reaction intermediates may interact only with the middle side part of cgreGFP. PMID:26867648

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

    NASA Astrophysics Data System (ADS)

    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.

  3. The minimal response to contact metamorphism by the Devonian Buchan Caves Limestone, Buchan Rift, Victoria, Australia

    USGS Publications Warehouse

    Barker, C.E.; Bone, Y.

    1995-01-01

    A 2.2 m thick, Late Eocene (?) dike that intruded the Devonian Buchan Caves Limestone, near Murrindal, Victoria, has produced a narrow contact aureole only centimeters wide in the adjacent host rock. The lack of response of the Buchan Caves Limestone to contact metamorphism is attributed to: 1) prior heating to near 200??C; and 2) the fact that the dike intruded into cool, near surface, low-porosity rocks which may have been in the vadose zone. -from Authors

  4. Satellite-Based Assessment of the spatial extent of Aquatic Vegetation in Lake Victoria

    NASA Astrophysics Data System (ADS)

    Clark, W.; Aligeti, N.; Jeyaprakash, T.; Martins, M.; Stodghill, J.; Winstanley, H.

    2011-12-01

    Lake Victoria in Africa is the second largest freshwater lake in the world and is known for its abundance of aquatic wildlife. In particular over 200 different fish species are caught and sold by local fisherman. The lake is a major contributor to the local economy as a corridor of transportation, source of drinking water, and source of hydropower. However, the invasion of aquatic vegetation such as water hyacinth in the lake has disrupted each of these markets. Aquatic vegetation now covers a substantial area of the coastline blocking waterways, disrupting hydropower, hindering the collection of drinking water and decreasing the profitability of fishing. The vegetation serves as a habitat for disease carrying mosquitoes as well as snakes and snails that spread the parasitic disease bilharzia. The current control measures of invasive aquatic vegetation rely on biological, chemical and mechanical control. The objective of this study was to utilize remote sensing to map aquatic vegetation within Lake Victoria from 2000 to 2011. MODIS, Landsat 4-5TM, and Landsat 7-ETM imagery was employed to perform change detections in vegetation and identify the extent of aquatic vegetation throughout the years. The efficiency of containment efforts were evaluated and ideal time for application of such efforts were suggested. A methodology for aquatic vegetation surveillance was created. The results of this project were presented as a workshop to the Lake Victoria Fisheries Organization, SERVIR, and other partner organizations. The workshop provided instruction into the use of NASA and other satellite derived products. Time series animations of the spatial extent of aquatic vegetation within the lake were created. By identifying seasons of decreased aquatic vegetation, ideal times to employ control efforts were identified. SERVIR will subsequently utilize the methodologies and mapping results of this study to develop operational aquatic vegetation surveillance for Lake Victoria.

  5. Mapping dominant annual land cover from 2009 to 2013 across Victoria, Australia using satellite imagery.

    PubMed

    Sheffield, Kathryn; Morse-McNabb, Elizabeth; Clark, Rob; Robson, Susan; Lewis, Hayden

    2015-01-01

    There is a demand for regularly updated, broad-scale, accurate land cover information in Victoria from multiple stakeholders. This paper documents the methods used to generate an annual dominant land cover (DLC) map for Victoria, Australia from 2009 to 2013. Vegetation phenology parameters derived from an annual time series of the Moderate Resolution Imaging Spectroradiometer Vegetation Indices 16-day 250 m (MOD13Q1) product were used to generate annual DLC maps, using a three-tiered hierarchical classification scheme. Classification accuracy at the broadest (primary) class level was over 91% for all years, while it ranged from 72 to 81% at the secondary class level. The most detailed class level (tertiary) had accuracy levels ranging from 61 to 68%. The approach used was able to accommodate variable climatic conditions, which had substantial impacts on vegetation growth patterns and agricultural production across the state between both regions and years. The production of an annual dataset with complete spatial coverage for Victoria provides a reliable base data set with an accuracy that is fit-for-purpose for many applications. PMID:26602009

  6. Mapping dominant annual land cover from 2009 to 2013 across Victoria, Australia using satellite imagery.

    PubMed

    Sheffield, Kathryn; Morse-McNabb, Elizabeth; Clark, Rob; Robson, Susan; Lewis, Hayden

    2015-01-01

    There is a demand for regularly updated, broad-scale, accurate land cover information in Victoria from multiple stakeholders. This paper documents the methods used to generate an annual dominant land cover (DLC) map for Victoria, Australia from 2009 to 2013. Vegetation phenology parameters derived from an annual time series of the Moderate Resolution Imaging Spectroradiometer Vegetation Indices 16-day 250 m (MOD13Q1) product were used to generate annual DLC maps, using a three-tiered hierarchical classification scheme. Classification accuracy at the broadest (primary) class level was over 91% for all years, while it ranged from 72 to 81% at the secondary class level. The most detailed class level (tertiary) had accuracy levels ranging from 61 to 68%. The approach used was able to accommodate variable climatic conditions, which had substantial impacts on vegetation growth patterns and agricultural production across the state between both regions and years. The production of an annual dataset with complete spatial coverage for Victoria provides a reliable base data set with an accuracy that is fit-for-purpose for many applications.

  7. Urban eutrophication and its spurring conditions in the Murchison Bay of Lake Victoria.

    PubMed

    Kabenge, Martin; Wang, Hongtao; Li, Fengting

    2016-01-01

    The efficiency of Lake Victoria in providing its ecosystem services to riparian states, both immediate and along the Nile river basin, is strongly related to its water quality. Over the past few decades, eutrophication has increased in the lake arising from increased inflow of nutrients. This study was carried out in the Murchison Bay area of Lake Victoria with the aims of assessing the progress of eutrophication nutrient enrichment into the lake between 1990 and 2014. Using Landsat satellite floating algae index (FAI) products and data from laboratory analysis of water samples, the study revealed that floating algae reoccurred periodically with coverage varying between 1 and 18 km(2). The findings also indicated that the range of nitrate-nitrogen concentrations increased greatly with maximum concentrations recorded at 31.2 mg l(-1) in 2007 from 0.084 mg l(-1) in 1990. The soluble reactive phosphorus concentration range showed a maximum of 1.45 mg l(-1) in 2007 from 0.043 mg l(-1) in 1990. The chlorophyll levels increased from an average of 17 μg l(-1) in 1992 by threefold in 1996 but had however declined and halved in intensity by 2011. The eutrophication that has occurred in Lake Victoria over the past decades has been due to pollution from industrial, residential, and agricultural areas within the catchment. PMID:26531714

  8. Pervasive, tholeiitic refertilisation and heterogeneous metasomatism in Northern Victoria Land lithospheric mantle (Antarctica)

    NASA Astrophysics Data System (ADS)

    Pelorosso, Beatrice; Bonadiman, Costanza; Coltorti, Massimo; Faccini, Barbara; Melchiorre, Massimiliano; Ntaflos, Theodoros; Gregoire, Michel

    2016-04-01

    The petrology of peridotite xenoliths in the Cenozoic volcanics from Greene Point (Northern Victoria Land, Antarctica) provides new constraints on the characterisation of the lithospheric mantle beneath the West Antarctic Rift. Based on mineral major and trace element models, this mantle domain is proposed to represent a residuum after 10% and 20% partial melting. Moreover, melting models and isotopic results for Sr and Nd systematics highlight the substantial contribution of tholeiitic melts percolating through peridotites. Close correlation with trace element contents in clinopyroxene phenocrysts from Ferrar and Karoo tholeiites allows us to ascribe this refertilisation event to the Jurassic. This asthenospheric melt was also able to transfer a garnet signature to the Northern Victoria Land mantle segment. The rare presence of glass and secondary phases indicate that Greene Point xenoliths were heterogeneously affected by alkaline metasomatism, probably related to the West Antarctic Rift System opening; this has also been widely observed in other Northern Victoria Land localities (i.e., Baker Rocks). Temperature and fO2 were calculated (950 °C; Δlog fO2 (QFM), - 1.70 to - 0.39) at a fixed pressure of 15 kbar, confirming the tendency of the anhydrous Greene Point xenolith population to have higher equilibration temperatures and comparable redox conditions, compared to the nearby amphibole-bearing peridotites from Baker Rocks.

  9. Parameterization of the inherent optical properties of Murchison Bay, Lake Victoria

    NASA Astrophysics Data System (ADS)

    Okullo, Willy; Ssenyonga, Taddeo; Hamre, Børge; Frette, Øyvind; Sørensen, K.; Stamnes, Jakob J.; Steigen, Andreas; Stamnes, Knut

    2007-12-01

    Lake Victoria, Africa's largest freshwater lake, suffers greatly from negative changes in biomass of species of fish and also from severe eutrophication. The continuing deterioration of Lake Victoria's ecological functions has great long-term consequences for the ecosystem benefits it provides to the countries bordering its shores. However, knowledge about temporal and spatial variations of optical properties and how they relate to lake constituents is important for a number of reasons such as remote sensing, modeling of underwater light fields, and long-term monitoring of lake waters. Based on statistical analysis of data from optical measurements taken during half a year of weekly cruises in Murchison Bay, Lake Victoria, we present a three-component model for the absorption and a two-component model for the scattering of light in the UV and the visible regions of the solar spectrum along with tests of their ranges of validity. The three-component input to the model for absorption is the chlorophyll-a (Chl-a), total suspended materials concentrations, and yellow substance absorption, while the two-component input to the model for scattering is the Chl-a concentration and total suspended materials.

  10. Parameterization of the inherent optical properties of Murchison Bay, Lake Victoria.

    PubMed

    Okullo, Willy; Ssenyonga, Taddeo; Hamre, Børge; Frette, Oyvind; Sørensen, K; Stamnes, Jakob J; Steigen, Andreas; Stamnes, Knut

    2007-12-20

    Lake Victoria, Africa's largest freshwater lake, suffers greatly from negative changes in biomass of species of fish and also from severe eutrophication. The continuing deterioration of Lake Victoria's ecological functions has great long-term consequences for the ecosystem benefits it provides to the countries bordering its shores. However, knowledge about temporal and spatial variations of optical properties and how they relate to lake constituents is important for a number of reasons such as remote sensing, modeling of underwater light fields, and long-term monitoring of lake waters. Based on statistical analysis of data from optical measurements taken during half a year of weekly cruises in Murchison Bay, Lake Victoria, we present a three-component model for the absorption and a two-component model for the scattering of light in the UV and the visible regions of the solar spectrum along with tests of their ranges of validity. The three-component input to the model for absorption is the chlorophyll-a (Chl-a), total suspended materials concentrations, and yellow substance absorption, while the two-component input to the model for scattering is the Chl-a concentration and total suspended materials.

  11. Mapping dominant annual land cover from 2009 to 2013 across Victoria, Australia using satellite imagery

    PubMed Central

    Sheffield, Kathryn; Morse-McNabb, Elizabeth; Clark, Rob; Robson, Susan; Lewis, Hayden

    2015-01-01

    There is a demand for regularly updated, broad-scale, accurate land cover information in Victoria from multiple stakeholders. This paper documents the methods used to generate an annual dominant land cover (DLC) map for Victoria, Australia from 2009 to 2013. Vegetation phenology parameters derived from an annual time series of the Moderate Resolution Imaging Spectroradiometer Vegetation Indices 16-day 250 m (MOD13Q1) product were used to generate annual DLC maps, using a three-tiered hierarchical classification scheme. Classification accuracy at the broadest (primary) class level was over 91% for all years, while it ranged from 72 to 81% at the secondary class level. The most detailed class level (tertiary) had accuracy levels ranging from 61 to 68%. The approach used was able to accommodate variable climatic conditions, which had substantial impacts on vegetation growth patterns and agricultural production across the state between both regions and years. The production of an annual dataset with complete spatial coverage for Victoria provides a reliable base data set with an accuracy that is fit-for-purpose for many applications. PMID:26602009

  12. Urban eutrophication and its spurring conditions in the Murchison Bay of Lake Victoria.

    PubMed

    Kabenge, Martin; Wang, Hongtao; Li, Fengting

    2016-01-01

    The efficiency of Lake Victoria in providing its ecosystem services to riparian states, both immediate and along the Nile river basin, is strongly related to its water quality. Over the past few decades, eutrophication has increased in the lake arising from increased inflow of nutrients. This study was carried out in the Murchison Bay area of Lake Victoria with the aims of assessing the progress of eutrophication nutrient enrichment into the lake between 1990 and 2014. Using Landsat satellite floating algae index (FAI) products and data from laboratory analysis of water samples, the study revealed that floating algae reoccurred periodically with coverage varying between 1 and 18 km(2). The findings also indicated that the range of nitrate-nitrogen concentrations increased greatly with maximum concentrations recorded at 31.2 mg l(-1) in 2007 from 0.084 mg l(-1) in 1990. The soluble reactive phosphorus concentration range showed a maximum of 1.45 mg l(-1) in 2007 from 0.043 mg l(-1) in 1990. The chlorophyll levels increased from an average of 17 μg l(-1) in 1992 by threefold in 1996 but had however declined and halved in intensity by 2011. The eutrophication that has occurred in Lake Victoria over the past decades has been due to pollution from industrial, residential, and agricultural areas within the catchment.

  13. Characterization of bacterial communities in lithobionts and soil niches from Victoria Valley, Antarctica.

    PubMed

    Van Goethem, Marc W; Makhalanyane, Thulani P; Valverde, Angel; Cary, Stephen C; Cowan, Don A

    2016-04-01

    Here we provide the first exploration of microbial diversity from three distinct Victoria Valley edaphic habitats, namely lithobionts (hypoliths, endoliths) and surface soils. Using a combination of terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene amplicon pyrosequencing we assess community structure and diversity patterns, respectively. Our analysis revealed that habitat type (endolithic versus hypolithic versus surface soils) significantly influenced bacterial community composition, even though dominant phyla such as Actinobacteria (41% of total reads) were common to all samples. Consistent with previous surveys in other Dry Valley ecosystems, we found that lithobionts were colonized by a few highly dominant phylotypes (such asGemmatimonasandLeptolyngbya). Our analyses also show that soil bacteria were more diverse and evenly distributed than initially expected based on previous evidence. In contrast to total bacteria, the distribution of Cyanobacteria was not strongly influenced by habitat type, although soil- and endolith-specific cyanobacterial lineages were found. The detection of cyanobacterial lineages in these habitats appears to be influenced by the dispersal of aquatic inocula from lacustrine communities or benthic mats which are abundant in Victoria Valley. Together, our results provide insights into the phylogenetic variation and community structure across niche habitats in Victoria Valley.

  14. Characterization of bacterial communities in lithobionts and soil niches from Victoria Valley, Antarctica.

    PubMed

    Van Goethem, Marc W; Makhalanyane, Thulani P; Valverde, Angel; Cary, Stephen C; Cowan, Don A

    2016-04-01

    Here we provide the first exploration of microbial diversity from three distinct Victoria Valley edaphic habitats, namely lithobionts (hypoliths, endoliths) and surface soils. Using a combination of terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene amplicon pyrosequencing we assess community structure and diversity patterns, respectively. Our analysis revealed that habitat type (endolithic versus hypolithic versus surface soils) significantly influenced bacterial community composition, even though dominant phyla such as Actinobacteria (41% of total reads) were common to all samples. Consistent with previous surveys in other Dry Valley ecosystems, we found that lithobionts were colonized by a few highly dominant phylotypes (such asGemmatimonasandLeptolyngbya). Our analyses also show that soil bacteria were more diverse and evenly distributed than initially expected based on previous evidence. In contrast to total bacteria, the distribution of Cyanobacteria was not strongly influenced by habitat type, although soil- and endolith-specific cyanobacterial lineages were found. The detection of cyanobacterial lineages in these habitats appears to be influenced by the dispersal of aquatic inocula from lacustrine communities or benthic mats which are abundant in Victoria Valley. Together, our results provide insights into the phylogenetic variation and community structure across niche habitats in Victoria Valley. PMID:26946500

  15. Trend and variability in observed hydrometeorological extremes in the Lake Victoria basin

    NASA Astrophysics Data System (ADS)

    Nyeko-Ogiramoi, P.; Willems, P.; Ngirane-Katashaya, G.

    2013-05-01

    SummaryIn the Lake Victoria basin hydrology, trend analysis has mainly been limited to the mean of the hydrological variable without explicit consideration of extremes, which are very crucial in understanding the behaviour of disastrous hydrometeorological events. Since the effects of climate change are unleashed, more through the occurrence of extremes, analysis of both monotonic and cyclic trends in hydrological extremes is very crucial. The presence of a significant linear trend, in a long-term hydrometeorological record of extremes, may provide evidence of a shift from the natural trend to that which is enhanced by, for example, anthropogenic forcing. In addition, cyclic trends analysis of hydrological extremes provides information on the cyclic behaviour of the extreme anomalies that have occurred over and above the natural climate variability and may link them to past consequences and their drivers. Analysis of long term records of extremes for rainfall, temperature and streamflows for selected stations in the Lake Victoria basin, were carried out based on a linear trend test, to detect significant monotonic trends, and quantile perturbation analysis, to detect significant temporal extreme anomalies. In addition, correlations between change in rainfall extremes and that for the other extremes, as well as sunspot maxima, were investigated. The findings indicated that extremes in the Lake Victoria basin are, generally, experiencing positive linear trends. Albeit positive trend was generally demonstrated, the presence of significant linear trend was manifested in the extremes of the data obtained from the stations located in the northern and eastern parts of the Lake Victoria basin. This may suggest that the monotony in the positive trend is a result of an ever increasing and consistent external enhancement of the natural climate agitation. The latter has implications for flood risks if the trend persists in the near future. The cyclic analysis of the behaviour

  16. Imaging the static dielectric constant in vitro and in living cells by a bioconjugable GFP chromophore analog.

    PubMed

    Signore, Giovanni; Abbandonato, Gerardo; Storti, Barbara; Stöckl, Martin; Subramaniam, Vinod; Bizzarri, Ranieri

    2013-02-28

    A fluorescent probe structurally similar to the GFP chromophore is demonstrated to report the local static dielectric constant. This probe can be chemically functionalized for selective targeting at the intracellular level.

  17. Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants.

    PubMed

    Ckurshumova, Wenzislava; Caragea, Adriana E; Goldstein, Rochelle S; Berleth, Thomas

    2011-09-01

    Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

  18. Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants.

    PubMed

    Ckurshumova, Wenzislava; Caragea, Adriana E; Goldstein, Rochelle S; Berleth, Thomas

    2011-09-01

    Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development. PMID:21772029

  19. From jellyfish to biosensors: the use of fluorescent proteins in plants.

    PubMed

    Voss, Ute; Larrieu, Antoine; Wells, Darren M

    2013-01-01

    The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors. PMID:24166435

  20. GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

    PubMed Central

    Rodriguez-Garcia, Arancha; Rojo-Ruiz, Jonathan; Navas-Navarro, Paloma; Aulestia, Francisco Javier; Gallego-Sandin, Sonia; Garcia-Sancho, Javier; Alonso, Maria Teresa

    2014-01-01

    Genetically encoded calcium indicators allow monitoring subcellular Ca2+ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca2+ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg2+, tunable Ca2+ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca2+ indicators for organelles and becomes a valuable tool for in vivo Ca2+ imaging applications. PMID:24501126

  1. Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

    PubMed Central

    Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

    2014-01-01

    In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Human CYP1A1GFP expression in transgenic mice serves as a biomarker for environmental toxicant exposure.

    PubMed

    Operaña, Theresa N; Nguyen, Nghia; Chen, Shujuan; Beaton, Deirdre; Tukey, Robert H

    2007-01-01

    The human CYP1A1 gene is regulated by the aryl hydrocarbon receptor (AhR), and induction of CYP1A1 is known to play an important role in xenobiotic metabolism. To examine the regulation of human CYP1A1 in vivo, we created a transgenic mouse strain (Tg-CYP1A1(GFP)) expressing a chimeric gene consisting of the entire human CYP1A1 gene (15 kb) fused with a GFP reporter gene. The treatment of Tg-CYP1A1(GFP) mice with a single intraperitoneal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[a]pyrene (B[a]P) led to the induction of CYP1A1(GFP) in both the liver and the lung as determined by fluorescence and Western blot analysis. The localization of induced fluorescence in liver also demonstrated the usefulness of cultured hepatocytes in examining the actions of AhR agonists toward induction of CYP1A1(GFP). Other routes of B[a]P administration, such as by oral exposure at 100 mg/kg for 3 days, led to reduced induction of CYP1A1(GFP) in liver and lung. In liver, expression of CYP1A1(GFP) was a sensitive marker for oral exposure, while mouse CYP1A1 was not induced at these doses. While first pass metabolism of B[a]P in the gastrointestinal tract reduces the potential of the AhR to induce CYP1A1(GFP) in the liver, adequate concentrations reach the hepatic circulation as demonstrated by induction of human UGT1A proteins in transgenic mice that express the human UGT1 locus. The capability to identify fluorescently labeled CYP1A1 in vivo provides a sensitive measurement of gene response and links exposure to potential environmental toxicants and activation of the AhR. PMID:17065433

  4. A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

    PubMed Central

    Arias-Barreiro, Carlos R.; Okazaki, Keisuke; Koutsaftis, Apostolos; Inayat-Hussain, Salmaan H.; Tani, Akio; Katsuhara, Maki; Kimbara, Kazuhide; Mori, Izumi C.

    2010-01-01

    A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10−7 (arsenite), 1.0 × 10−4 (naphthalene), 1.0 × 10−4 (Cu2+), 3.8 × 10−4 (H2O2), 1.0 × 10−3 (Cd2+), 1.0 × 10−3 (Zn2+), 1.0 × 10−2 (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb2+), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation. PMID:22163550

  5. Differential localisation of GFP fusions to cytoskeleton-binding proteins in animal, plant, and yeast cells. Green-fluorescent protein.

    PubMed

    Timmers, A C J; Niebel, A; Balagué, C; Dagkesamanskaya, A

    2002-10-01

    The structure and functioning of the cytoskeleton is controlled and regulated by cytoskeleton-associated proteins. Fused to the green-fluorescent protein (GFP), these proteins can be used as tools to monitor changes in the organisation of the cytoskeleton in living cells and tissues in different organisms. Since the localisation of a specific cytoskeleton protein may indicate a particular function for the associated cytoskeletal element, studies of cytoskeleton-binding proteins fused to GFP may provide insight into the organisation and functioning of the cytoskeleton. In this article, we focused on two animal proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae). Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells. Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover. Chemical fixation was found to drastically alter the distribution of plastin-GFP. Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules. Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast. We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about cytoskeleton function in eukaryotic cells. PMID:12417938

  6. CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.

    PubMed

    Auer, Thomas O; Duroure, Karine; Concordet, Jean-Paul; Del Bene, Filippo

    2014-12-01

    Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.

  7. Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum.

    PubMed

    Fang, Wei; Bonaffini, Sarah; Zou, Jian; Wang, Xiaolei; Zhang, Cen; Tsujimura, Taro; Kawamura, Shoji; Wei, Xiangyun

    2013-01-01

    Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR(RH2)-RH2-1 or LCR(RH2)-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR(RH2)-RH2-1:GFP)(pt112) and Tg(LCR(RH2)-RH2-2:GFP)(pt115) transgenic zebrafish lines express GFP in the pineal gland and certain types of photoreceptors. In addition, some of these lines also express GFP in the hatching gland, optic tectum, or olfactory bulb. Some of the expression patterns differ significantly from previously published similar transgenic fish lines, making them useful tools for studying the development of the corresponding tissues and organs. In addition, the variations of GFP expression among different lines corroborate the notion that transgenic expression is often subjected to position effect, thus emphasizing the need for careful verification of expression patterns when transgenic animal models are utilized for research.

  8. Isolation, plant colonization potential, and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp. Ph6-gfp

    NASA Astrophysics Data System (ADS)

    Sun, Kai; Liu, Juan; Gao, Yanzheng; Jin, Li; Gu, Yujun; Wang, Wanqing

    2014-06-01

    This investigation provides a novel method of endophyte-aided removal of polycyclic aromatic hydrocarbons (PAHs) from plant bodies. A phenanthrene-degrading endophytic bacterium Pseudomonas sp. Ph6 was isolated from clover (Trifolium pratense L.) grown in a PAH-contaminated site. After being marked with the GFP gene, the colonization and distribution of strain Ph6-gfp was directly visualized in plant roots, stems, and leaves for the first time. After ryegrass (Lolium multiflorum Lam.) roots inoculation, strain Ph6-gfp actively and internally colonized plant roots and transferred vertically to the shoots. Ph6-gfp had a natural capacity to cope with phenanthrene in vitro and in planta. Ph6-gfp degraded 81.1% of phenanthrene (50 mg.L-1) in a culture solution within 15 days. The inoculation of plants with Ph6-gfp reduced the risks associated with plant phenanthrene contamination based on observations of decreased concentration, accumulation, and translocation factors of phenanthrene in ryegrass. Our results will have important ramifications in the assessment of the environmental risks of PAHs and in finding ways to circumvent plant PAH contamination.

  9. Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas.

    PubMed

    Shen, B-C; Stewart, C N; Zhang, M-Q; Le, Y-T; Tang, Z-X; Mi, X-C; Wei, W; Ma, K-P

    2006-09-01

    Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

  10. Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

    PubMed Central

    Bleckmann, Maren; Schmelz, Stefan; Schinkowski, Christian; Scrima, Andrea

    2016-01-01

    ABSTRACT Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal‐cell biotechnology. Difficult‐to‐express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full‐length protein constructs. Post‐translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time‐, labor‐, and cost‐intensive. It is clearly desirable to find an automated and miniaturized fast multi‐sample screening method for protein expression in such systems. With this in mind, in this paper a high‐throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide‐binding Oligomerization Domain‐containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid‐based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in

  11. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

    PubMed

    Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

    2015-02-01

    Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

  12. GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

    PubMed Central

    Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

    2008-01-01

    Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

  13. Analysis of cargo transport by IFT and GFP imaging of IFT in Chlamydomonas.

    PubMed

    Diener, Dennis

    2009-01-01

    Chlamydomonas reinhardtii is the organism in which intraflagellar transport (IFT) was first visualized and in which the composition of IFT particles was originally elucidated. As the universality of IFT among ciliated/flagellated cells was uncovered, the diversity of organisms used to study IFT has grown. Still, because of the ease of isolation of flagella from Chlamydomonas and the battery of temperature-sensitive mutants affecting IFT proteins and motors, this unicellular alga remains the principal model for biochemical studies of IFT motors and cargo; furthermore, the long, exposed flagella of this cell are ideally suited for observing IFT in real time with GFP-tagged components of IFT.

  14. Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene

    PubMed Central

    Stefaniuk, Marzena; Gualda, Emilio J.; Pawlowska, Monika; Legutko, Diana; Matryba, Paweł; Koza, Paulina; Konopka, Witold; Owczarek, Dorota; Wawrzyniak, Marcin; Loza-Alvarez, Pablo; Kaczmarek, Leszek

    2016-01-01

    Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy. PMID:27312902

  15. Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.

    PubMed

    Stefaniuk, Marzena; Gualda, Emilio J; Pawlowska, Monika; Legutko, Diana; Matryba, Paweł; Koza, Paulina; Konopka, Witold; Owczarek, Dorota; Wawrzyniak, Marcin; Loza-Alvarez, Pablo; Kaczmarek, Leszek

    2016-01-01

    Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy. PMID:27312902

  16. High prevalence of non-synonymous substitutions in mtDNA of cichlid fishes from Lake Victoria.

    PubMed

    Shirai, Kazumasa; Inomata, Nobuyuki; Mizoiri, Shinji; Aibara, Mitsuto; Terai, Yohey; Okada, Norihiro; Tachida, Hidenori

    2014-12-01

    When a population size is reduced, genetic drift may fix slightly deleterious mutations, and an increase in nonsynonymous substitution is expected. It has been suggested that past aridity has seriously affected and decreased the populations of cichlid fishes in Lake Victoria, while geographical studies have shown that the water levels in Lake Tanganyika and Lake Malawi have remained fairly constant. The comparably stable environments in the latter two lakes might have kept the populations of cichlid fishes large enough to remove slightly deleterious mutations. The difference in the stability of cichlid fish population sizes between Lake Victoria and the Lakes Tanganyika and Malawi is expected to have caused differences in the nonsynonymous/synonymous ratio, ω (=dN/dS), of the evolutionary rate. Here, we estimated ω and compared it between the cichlids of the three lakes for 13 mitochondrial protein-coding genes using maximum likelihood methods. We found that the lineages of the cichlids in Lake Victoria had a significantly higher ω for several mitochondrial loci. Moreover, positive selection was indicated for several codons in the mtDNA of the Lake Victoria cichlid lineage. Our results indicate that both adaptive and slightly deleterious molecular evolution has taken place in the Lake Victoria cichlids' mtDNA genes, whose nonsynonymous sites are generally conserved. PMID:25241383

  17. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

    PubMed

    van der Schaar, H M; Melia, C E; van Bruggen, J A C; Strating, J R P M; van Geenen, M E D; Koster, A J; Bárcena, M; van Kuppeveld, F J M

    2016-01-01

    Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for

  18. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

    PubMed Central

    van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.

    2016-01-01

    ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition

  19. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

    PubMed

    van der Schaar, H M; Melia, C E; van Bruggen, J A C; Strating, J R P M; van Geenen, M E D; Koster, A J; Bárcena, M; van Kuppeveld, F J M

    2016-01-01

    Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for

  20. Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

    PubMed

    Sivakumar, Krishnakumar; Mukherjee, Manisha; Cheng, Hsin-I; Zhang, Yingdan; Ji, Lianghui; Cao, Bin

    2015-03-01

    Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

  1. Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice

    PubMed Central

    Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki

    2016-01-01

    Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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. PMID:19269340

  4. Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP.

    PubMed

    Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi; Miyata, Maiko; Huang, Peng; Ishiguro, Naoki; Hamaguchi, Michinari; Iwamoto, Takashi

    2008-08-01

    Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.

  5. Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP

    SciTech Connect

    Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi; Miyata, Maiko; Huang, Peng; Ishiguro, Naoki; Hamaguchi, Michinari; Iwamoto, Takashi

    2008-08-08

    Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.

  6. Chromatin movement visualized with photoactivable GFP-labeled histone H4.

    PubMed

    Wiesmeijer, Karien; Krouwels, Ilke M; Tanke, Hans J; Dirks, Roeland W

    2008-01-01

    The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.

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

    PubMed Central

    Shrivats, Arun R.; Mishina, Yuji; Averick, Saadyah; Matyjaszewski, Krzysztof; Hollinger, Jeffrey O.

    2016-01-01

    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. PMID:27280121

  8. Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

    NASA Technical Reports Server (NTRS)

    Granger, C. L.; Cyr, R. J.

    2000-01-01

    Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

  9. Short Hydrogen Bonds and Proton Delocalization in Green Fluorescent Protein (GFP).

    PubMed

    Oltrogge, Luke M; Boxer, Steven G

    2015-06-24

    Short hydrogen bonds and specifically low-barrier hydrogen bonds (LBHBs) have been the focus of much attention and controversy for their possible role in enzymatic catalysis. The green fluorescent protein (GFP) mutant S65T, H148D has been found to form a very short hydrogen bond between Asp148 and the chromophore resulting in significant spectral perturbations. Leveraging the unique autocatalytically formed chromophore and its sensitivity to this interaction we explore the consequences of proton affinity matching across this putative LBHB. Through the use of noncanonical amino acids introduced through nonsense suppression or global incorporation, we systematically modify the acidity of the GFP chromophore with halogen substituents. X-ray crystal structures indicated that the length of the interaction with Asp148 is unchanged at ∼2.45 Å while the absorbance spectra demonstrate an unprecedented degree of color tuning with increasing acidity. We utilized spectral isotope effects, isotope fractionation factors, and a simple 1D model of the hydrogen bond coordinate in order to gain insight into the potential energy surface and particularly the role that proton delocalization may play in this putative short hydrogen bond. The data and model suggest that even with the short donor-acceptor distance (∼2.45 Å) and near perfect affinity matching there is not a LBHB, that is, the barrier to proton transfer exceeds the H zero-point energy. PMID:27162964

  10. Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps

    PubMed Central

    Hudson, Andrew M.; Petrella, Lisa N.; Tanaka, Akemi J.; Cooley, Lynn

    2008-01-01

    Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. We have used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, we were able to use the Flp/FRT system to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle. PMID:18199432

  11. Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection.

    PubMed

    Chalupowicz, L; Zellermann, E-M; Fluegel, M; Dror, O; Eichenlaub, R; Gartemann, K-H; Savidor, A; Sessa, G; Iraki, N; Barash, I; Manulis-Sasson, S

    2012-01-01

    The vascular pathogen Clavibacter michiganensis subsp. michiganensis is responsible for bacterial wilt and canker of tomato. Pathogenicity of this bacterium is dependent on plasmid-borne virulence factors and serine proteases located on the chromosomal chp/tomA pathogenicity island (PAI). In this study, colonization patterns and movement of C. michiganensis subsp. michiganensis during tomato infection was examined using a green fluorescent protein (GFP)-labeled strain. A plasmid expressing GFP in C. michiganensis subsp. michiganensis was constructed and found to be stable in planta for at least 1 month. Confocal laser-scanning microscopy (CLSM) of inoculated stems showed that the pathogen extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem. Acropetal movement of the wild-type strain C. michiganensis subsp. michiganensis NCPPB382 (Cmm382) in tomato resulted in an extensive systemic colonization of the whole plant reaching the apical region after 15 days, whereas Cmm100 (lacking the plasmids pCM1 and pCM2) or Cmm27 (lacking the chp/tomA PAI) remained confined to the area surrounding of the inoculation site. Cmm382 formed biofilm-like structures composed of large bacterial aggregates on the interior of xylem walls as observed by CLSM and scanning electron microscopy. These findings suggest that virulence factors located on the chp/tomA PAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates.

  12. The fluorescent photobleaching properties of GFP expressed in human lung cancer cells

    NASA Astrophysics Data System (ADS)

    Jin, Ying; Xing, Da

    2003-12-01

    The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the dicistronic expression vector (pEGFP-C1) was used to transfected into human lung cancer cell line (ASTC-a-1) and a positive clone which stably expressed GFP in high level was obtained. After more than three months' passengers, the cells were also remained the strong fluorescence under fluorescent microscope. The results showed that the green fluorescent protein expressed in tumor cells was also photobleached under intense irradiation (approximately 488 nm) and the degree of photobleaching varied with the difference of the intensity of the excitation. Using different interdiction parcel (None, ND4, ND8, ND16), there were significant differences in photobleaching among the different excitation. The photobleaching was also affected by the time length of excitation, and the intensity of fluorescence was obviously decreased along with the increasing of excitation time, especially to stronger excitation.

  13. Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

    PubMed

    Maximova, S; Miller, C; Antúnez de Mayolo, G; Pishak, S; Young, A; Guiltinan, M J

    2003-06-01

    We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA. PMID:12789505

  14. Stable isotope paleoecology of Late Pleistocene Middle Stone Age humans from the Lake Victoria basin, Kenya.

    PubMed

    Garrett, Nicole D; Fox, David L; McNulty, Kieran P; Faith, J Tyler; Peppe, Daniel J; Van Plantinga, Alex; Tryon, Christian A

    2015-05-01

    Paleoanthropologists have long argued that environmental pressures played a key role in human evolution. However, our understanding of how these pressures mediated the behavioral and biological diversity of early modern humans and their migration patterns within and out of Africa is limited by a lack of archaeological evidence associated with detailed paleoenvironmental data. Here, we present the first stable isotopic data from paleosols and fauna associated with Middle Stone Age (MSA) sites in East Africa. Late Pleistocene (∼100-45 ka, thousands of years ago) sediments on Rusinga and Mfangano Islands in eastern Lake Victoria (Kenya) preserve a taxonomically diverse, non-analog faunal community associated with MSA artifacts. We analyzed the stable carbon and oxygen isotope composition of paleosol carbonate and organic matter and fossil mammalian tooth enamel, including the first analyses for several extinct bovids such as Rusingoryx atopocranion, Damaliscus hypsodon, and an unnamed impala species. Both paleosol carbonate and organic matter data suggest that local habitats associated with human activities were primarily riverine woodland ecosystems. However, mammalian tooth enamel data indicate that most large-bodied mammals consumed a predominantly C4 diet, suggesting an extensive C4 grassland surrounding these riverine woodlands in the region at the time. These data are consistent with other lines of paleoenvironmental evidence that imply a substantially reduced Lake Victoria at this time, and demonstrate that C4 grasslands were significantly expanded into equatorial Africa compared with their present distribution, which could have facilitated dispersal of human populations and other biotic communities. Our results indicate that early populations of Homo sapiens from the Lake Victoria region exploited locally wooded and well-watered habitats within a larger grassland ecosystem. PMID:25805041

  15. Irrigator responses to groundwater resource management in northern Victoria, southeastern Australia

    NASA Astrophysics Data System (ADS)

    Gill, Bruce C.; Webb, John; Wilkinson, Roger; Cherry, Don

    2014-10-01

    In northern Victoria, farmers are the biggest users of groundwater and therefore the main stakeholders in plans that seek to sustainably manage the resource. Interviews with 30 irrigation farmers in two study areas, analysed using qualitative social research methods, showed that the overwhelming majority of groundwater users agreed with the need for groundwater management and thought that the current plans had achieved sustainable resource use. The farmers also expressed a strong need for clear technical explanations for management decisions, in particular easily understood water level data. The social licence to implement the management plans arose through effective consultation with the community during plan development. Several additional factors combined to gain acceptance for the plans: good data on groundwater usage and aquifer levels is available; irrigation farmers had been exposed to usage restrictions since the late 1990s; an ‘adaptive’ management approach is in use which allowed refinements to be readily incorporated and fortuitously, plan development coincided with the 1998-2009 drought, when declines in groundwater levels reinforced the usefulness of the plans. The imposition of a nation-wide water use reduction plan in 2012 had relatively little impact in Victoria because of the early implementation of effective groundwater management plans. However, economic difficulties that reduce groundwater users’ capacity to pay groundwater management charges mean that the future of the plans in Victoria is not assured. Nevertheless, the high level of trust that exists between Victorian irrigation farmers and the management agencies suggests that the continued use of a consultative approach will continue to produce workable outcomes. Lessons from the Victorian experience may be difficult to apply in other areas of groundwater use in Australia and overseas, where there may be a quite different history of development and culture of groundwater management.

  16. Population genetic structure of Anopheles gambiae mosquitoes on Lake Victoria islands, west Kenya

    PubMed Central

    Chen, Hong; Minakawa, Noboru; Beier, John; Yan, Guiyun

    2004-01-01

    Background Understanding the genetic structure of island Anopheles gambiae populations is important for the current tactics in mosquito control and for the proposed strategy using genetically-modified mosquitoes (GMM). Genetically-isolated mosquito populations on islands are a potential site for testing GMM. The objective of this study was to determine the genetic structure of A. gambiae populations on the islands in Lake Victoria, western Kenya. Methods The genetic diversity and the population genetic structures of 13 A. gambiae populations from five islands on Lake Victoria and six villages from the surrounding mainland area in the Suba District were examined using six microsatellite markers. The distance range of sampling sites varied between 2.5 and 35.1 km. Results A similar level of genetic diversity between island mosquito populations and adjacent mainland populations was found. The average number of alleles per locus was 7.3 for the island populations and 6.8 for the mainland populations. The average observed heterozygosity was 0.32 and 0.28 for the island and mainland populations, respectively. A low but statistically significant genetic structure was detected among the island populations (FST = 0.019) and between the island and mainland populations (FST = 0.003). A total of 12 private alleles were found, and nine of them were from the island populations. Conclusion A level of genetic differentiation between the island and mainland populations was found. Large extent of gene flow between the island and mainland mosquito populations may result from wind- or human-assisted dispersal. Should the islands on Lake Victoria be used as a trial site for the release program of GMM, mosquito dispersal between the islands and between the island and the mainland should be vigorously monitored. PMID:15581429

  17. A review of mercury in Lake Victoria, East Africa: implications for human and ecosystem health.

    PubMed

    Campbell, Linda; Dixon, D G; Hecky, R E

    2003-01-01

    Lake Victoria, East Africa, has been the site of many recent studies measuring mercury (Hg) concentrations in water, fish, sediment, soil, and humans. Most of these studies were motivated by concerns about Hg contamination from processing of gold ore on the southern shores. Total Hg (THg) concentrations in fish were usually below permissible World Health Organization (WHO) concentrations and international marketing limits and do not threaten the lucrative export industry. Nile perch 3-10 kg and most >10 kg had THg concentrations above the WHO threshold concentrations for at-risk groups (200 ng/g). Elevated THg concentrations in large Nile perch are not of major concern because Nile perch are rarely consumed by the people living on Lake Victoria and very large Nile perch are becoming increasingly rare in catches. Water THg concentrations were below Canadian drinking water guidelines but were elevated relative to those in the northern Great Lakes. Sediment and soil THg concentrations were within inter-national guidelines and are comparable to those in northern latitudes but are lower than those in the Amazon basin. Biomass burning and soil erosion are estimated to be the major sources of THg for the lake and probably constitute a larger source of THg than gold mining in Tanzania.THg concentrations in urine and hair from human volunteers indicate that while gold miners and frequent skin-bleaching cream users are at risk of inorganic mercury poisoning, the rest of the population, including fishermen, is not. Human exposure assessments demonstrated that fish consumption and soil geophagy constitute major sources of THg for humans, but the total estimated daily intake of THg was below the Health Canada tolerable daily intake (TDI) limits. The use of beauty creams containing high inorganic Hg concentrations, however, caused the estimated THg exposure to exceed the TDI. The high THg content in the hair of regular cream users supports this assessment. The nutritional

  18. A new species of Anabarhynchus (Diptera: Therevidae) from an ocean beach in south east Victoria

    PubMed Central

    Yeates, David K.

    2014-01-01

    Abstract Anabarhynchus Macquart 1848 is a large genus of the Therevidae (Diptera) that are endemic to Australasia with a couple of described species from Melanesia. We describe and illustrate Anabarhynchus oceanus sp. n., a species found on ocean beaches in eastern Victoria, Australia. The species shares most characters with the monobasic Anabarhynchus kampmeierae species group of Lyneborg (2001), but also shares a unique feature of the male genitalia with the endemic New Zealand genus Megathereva Lyneborg, 1992. This new species brings the total number of described Australian species in the genus to 113. PMID:25349526

  19. A cross-national comparison of school drug policies in Washington State, United States, and Victoria, Australia.

    PubMed

    Beyers, Jennifer M; Evans-Whipp, Tracy; Mathers, Megan; Toumbourou, John W; Catalano, Richard F

    2005-04-01

    Using mail survey data collected from primary and secondary school administrators in Washington State, United States, and in Victoria, Australia, this study compared aspects of the school drug policy environment in the 2 states. Documented substance-use policies were prevalent in Washington and Victoria but less prevalent.in primary schools, especially in Victoria. Victorian school policy-setting processes were significantly more likely to involve teachers, parents, and students than processes in Washington schools. Consistent with expectations based on their respective national drug policy frameworks, school drug policies in Washington schools were more oriented toward total abstinence and more frequently enforced with harsh punishment (such as expulsion or calling law enforcement), whereas policies in Victorian schools were more reflective of harm-minimization principles. Within both states, however, schools more regularly used harsh punishment and remediation consequences for alcohol and illicit-drug violations compared to tobacco policy violations, which were treated more leniently.

  20. VICTORIA: A mechanistic model of radionuclide behavior in the reactor coolant system under severe accident conditions. Revision 1

    SciTech Connect

    Heams, T J; Williams, D A; Johns, N A; Mason, A; Bixler, N E; Grimley, A J; Wheatley, C J; Dickson, L W; Osborn-Lee, I; Domagala, P; Zawadzki, S; Rest, J; Alexander, C A; Lee, R Y

    1992-12-01

    The VICTORIA model of radionuclide behavior in the reactor coolant system (RCS) of a light water reactor during a severe accident is described. It has been developed by the USNRC to define the radionuclide phenomena and processes that must be considered in systems-level models used for integrated analyses of severe accident source terms. The VICTORIA code, based upon this model, predicts fission product release from the fuel, chemical reactions involving fission products, vapor and aerosol behavior, and fission product decay heating. Also included is a detailed description of how the model is implemented in VICTORIA, the numerical algorithms used, and the correlations and thermochemical data necessary for determining a solution. A description of the code structure, input and output, and a sample problem are provided.

  1. QM/MM study on the light emitters of aequorin chemiluminescence, bioluminescence, and fluorescence: a general understanding of the bioluminescence of several marine organisms.

    PubMed

    Chen, Shu-Feng; Ferré, Nicolas; Liu, Ya-Jun

    2013-06-24

    Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF-hand calcium-binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H-bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine-type luciferases).

  2. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  3. Influence of the Pearl River estuary and vertical mixing in Victoria Harbor on water quality in relation to eutrophication impacts in Hong Kong waters.

    PubMed

    Yin, Kedong; Harrison, Paul J

    2007-06-01

    This study presents water quality parameters such as nutrients, phytoplankton biomass and dissolved oxygen based on 11 years of water quality data in Victoria Harbor and examined how the Pearl River estuary discharge in summer and year round sewage discharge influenced these parameters. Nutrients in Victoria Harbor were strongly influenced by both the Pearl River and sewage effluent, as indicated by the high NO(3) inputs from the Pearl River in summer and higher NH(4) and PO(4) in Victoria Harbor than both its sides. N:P ratios were low in the dry season, but increased to >16:1 in the wet season, suggesting that P is potentially the most limiting nutrient in this area during the critical period in the summer. Although there were generally high nutrients, the phytoplankton biomass was not as high as one would expect in Victoria Harbor. In fact, there were high concentrations of chl near the bottom well below the photic zone. Salinity near the bottom was lower in Victoria Harbor than at the two entrances to Victoria Harbor, suggesting strong vertical mixing within Victoria Harbor. Therefore, strong vertical mixing and horizontal advection appear to play an important role in significantly reducing eutrophication impacts in Victoria Harbor. Consequently, dissolved oxygen near the bottom was low in summer, but only occasionally dipped to 2 mgL(-1) despite the high organic loading from sewage effluent.

  4. Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line

    PubMed Central

    Smiley, Sheila; Nickerson, Philip E.; Comanita, Lacrimioara; Daftarian, Narsis; El-Sehemy, Ahmed; Tsai, En Leh Samuel; Matan-Lithwick, Stuart; Yan, Keqin; Thurig, Sherry; Touahri, Yacine; Dixit, Rajiv; Aavani, Tooka; De Repentingy, Yves; Baker, Adam; Tsilfidis, Catherine; Biernaskie, Jeff; Sauvé, Yves; Schuurmans, Carol; Kothary, Rashmi; Mears, Alan J.; Wallace, Valerie A.

    2016-01-01

    We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration. PMID:26965927

  5. Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein.

    PubMed

    Oda, H; Tsukita, S

    1999-04-01

    Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, we constructed Dalpha-catenin fused with a green fluorescent protein (Dalpha-catenin-GFP), and found that it targeted apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, we examined the dynamic performance of AJs containing Dalpha-catenin-GFP in epithelial morphogenetic movements. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dalpha-catenin-GFP occurred concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behaviour of AJs was concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension could be followed. Finally, we utilized Dalpha-catenin-GFP to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution.

  6. Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells.

    PubMed

    Choi, WooJae; Yum, SooYoung; Lee, SongJeon; Lee, WonWu; Lee, JiHyun; Kim, SeokJoong; Koo, OkJae; Lee, ByeongChun; Jang, Goo

    2015-12-01

    Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6-203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science. PMID:25424059

  7. Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap

    NASA Astrophysics Data System (ADS)

    Galan, Jacob A.; Paris, Leela L.; Zhang, Hua-jie; Adler, Jacob; Geahlen, Robert L.; Tao, W. Andy

    2011-02-01

    Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein-protein interactions using anti-GFP single-chain antibody VHH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain VHH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.

  8. Absorption capacity of renal proximal tubular cells studied by combined injections of YFP and GFP in Rana temporaria L.

    PubMed

    Prutskova, N P; Seliverstova, E V

    2013-09-01

    The capacity for protein reabsorption in the renal proximal tubule (PT) was studied in Rana temporaria frogs by separate, simultaneous and sequential introduction of yellow fluorescent protein (YFP) and green fluorescent protein (GFP). The uptake patterns of YFP and GFP in PT epithelial cells were investigated 15-120min after their bolus intravenous and intraperitoneal injection. As shown by confocal microscopy, the tubular uptake of YFP and GFP was time- and dose-dependent. These proteins are absorbed in similar way and can be accumulated in the same endocytic vesicles after their combined injections. When GFP was injected 30 and 90min before YFP, and vice versa, the number of vesicles with pre-injected protein increased and the percentage of vesicles with colocalized GFP and YFP reduced. At the same time, the uptake rate of a protein injected later progressively and significantly decreased. Subcellular localization of endocytic receptors, megalin and cubilin, in renal PT cells after intravenous YFP introduction were revealed by immunofluorescent microscopy. Colocalization of internalized YFP with megalin or cubilin in the endocytic vesicles was demonstrated. The data suggest the possibility of protein uptake by receptor-mediated endocytosis and the existence of a mechanism limiting the protein absorption rate in wintering frogs.

  9. Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein.

    PubMed

    Weston, Chris J; Shepherd, Emma L; Adams, David H

    2013-06-01

    Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.

  10. Risk factors for severe injury in cyclists involved in traffic crashes in Victoria, Australia.

    PubMed

    Boufous, Soufiane; de Rome, Liz; Senserrick, Teresa; Ivers, Rebecca

    2012-11-01

    This study examines the impact of cyclist, road and crash characteristics on the injury severity of cyclists involved in traffic crashes reported to the police in Victoria, Australia between 2004 and 2008. Logistic regression analysis was carried out to identify predictors of severe injury (serious injury and fatality) in cyclist crashes reported to the police. There were 6432 cyclist crashes reported to the police in Victoria between 2004 and 2008 with 2181 (33.9%) resulting in severe injury of the cyclist involved. The multivariate analysis found that factors that increase the risk of severe injury in cyclists involved in traffic crashes were age (50 years and older), not wearing a helmet, riding in the dark on unlit roads, riding on roads zoned 70 km/h or above, on curved sections of the road, in rural locations and being involved in head-on collisions as well as off path crashes, which include losing control of vehicle, and on path crashes which include striking the door of a parked vehicle. While this study did not test effectiveness of preventative measures, policy makers should consider implementation of programs that address these risk factors including helmet programs and environmental modifications such as speed reduction on roads that are frequented by cyclists. PMID:23036419

  11. Mantle metasomatism beneath western Victoria, Australia. I. Metasomatic processes in Cr-diopside lherzolites

    SciTech Connect

    O'Reilly, S.Y.; Griffin, W.L.

    1988-02-01

    Most Cr-diopside spinel lherzolite xenoliths from Bullenmerri and Gnotuk Maars, western Victoria, show modal metasomatism involving the growth of amphibole +- mica +- apatite at the expense of primary pyroxenes + spinel. The metasomatism is attributed to CO/sub 2/-rich fluids, observed in fluid inclusions. REE patterns of anhydrous lherolites range from LREE-depleted ((LaYb)/sub n/ approx. = 0.3) to LREE-enriched ((LaYb)/sub n/ = 30-60), and show an inverse correlation of NdSm with CaO. Amphibole-rich peridotites are enriched in LREE ((LaYb)/sub n/ = 10-30), Ar and Ta, with high KRb. Mica-rich rocks are enriched in K, Rb, Ba, Ta and Ti, with low KRb. Introduction of apatite leads to high ..sigma..REE (with (LaYb)/sub n/ = 40-100), Sr, U and Th contents. The distribution of trace and minor elements in the ilherzolites is thus controlled by the crystal chemistry of the primary and metasomatic phases. Micaceous xenoliths may be derived from thin selvedges on pyroxenite veins. Abundant amphibole lherzolites may form a matrix enclosing relic volumes of anhydrous lherzolites showing varying degrees of cryptic metasomatism. The overall pattern of trace-element enrichment in the mantle beneath Victoria will depend on the volumetric proportions of these rock types.

  12. Hydrology or floristics? Mapping and classification of wetlands in Victoria, Australia, and implications for conservation planning.

    PubMed

    Robertson, Hugh A; Fitzsimons, James A

    2004-10-01

    A national approach to the conservation of biodiversity in Australia's freshwater ecosystems is a high priority. This requires a consistent and comprehensive system for the classification, inventory, and assessment of wetland ecosystems. This paper, using the State of Victoria as a case study, compares two classification systems that are commonly utilized to delineate and map wetlands--one based on hydrology (Victorian Wetland Database [VWD]) and one based on indigenous vegetation types and other natural features (Ecological Vegetation Classes [EVC]). We evaluated the extent of EVC mapping of wetlands relative to the VWD classification system using a number of datasets within a geographical information system. There were significant differences in the coverage of extant EVCs across bioregions, different-sized wetlands, and VWD wetland types. Resultant depletion levels were markedly different when examined using the two systems, with depletion levels, and therefore perceived conservation status, of EVCs being significantly higher. Although there is little doubt that many wetland ecosystems in Victoria are in fact threatened, the extent of this threat cannot accurately be determined by relying on the EVC mapping as it currently stands. The study highlighted the significant impact wetland classification methods have in determining the conservation status of freshwater ecosystems.

  13. Rural Health and Spiritual Care Development: A Review of Programs across Rural Victoria, Australia.

    PubMed

    Carey, Lindsay B; Hennequin, Christine; Krikheli, Lillian; O'Brien, Annette; Sanchez, Erin; Marsden, Candace R

    2016-06-01

    Given declining populations in rural areas and diminishing traditional religious support, this research explores whether spiritual care education programs would be beneficial for and appreciated by those working in rural health and/or community organizations. An overview of literature identified three dominant rural health issues affecting the provision of spiritual care in rural areas, namely the disparity between rural and urban areas in terms of resources, the lack of access to services, plus the need for education and training within rural areas. Spiritual Health Victoria Incorporated (Victoria, Australia) sought to address these issues with the implementation of a variety of spiritual education programs within rural areas. Results of an evaluation of these programs are presented specifying participant demographics, reasons why participants attended, their evaluation of the program and any recommendations for future programs. In overall terms, the results indicated that at least 90% of participants favorably rated their attended program as either 'very good' or 'good' and indicated that the main reason for their attendance was to develop their own education and/or practice of spiritual care within their rural context for the benefit of local constituents. Several recommendations are made for future programs.

  14. Household use of and satisfaction with alternative water sources in Victoria Australia.

    PubMed

    Hurlimann, Anna

    2011-10-01

    Climate change is increasing the variability of rainfall, and thus the availability of water supplies in many areas of the world. These impacts are already being felt in the state of Victoria, Australia where a 12 year drought period was recently experienced. Restrictions to water use have been implemented, as one component of a broad policy approach to manage the drought. While anecdotal evidence suggests that the substitution of centralised water supplies is occurring, this has not been proven empirically. This paper reports results from a survey of households in Victoria regarding their use of alternative water sources. The study found that substitution is occurring. Garden watering is the purpose which has the highest rate of alternative water source use. In total 41.6% of respondents always, and 33.2% sometimes use an alternative water source for garden watering. The most commonly used alternative source of water for garden watering is water previously used in the laundry (30.7%). The alternative source of water used was found to vary depending on the purpose of the water use. High levels of satisfaction were found for all alternative water sources used. Several barriers were found to the use of alternative water sources, the main of which were: inflexibility of existing infrastructure, cost, policy, and housing status. The results have implications for water retailers, policy makers and governments in locations facing water shortage.

  15. Stretching out the Australasian microtektite strewn field in Victoria Land Transantarctic Mountains

    NASA Astrophysics Data System (ADS)

    Folco, Luigi; D'Orazio, Massimo; Gemelli, Maurizio; Rochette, Pierre

    2016-06-01

    Petrographic and geochemical studies of microtektites collected in newly explored summit plateaus of the Transantarctic Mountains (i.e., Schroeder Spur, Killer Nunatak, Miller Butte in the inland catchment of the Rennick Glacier, and Allan Hills, in the inland catchment of the Mackay-David Glaciers) document a regional distribution of Australasian microtektites in Victoria Land. A geochemical comparison with Australasian microtektites from deep sea sediments at lower latitudes identifies a possible projectile geochemical signature for the first time, and confirms that Transantarctic Mountains microtektites experienced higher thermal regimes. Ballistic calculations reveal that the extraordinary distance of the Transantarctic Mountains microtektites from the hypothetical impact location in Indochina (∼11,000 km) could be more efficiently attained at relatively low ejection angles (20°-40°). Finally, the occurrence of Australasian microtektites (∼0.8 Ma old) on specific glacial surfaces of the Antarctic bedrock constrains the glacial history of the East Antarctic Ice Sheet in Victoria Land. In particular, data from Allan Hills supports a glaciological scenario envisaging an extremely stable East Antarctic Ice Sheet over at least the last ∼1 Ma in the inland catchment of the Mackay/David glaciers. This is consistent with the large accumulation of meteorites in the adjacent blue ice fields.

  16. Sunspots, El Niño, and the levels of Lake Victoria, East Africa

    NASA Astrophysics Data System (ADS)

    Stager, J. Curt; Ruzmaikin, Alexander; Conway, Declan; Verburg, Piet; Mason, Peter J.

    2007-08-01

    An association of high sunspot numbers with rises in the level of Lake Victoria, East Africa, has been the focus of many investigations and vigorous debate during the last century. In this paper, we show that peaks in the ~11-year sunspot cycle were accompanied by Victoria level maxima throughout the 20th century, due to the occurrence of positive rainfall anomalies ~1 year before solar maxima. Similar patterns also occurred in at least five other East African lakes, which indicates that these sunspot-rainfall relationships were broadly regional in scale. Although irradiance fluctuations associated with the sunspot cycle are weak, their effects on tropical rainfall could be amplified through interactions with sea surface temperatures and atmospheric circulation systems, including ENSO. If this Sun-rainfall relationship persists in the future, then sunspot cycles can be used for long-term prediction of precipitation anomalies and associated outbreaks of insect-borne disease in much of East Africa. In that case, unusually wet rainy seasons and Rift Valley Fever epidemics should occur a year or so before the next solar maximum, which is expected to occur in 2011-2012 AD.

  17. Factors influencing waste separation and utilization among households in the Lake Victoria crescent, Uganda.

    PubMed

    Ekere, William; Mugisha, Johnny; Drake, Lars

    2009-12-01

    Wastes, which are the by-products of consumption, are a growing problem in the urban and peri-urban areas of the Lake Victoria region largely due to high urban population growth rates, consumption habits, low collection rates and hence waste accumulation. Whereas the biodegradable proportion is high and could be reutilized, a few have tapped the economic potential of this waste. This study was conducted to explore the potential alternatives and determinants of waste separation and utilization among urban and peri-urban households in the Lake Victoria crescent. A random sample of households in five urban and peri-urban areas of the crescent were selected and surveyed. Logit models were used to establish the factors influencing waste separation and utilization in urban and peri-urban areas of the lake crescent. Results indicate that, gender, peer influence, land size, location of household and membership of environmental organization explain household waste utilization and separation behaviour. Campaigns for waste separation and reuse should be focused in the peri-urban areas where high volumes of wastes are generated and accumulate. Social influence or pressure should be used to encourage more waste reuse and separation.

  18. Results of high-resolution echo-sounding of Lake Victoria

    NASA Astrophysics Data System (ADS)

    Scholz, C. A.; Rosendahl, B. R.; Versfelt, J. W.; Rach, N.

    About 1800 km of reconnaissance echo-sounder data have been collected from Lake Victoria. The profiles show a maximum open-basin thickness of 8 m oatest Pleistocene and Holocene fine-grained muds. Their distribution mimics bathymetry, except for locally thicker patches around bathymetric highs, which serve as current and seiche breaks. The transparent sediment blanket overlies an acoustic basement that ranges from crystalline basement to a late-Tertiary, boulder-studded peneplain to coarser-grained lacustrine sediments and dewatered fine-grained mudstones, depending upon position in the lake. Correlation to discontinuities in core data suggest that the boundary between the fine-grained, acoustically transparent muds and acoustic basement represents a 14,000 year old desiccation surface of essentially lake-wide extent. Many curious signatures are noted in the Lake Victoria echograms, including spiky diffractions that may represent buried boulders or gas escape structures; megaripples and sediment wave bedforms associated with current activity in the SW corner of the lake; and a tilted-block terrain in the Speke Gulf that is probably caused by recent submergence of subaerially eroded Archean crystalline rocks along uniformly-spaced joints and fractures.

  19. Household use of and satisfaction with alternative water sources in Victoria Australia.

    PubMed

    Hurlimann, Anna

    2011-10-01

    Climate change is increasing the variability of rainfall, and thus the availability of water supplies in many areas of the world. These impacts are already being felt in the state of Victoria, Australia where a 12 year drought period was recently experienced. Restrictions to water use have been implemented, as one component of a broad policy approach to manage the drought. While anecdotal evidence suggests that the substitution of centralised water supplies is occurring, this has not been proven empirically. This paper reports results from a survey of households in Victoria regarding their use of alternative water sources. The study found that substitution is occurring. Garden watering is the purpose which has the highest rate of alternative water source use. In total 41.6% of respondents always, and 33.2% sometimes use an alternative water source for garden watering. The most commonly used alternative source of water for garden watering is water previously used in the laundry (30.7%). The alternative source of water used was found to vary depending on the purpose of the water use. High levels of satisfaction were found for all alternative water sources used. Several barriers were found to the use of alternative water sources, the main of which were: inflexibility of existing infrastructure, cost, policy, and housing status. The results have implications for water retailers, policy makers and governments in locations facing water shortage. PMID:21715083

  20. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells.

    PubMed

    Peddie, Christopher J; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O'Toole, Peter; Larijani, Banafshe; Collinson, Lucy M

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure.

  1. Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

    PubMed Central

    Zou, Jian; Wei, Xiangyun

    2010-01-01

    Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. PMID:20689504

  2. Fluorescence-Activated Cell Sorting and Gene Expression Profiling of GFP-Positive Cells from Transgenic Zebrafish Lines.

    PubMed

    Tanabe, Hideyuki; Seki, Masahide; Itoh, Mari; Deepak, Ailani; Lal, Pradeep; Horiuchi, Terumi; Suzuki, Yutaka; Kawakami, Koichi

    2016-01-01

    Gene expression profiling is a useful approach for deeper understanding of the specificity of cells, tissues, and organs in the transcriptional level. Recent development of high-throughput next-generation sequence (NGS) allows the RNA-seq method for this profiling. This method provides precise information of transcripts about the quantitation and the structure such as the splicing variants. In this chapter, we describe a method for gene expression profiling of GFP-positive cells from transgenic zebrafish by RNA-seq. We labeled specific cells in the brain with GFP by crossing a Gal4 driver line with the UAS:GFP line, isolated those cells by fluorescence-activated cell sorting (FACS), and analyzed by RNA-seq. PMID:27464803

  3. Ultrafast Electronic and Vibrational Dynamics of Stabilized A State Mutants of the Green Fluorescent Protein (GFP): Snipping the Proton Wire

    PubMed Central

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

    2008-01-01

    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. PMID:19554079

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

    NASA Astrophysics Data System (ADS)

    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.

  5. A linked series of laboratory exercises in molecular biology utilizing bioinformatics and GFP.

    PubMed

    Medin, Carey L; Nolin, Katie L

    2011-01-01

    Molecular biologists commonly use bioinformatics to map and analyze DNA and protein sequences and to align different DNA and protein sequences for comparison. Additionally, biologists can create and view 3D models of protein structures to further understand intramolecular interactions. The primary goal of this 10-week laboratory was to introduce the importance of bioinformatics in molecular biology. Students employed multiprimer, site-directed mutagenesis to create variant colors from a plasmid expressing green fluorescent protein (GFP). Isolated mutant plasmid from Escherichia coli showing changes in fluorescence were sequenced. Students used sequence alignment tools, protein translator tools, protein modeling, and visualization to analyze the potential effect of their mutations within the protein structure. This laboratory linked molecular techniques and bioinformatics to promote and expand the understanding of experimental results in an upper-level undergraduate laboratory course. PMID:22081550

  6. Modular multiplication in GF(p) for public-key cryptography

    NASA Astrophysics Data System (ADS)

    Olszyna, Jakub

    Modular multiplication forms the basis of modular exponentiation which is the core operation of the RSA cryptosystem. It is also present in many other cryptographic algorithms including those based on ECC and HECC. Hence, an efficient implementation of PKC relies on efficient implementation of modular multiplication. The paper presents a survey of most common algorithms for modular multiplication along with hardware architectures especially suitable for cryptographic applications in energy constrained environments. The motivation for studying low-power and areaefficient modular multiplication algorithms comes from enabling public-key security for ultra-low power devices that can perform under constrained environments like wireless sensor networks. Serial architectures for GF(p) are analyzed and presented. Finally proposed architectures are verified and compared according to the amount of power dissipated throughout the operation.

  7. Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery

    NASA Astrophysics Data System (ADS)

    Hao, Yuzhi; Xu, Peng; He, Chuan; Yang, Xiaoyan; Huang, Min; Xing, James; Chen, Jie

    2011-07-01

    1-ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

  8. GFP-complementation assay to detect functional CPP and protein delivery into living cells

    PubMed Central

    Milech, Nadia; Longville, Brooke AC; Cunningham, Paula T; Scobie, Marie N; Bogdawa, Heique M; Winslow, Scott; Anastasas, Mark; Connor, Theresa; Ong, Ferrer; Stone, Shane R; Kerfoot, Maria; Heinrich, Tatjana; Kroeger, Karen M; Tan, Yew-Foon; Hoffmann, Katrin; Thomas, Wayne R; Watt, Paul M; Hopkins, Richard M

    2015-01-01

    Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease. PMID:26671759

  9. Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery.

    PubMed

    Hao, Yuzhi; Xu, Peng; He, Chuan; Yang, Xiaoyan; Huang, Min; Xing, James; Chen, Jie

    2011-07-15

    1-Ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

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

    PubMed Central

    Armesto, Cecilia; Maia, Fernanda Gonçalves Martins; de Abreu, Mário Sobral; Figueira, Antonia dos Reis; da Silva, Bruno Marques; Monteiro, Fernando Pereira

    2012-01-01

    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 × 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. PMID:24031947

  11. Quantitative GFP fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish

    SciTech Connect

    Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Kim, Dong-Jae; Na, Yi-Rang; Noh, Kyoung-Jin; Park, Sung-Hoon; Lee, Hyun-Kyoung; Lee, Byoung-Hee; Ryu, Doug-Young; Park, Jae-Hak

    2007-12-01

    In transgenic zebrafish (Danio rerio), green fluorescent protein (GFP) is a promising marker for environmental pollutants. In using GFP, one of the obstacles which we faced was how to compare toxicity among different toxicants or among a specific toxicant in different model species with the intensity of GFP expression. Using a fluorescence detection method, we first validated our method for estimating the amount of GFP fluorescence present in transgenic fish, which we used as an indicator of developmental toxicity caused by the well-known toxicant, arsenite. To this end, we developed mosaic transgenic zebrafish with the human heat shock response element (HSE) fused to the enhanced GFP (EGFP) reporter gene to indicate exposure to arsenite. We confirmed that EGFP expression sites correlate with gross morphological disruption caused by arsenite exposure. Arsenite (300.0 {mu}M) caused stronger EGFP fluorescence intensity and quantity than 50.0 {mu}M and 10.0 {mu}M arsenite in our transgenic zebrafish. Furthermore, arsenite-induced apoptosis was demonstrated by TUNEL assay. Apoptosis was inhibited by the antioxidant, N-acetyl-cystein (NAC) in this transgenic zebrafish. The distribution of TUNEL-positive cells in embryonic tissues was correlated with the sites of arsenite toxicity and EGFP expression. The EGFP values quantified using the standard curve equation from the known GFP quantity were consistent with the arsenite-induced EGFP expression pattern and arsenite concentration, indicating that this technique can be a reliable and applicable measurement. In conclusion, we propose that fluorescence-based EGFP quantification in transgenic fish containing the hsp70 promoter-EGFP reporter-gene construct is a useful indicator of development toxicity caused by arsenite.

  12. Identification of Coilin Mutants in a Screen for Enhanced Expression of an Alternatively Spliced GFP Reporter Gene in Arabidopsis thaliana

    PubMed Central

    Kanno, Tatsuo; Lin, Wen-Dar; Fu, Jason L.; Wu, Ming-Tsung; Yang, Ho-Wen; Lin, Shih-Shun; Matzke, Antonius J. M.; Matzke, Marjori

    2016-01-01

    Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles. The molecular function of coilin, particularly of the substantial nucleoplasmic fraction, remains uncertain. We identified coilin loss-of-function mutations in a genetic screen for mutants showing either reduced or enhanced expression of an alternatively spliced GFP reporter gene in Arabidopsis thaliana. The coilin mutants feature enhanced GFP fluorescence and diminished Cajal bodies compared with wild-type plants. The amount of GFP protein is several-fold higher in the coilin mutants owing to elevated GFP transcript levels and more efficient splicing to produce a translatable GFP mRNA. Genome-wide RNA-sequencing data from two distinct coilin mutants revealed a small, shared subset of differentially expressed genes, many encoding stress-related proteins, and, unexpectedly, a trend toward increased splicing efficiency. These results suggest that coilin attenuates splicing and modulates transcription of a select group of genes. The transcriptional and splicing changes observed in coilin mutants are not accompanied by gross phenotypic abnormalities or dramatically altered stress responses, supporting a role for coilin in fine tuning gene expression. Our GFP reporter gene provides a sensitive monitor of coilin activity that will facilitate further investigations into the functions of this enigmatic protein. PMID:27317682

  13. Identification of Coilin Mutants in a Screen for Enhanced Expression of an Alternatively Spliced GFP Reporter Gene in Arabidopsis thaliana.

    PubMed

    Kanno, Tatsuo; Lin, Wen-Dar; Fu, Jason L; Wu, Ming-Tsung; Yang, Ho-Wen; Lin, Shih-Shun; Matzke, Antonius J M; Matzke, Marjori

    2016-08-01

    Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles. The molecular function of coilin, particularly of the substantial nucleoplasmic fraction, remains uncertain. We identified coilin loss-of-function mutations in a genetic screen for mutants showing either reduced or enhanced expression of an alternatively spliced GFP reporter gene in Arabidopsis thaliana The coilin mutants feature enhanced GFP fluorescence and diminished Cajal bodies compared with wild-type plants. The amount of GFP protein is several-fold higher in the coilin mutants owing to elevated GFP transcript levels and more efficient splicing to produce a translatable GFP mRNA. Genome-wide RNA-sequencing data from two distinct coilin mutants revealed a small, shared subset of differentially expressed genes, many encoding stress-related proteins, and, unexpectedly, a trend toward increased splicing efficiency. These results suggest that coilin attenuates splicing and modulates transcription of a select group of genes. The transcriptional and splicing changes observed in coilin mutants are not accompanied by gross phenotypic abnormalities or dramatically altered stress responses, supporting a role for coilin in fine tuning gene expression. Our GFP reporter gene provides a sensitive monitor of coilin activity that will facilitate further investigations into the functions of this enigmatic protein. PMID:27317682

  14. Coastal Louisiana Wetlands Restoration Monitoring with Global Fiducials Program (GFP) Imagery

    NASA Astrophysics Data System (ADS)

    Fisher, G.

    2012-12-01

    Coastal Louisiana has experienced dramatic landscape change over the past century due to human induced changes to the environment as well as an onslaught of major coastal storms. Coastal Louisiana loses on average 25-35 square miles of land per year. The USGS has partnered with the National Oceanographic and Atmospheric Administration (NOAA) - National Marine Fisheries Service to provide cyclical remote sensing data for selected restoration sites along the coast of Louisiana. Three of these sites are actively maintained in the GFP archive - Atchafalaya River Delta, East Timbalier Island, and Pecan Island. These three sites coincide with NOAA restoration sites that have been monitored since early 2000. The GFP has provided a consistent set of remote sensing data that has greatly benefited the long-term monitoring of these restoration sites. Long-term monitoring of these sites includes both pre- and post-hurricane season data collection used to identify landscape change along the coast. The long-term monitoring also has helped to identify areas of success in the restoration projects, as well as areas that have continued to decline in spite of restoration efforts. These three sites are significant to the program because they provide a variety of coastal landscape types: an open water barrier island environment at East Timbalier Island; coastal wetlands at Pecan Island, which have experienced subsidence of the marsh and convergence to an open water environment; and a deltaic marsh environment at Atchafalaya River Delta. Long-term monitoring of these sites has provided a wealth of knowledge about the changes occurring, as well as a valuable tool for reliable shoreline measurements. Continued monitoring is necessary to accurately assess the condition of these areas as environmental conditions continue to shape the landscape.

  15. Generation of knockout mice expressing a GFP-reporter under the control of the Lmx1a locus.

    PubMed

    Griesel, Gundula; Krug, Christian; Yurlova, Larisa; Diaconu, Mihaela; Mansouri, Ahmed

    2011-01-01

    Lmx1a is a member of the LIM homeodomain containing transcription factors and plays an important role during embryonic development. Specifically, it is required for the proper formation of several structures in the central nervous system, such as the roof plate, the cerebellum, and the inner ear. All these defects may contribute to the neurological phenotype observed in dreher mice, lacking functional Lmx1a protein. Interestingly, this factor was also found to promote midbrain dopaminergic neuron fate. We have introduced Green fluorescent protein (GFP) coding sequences into the Lmx1a locus by homologous recombination, and created knockout mice where GFP recapitulates the Lmx1a endogenous expression pattern.

  16. The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

    PubMed

    Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

    2013-03-01

    Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

  17. Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS)

    PubMed Central

    Hendriks, William T.; Bros-Facer, Virginie; van Minnen, Jan; Martin, Joanne E.; Jackson, Graham S.; Greensmith, Linda; Schiavo, Giampietro; Fisher, Elizabeth M. C.

    2010-01-01

    Background Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts. Methodology/Principal Findings Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP. Conclusions/Significance Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag. PMID:20221404

  18. Complete Genome Sequence of Cryobacterium arcticum Strain PAMC 27867, Isolated from a Sedimentary Rock Sample in Northern Victoria Land, Antarctica

    PubMed Central

    Lee, Jaejin; Cho, Ahnna; Yang, Jae Young; Woo, Jusun; Lee, Hong Kum; Hong, Soon Gyu

    2016-01-01

    Cryobacterium arcticum PAMC 27867, a psychrotolerant, Gram-positive bacterium, was isolated from a sedimentary rock sample collected at Eureka Spurs in northern Victoria Land, Antarctica. Here, we report the genome sequence of C. arcticum PAMC 27867. PMID:27587812

  19. 78 FR 60826 - Foreign-Trade Zone 155-Calhoun/Victoria Counties, Texas; Authorization of Production Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-02

    ... (78 FR 35604, 06/13/2013). The FTZ Board has determined that no further review of the activity is... Production Activity; Caterpillar, Inc. (Excavator and Frame Assembly Production); Victoria, Texas On May 29... proposed production activity to the Foreign-Trade Zones (FTZ) Board on behalf of Caterpillar, Inc.,...

  20. 78 FR 35604 - Foreign-Trade Zone (FTZ) 155-Calhoun/Victoria Counties, Texas; Notification of Proposed...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-13

    ... Proposed Production Activity; Caterpillar, Inc.; (Excavator and Frame Assembly Production), Victoria, Texas... proposed production activity to the FTZ Board on behalf of Caterpillar, Inc. (Caterpillar), located in... CFR 400.22) was received on May 29, 2013. The Caterpillar facility is located within FTZ 155, Site...

  1. Three Decades of Implementation of School-Based Management in the Australian Capital Territory and Victoria in Australia

    ERIC Educational Resources Information Center

    Gammage, David T.

    2008-01-01

    Purpose: The purpose of this paper is to explore how the process of implementation of school-based management (SBM) has worked within the public school systems in the Australian Capital Territory (ACT) and Victoria in Australia. The period covered was 1976-2006. Design/methodology/approach: The approach adopted was the mixed methodology which…

  2. Prevalence of Substance Use and Delinquent Behavior in Adolescents from Victoria, Australia and Washington State, United States

    ERIC Educational Resources Information Center

    McMorris, Barbara J.; Hemphill, Sheryl A.; Toumbourou, John W.; Catalano, Richard F.; Patton, George C.

    2007-01-01

    This article compares prevalence estimates of substance use and delinquent behavior in Washington State, United States and Victoria, Australia, two states chosen for their different policy environments around problem behavior. Few comparisons of international differences on rates of multiple problem behavior exist, and most are based on methods…

  3. Rates of Student-Reported Antisocial Behavior, School Suspensions, and Arrests in Victoria, Australia and Washington State, United States

    ERIC Educational Resources Information Center

    Hemphill, Sheryl A.; McMorris, Barbara J.; Toumbourou, John W.; Herrenkohl, Todd I.; Catalano, Richard F.; Mathers, Megan

    2007-01-01

    Background: Few methodologically rigorous international comparisons of student-reported antisocial behavior have been conducted. This paper examines whether there are differences in the frequency of both antisocial behavior and societal responses to antisocial behavior in Victoria, Australia and Washington State, United States. These 2 states were…

  4. BC College Transfer Credit Evaluation: An Analysis of Students Entering the University of Victoria, Winter 1998/99 Session.

    ERIC Educational Resources Information Center

    Victoria Univ. (British Columbia).

    This report documents the results of the transcript evaluation process for British Columbia (BC) college transfer students who applied to the University of Victoria (UVic) for the winter 1998/99 session. It examines the extent to which BC college credits were transferable/transferred to UVic and the reasons why they were sometimes not…

  5. Complete Genome Sequence of Cryobacterium arcticum Strain PAMC 27867, Isolated from a Sedimentary Rock Sample in Northern Victoria Land, Antarctica.

    PubMed

    Lee, Jaejin; Cho, Ahnna; Yang, Jae Young; Woo, Jusun; Lee, Hong Kum; Hong, Soon Gyu; Kim, Ok-Sun

    2016-01-01

    Cryobacterium arcticum PAMC 27867, a psychrotolerant, Gram-positive bacterium, was isolated from a sedimentary rock sample collected at Eureka Spurs in northern Victoria Land, Antarctica. Here, we report the genome sequence of C. arcticum PAMC 27867. PMID:27587812

  6. The Discourse of Public Education: An Urban Campaign for a Local Public High School in Melbourne, Victoria

    ERIC Educational Resources Information Center

    Rowe, Emma E.

    2014-01-01

    This paper explores the metonymic slippage surrounding the discourse of public education, through observations and interviews with Lawson High School active campaigners in the state of Victoria, Australia. The notion of campaigning for public education has become an ever-present issue on an international scale, and this article aims to contribute…

  7. Labor Pains: Implementing the Australian Labor Party's Policy of 'District Provision and School Reorganisation' in Victoria, 1989-92.

    ERIC Educational Resources Information Center

    Gronn, Peter

    1994-01-01

    Consent engineering is a consultative approach used by public policy-makers to secure the legitimacy of proposed policy changes by involving representative interest groups in decision making. Using the example of five reorganizing secondary schools in Victoria, Australia, this article outlines the circumstances obviating consent engineering at the…

  8. University of Victoria Genome British Columbia Proteomics Centre Partners with CPTAC - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    University of Victoria Genome British Columbia Proteomics Centre, a leader in proteomic technology development, has partnered with the U.S. National Cancer Institute (NCI) to make targeted proteomic assays accessible to the community through NCI’s CPTAC Assay Portal.

  9. Language in the Workplace Project and Workplace Communication for Skilled Migrants Course at Victoria University of Wellington, New Zealand

    ERIC Educational Resources Information Center

    de Bres, Julia; Holmes, Janet; Joe, Angela; Marra, Meredith; Newton, Jonathan; Riddiford Nicky; Vine, Bernadette

    2009-01-01

    The School of Linguistics and Applied Language Studies (LALS) at Victoria University of Wellington conducts research and teaching in Linguistics, Applied Linguistics, Writing and Deaf Studies. It incorporates a Deaf Studies Research Unit, which undertakes research on topics relating to deaf people and their language in New Zealand, and the New…

  10. Characterization of Vibrio cholerae bacteriophages isolated from the environmental waters of the Lake Victoria region of Kenya.

    PubMed

    Maina, Alice Nyambura; Mwaura, Francis B; Oyugi, Julius; Goulding, David; Toribio, Ana L; Kariuki, Samuel

    2014-01-01

    Over the last decade, cholera outbreaks have become common in some parts of Kenya. The most recent cholera outbreak occurred in Coastal and Lake Victoria region during January 2009 and May 2010, where a total of 11,769 cases and 274 deaths were reported by the Ministry of Public Health and Sanitation. The objective of this study is to isolate Vibrio cholerae bacteriophages from the environmental waters of the Lake Victoria region of Kenya with potential for use as a biocontrol for cholera outbreaks. Water samples from wells, ponds, sewage effluent, boreholes, rivers, and lakes of the Lake Victoria region of Kenya were enriched for 48 h at 37 °C in broth containing a an environmental strain of V. cholerae. Bacteriophages were isolated from 5 out of the 42 environmental water samples taken. Isolated phages produced tiny, round, and clear plaques suggesting that these phages were lytic to V. cholerae. Transmission electron microscope examination revealed that all the nine phages belonged to the family Myoviridae, with typical icosahedral heads, long contractile tails, and fibers. Head had an average diameter of 88.3 nm and tail of length and width 84.9 and 16.1 nm, respectively. Vibriophages isolated from the Lake Victoria region of Kenya have been characterized and the isolated phages may have a potential to be used as antibacterial agents to control pathogenic V. cholerae bacteria in water reservoirs.

  11. "Comfortably British" to "Fundamentally Economic"? The Effects of Language Policies on Year 12 Language Candidature in Victoria

    ERIC Educational Resources Information Center

    Baldwin, Jennifer

    2011-01-01

    This article seeks to explore how various language policies may have impacted Year 12 language candidature in Victoria. Australian Federal and Victorian State Government language policies are but one of the influences yet, it may be argued, the most significant influence. These stand alongside waves of immigration, global events and conflicts, and…

  12. "Why Has It Only become an Issue Now?": Young Drug Users' Perceptions of Drug Driving in Melbourne, Victoria

    ERIC Educational Resources Information Center

    Wilson, Laura Ann; Wilson, Dean

    2010-01-01

    Preliminary research into drug-user perceptions of drug driving was undertaken with a sample group of drug users aged 18 to 24 from Melbourne, Victoria. Eleven males and nine females participated in semi-structured interviews and completed self-report surveys. Participants discussed their drug driving and their perceptions of the likelihood of…

  13. White men and weak masculinity: men in the public asylums in Victoria, Australia, and New Zealand, 1860s-1900s.

    PubMed

    Coleborne, Catharine

    2014-12-01

    This article reveals a set of formulations of masculine identity through the fragments of extant casebook evidence from nineteenth-century psychiatric institutions in Victoria, Australia, and Auckland, New Zealand. It shows that some patterns in the identification of masculinity and insanity emerge, also highlighting the relevance of individual stories and 'cases' to fully understand how masculine identities were fashioned through medical institutional language.

  14. "Why Didn't They Teach Us Any of This before?": Youth Appraisal of Turkish Provision in Victoria

    ERIC Educational Resources Information Center

    Beykont, Zeynep F.

    2012-01-01

    This article examines youth assessment of the quality and success of languages provision. The discussion draws on data collected from students and graduates of Victoria's 16 secondary Turkish programs in large-scale surveys (n=858) and follow-up interviews (n=177). Surveys revealed that upper secondary Turkish classrooms serve predominantly…

  15. Marching Backwards into the Future: The Introduction of the English Creative Music Movement in State Secondary Schools in Victoria, Australia

    ERIC Educational Resources Information Center

    Burke, Harry

    2014-01-01

    In 1910, Victoria established an elite form of state secondary education that remained essentially unchanged until the introduction of a progressive curriculum during the late 1960s. This radical and voluntary curriculum introduced child-centred learning and personal development skills to state secondary schools. Many state secondary music…

  16. Rips, Currents and Snags: Investigating the Delivery of Educational Goals for Young Australians in the Region of Gippsland, Victoria

    ERIC Educational Resources Information Center

    Lynch, Timothy

    2012-01-01

    Monash University (Gippsland campus) is situated in Churchill, Latrobe Valley, located in central Gippsland, eastern Victoria. A large percentage of the Gippsland region comprises of a socio-economically disadvantaged population (Figure 1). In Semester One, 2011 as part of the Bachelor of Primary Education course at Monash, it was decided that a…

  17. 75 FR 71467 - Exelon Nuclear Texas Holdings, LLC, Early Site Permit Application for the Victoria County Station...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-23

    ..., BWR Projects Branch, Division of New Reactor Licensing, Office of New Reactors, U.S. Nuclear... From the Federal Register Online via the Government Publishing Office NUCLEAR REGULATORY COMMISSION Exelon Nuclear Texas Holdings, LLC, Early Site Permit Application for the Victoria County...

  18. Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock.

    PubMed Central

    Seehausen, Ole; Koetsier, Egbert; Schneider, Maria Victoria; Chapman, Lauren J; Chapman, Colin A; Knight, Mairi E; Turner, George F; van Alphen, Jacques J M; Bills, Roger

    2003-01-01

    Phylogenetic analyses based on mitochondrial (mt) DNA have indicated that the cichlid species flock of the Lake Victoria region is derived from a single ancestral species found in East African rivers, closely related to the ancestor of the Lake Malawi cichlid species flock. The Lake Victoria flock contains ten times less mtDNA variation than the Lake Malawi radiation, consistent with current estimates of the ages of the lakes. We present results of a phylogenetic investigation using nuclear (amplified fragment length polymorphism) markers and a wider coverage of riverine haplochromines. We demonstrate that the Lake Victoria-Edward flock is derived from the morphologically and ecologically diverse cichlid genus Thoracochromis from the Congo and Nile, rather than from the phenotypically conservative East African Astatotilapia. This implies that the ability to express much of the morphological diversity found in the species flock may by far pre-date the origin of the flock. Our data indicate that the nuclear diversity of the Lake Victoria-Edward species flock is similar to that of the Lake Malawi flock, indicating that the genetic diversity is considerably older than the 15 000 years that have passed since the lake began to refill. Most of this variation is manifested in trans-species polymorphisms, indicating very recent cladogenesis from a genetically very diverse founder stock. Our data do not confirm strict monophyly of either of the species flocks, but raise the possibility that these flocks have arisen from hybrid swarms. PMID:12590750

  19. Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae.

    PubMed

    Morales, Jorge; Chiu, Hsiling; Oo, Thiri; Plaza, Rosemary; Hoskins, Sally; Govind, Shubha

    2005-02-01

    Drosophila melanogaster larvae are attacked by virulent strains of parasitoid wasps. Females of Leptopilina heterotoma produce virus-like particles (VLPs) that efficiently destroy lamellocytes, a major larval immune effector cell type. We report here that L. victoriae, a closely related wasp species, also produces VLPs that trigger immune suppression responses in fly hosts. We compare the ability of immune suppression of the two parasitoids using a mutant host strain hopscotch(Tumorous-lethal) (hop(Tum-l)). hop(Tum-l) larvae have two defects of hematopoietic origin: overproliferation of hemocytes and constitutive encapsulation of self-tissue by lamellocytes. The encapsulation phenotype is suppressed weakly by L. victoriae and strongly by L. heterotoma. In vitro studies on hop(Tum-l) lamellocytes show that VLP-containing fluid from either wasp species induces lamellocyte lysis, but with different kinetics. Previously undocumented precursors of L. victoriae VLPs are synthesized in the long gland and are first visible within canals connecting secretory cells to the long gland lumen. VLP assembly occurs in the lumen. VLPs show multiple electron-dense projections surrounding a central core. Maturing particles appear segmented, singly or in arrays, embedded in the reservoir matrix. In sections, mature particles are pentagonal or hexagonal; the polygon vertices extending into spikes. Our results suggest that L. victoriae is likely to promote immune suppression by an active mechanism that is mediated by VLPs, similar to that used by L. heterotoma. PMID:15749103

  20. 'From the sphere of Sarah Gampism':The professionalisation of nursing and midwifery in the Colony of Victoria.

    PubMed

    Grehan, Madonna

    2004-09-01

    In the nineteenth century, while the Colony of Victoria was still a fledgling settlement, many of the hospitals of England, Scotland, Ireland, and Europe had instituted forms of nursing and midwifery training. When graduates of these training schemes emigrated to Australia with their knowledge, skills and experience, they found health practice to be haphazard and lacking in organisational standards. Individual immigrant women rose to prominence as managers of Victorian hospitals, and superintendents of homes for trained nurses. Through professional networks of their peers and compatriots, these women succeeded in placing the profession of nursing on a firm footing, and were instrumental in the emergence of professional organisations for trained nurses and midwives in Victoria, including the Melbourne District Nursing Society, the short-lived Nurses Association of Australasia (1892), and the Victorian Trained Nurses Association (1901). Their leadership was to have a profound influence on the way nursing and midwifery were regulated in twentieth century Victoria. In this historical review, we trace the movement to professionalise nursing and midwifery which emerged in the Colony of Victoria during the late nineteenth and early twentieth centuries.

  1. Extent and drainage status of organic soils in the Lake Victoria catchment

    NASA Astrophysics Data System (ADS)

    Barthelmes, Reni; Barthelmes, Alexandra; Joosten, Hans

    2016-04-01

    When considering peatlands and organic soils in the tropics, the huge areas in SE Asia prevail in public and scientific perception, whereas Africa has largely been out of focus. However, East Africa contains large areas of organic soils as well. They basically occur in the high altitudes of the uplifted flanks of the East African Rift System, isolated volcanoes and the Ethiopian highlands, in the Zambezian floodplains (e.g. Zambia), and in coastal environments (e.g. Mozambique and Madagascar). We used a mapping approach that integrates old field data and maps, specialized landscape and peatland-related knowledge, and modern RS and GIS techniques to elaborate a comprehensive and rather reliable overview of organic soils (incl. peatlands) in the Lake Victoria catchment. Maps at a scale of 1:25,000 have been prepared for Burundi, Kenya, Rwanda, Tanzania and Uganda. The land use intensity has been estimated for all organic soil areas based on satellite and aerial imagery. Feeding the Nile River, sustaining a fast growing and widely poor population and already facing climatic changes, organic soils of the Lake Victoria neighbouring countries are partially under heavy threat. We mapped 10,645 km2 of organic soils for the entire area of which 8,860 km2 (83.2%) seem to be in near natural condition. We assume slightly drainage and low degradation for 564 km2 (5.3%) and intensive drainage and heavy degradation for 1,222 km2 (11.5%). Degradation hotspot is Burundi with 522 km2 (79.5%) of heavily drained and degrading organic soils. This area assessment has been quite conservative to not overestimate the extent of organic soils. A reserve of 5-7,000 km2 of wetlands in the Lake Victoria catchment may include peatlands too, which needs to be confirmed in field surveys. Considering the key role of peatlands and organic soils for water provision and regulation and their rapid degradation due to drainage and inappropriate use, this inventory might be a step towards organic soil

  2. Risk factors for maternal morbidity in Victoria, Australia: a population-based study

    PubMed Central

    Lindquist, Anthea C; Kurinczuk, Jennifer J; Wallace, Euan M; Oats, Jeremy; Knight, Marian

    2015-01-01

    Objectives The aim of this analysis was to quantify the risk factors associated with maternal morbidity among women in Victoria, Australia, focusing particularly on sociodemographic factors. Design Case–control analysis. Participants Data on all maternities in Victoria from 1 January 2006 to 31 December 2008. Methods A case–control analysis was conducted using unconditional logistic regression to calculate adjusted ORs (aORs). Cases were defined as all women noted to have had a severe complication during the index pregnancy. Severe maternal morbidity was defined by the validated, composite Australian Maternal Morbidity Outcome Indicator. Socioeconomic position was defined by Socio-Economic Indices for Areas (SEIFA), specifically the Index of Relative Socioeconomic Disadvantage (IRSD), and other variables analysed were age, parity, Indigenous background, multiple pregnancy, country of birth, coexisting medical condition, previous caesarean section, spontaneous abortion or ectopic pregnancy. Results The study population comprised 211 060 women, including 1119 cases of severe maternal morbidity (0.53%). Compared with the highest IRSD quintile, the aOR for the 2nd quintile was 1.23 (95% CI 1.03 to 1.49), 0.98 (95% CI 0.79 to 1.21) for the 3rd quintile, 1.55 (95% CI 1.28 to 1.87) for the 4th and 1.21 (95% CI 1.00 to 1.47) for the lowest (most deprived) quintile. Indigenous status was associated with twice (aOR 2.02; 95% CI 1.32 to 3.09) the odds of being a case. Other risk factors for severe maternal morbidity were age ≥35 years (aOR 1.22; 95% CI 1.04 to 1.44), coexisting medical condition (aOR 1.39; 95% CI 1.16 to 1.65), multiple pregnancy (aOR 2.30; 95% CI 1.71 to 3.10), primiparity (aOR 1.36; 95% CI 1.18 to 1.57), previous caesarean section (aOR 1.79; 95% CI 1.53 to 2.10) and previous spontaneous miscarriage (aOR 1.25; 95% CI 1.08 to 1.44). Conclusions The findings from Victoria strongly suggest that social disadvantage needs to be acknowledged and further

  3. Supporting breastfeeding In Local Communities (SILC) in Victoria, Australia: a cluster randomised controlled trial

    PubMed Central

    McLachlan, Helen L; Forster, Della A; Amir, Lisa H; Cullinane, Meabh; Shafiei, Touran; Watson, Lyndsey F; Ridgway, Lael; Cramer, Rhian L; Small, Rhonda

    2016-01-01

    Objectives Breastfeeding has significant health benefits for mothers and infants. Despite recommendations from the WHO, by 6 months of age 40% of Australian infants are receiving no breast milk. Increased early postpartum breastfeeding support may improve breastfeeding maintenance. 2 community-based interventions to increase breastfeeding duration in local government areas (LGAs) in Victoria, Australia, were implemented and evaluated. Design 3-arm cluster randomised trial. Setting LGAs in Victoria, Australia. Participants LGAs across Victoria with breastfeeding initiation rates below the state average and > 450 births/year were eligible for inclusion. The LGA was the unit of randomisation, and maternal and child health centres in the LGAs comprised the clusters. Interventions Early home-based breastfeeding support by a maternal and child health nurse (home visit, HV) with or without access to a community-based breastfeeding drop-in centre (HV+drop-in). Main outcome measures The proportion of infants receiving ‘any’ breast milk at 3, 4 and 6 months (women's self-report). Findings 4 LGAs were randomised to the comparison arm and provided usual care (n=41 clusters; n=2414 women); 3 to HV (n=32 clusters; n=2281 women); and 3 to HV+drop-in (n=26 clusters; 2344 women). There was no difference in breastfeeding at 4 months in either HV (adjusted OR 1.04; 95% CI 0.84 to 1.29) or HV+drop-in (adjusted OR 0.92; 95% CI 0.78 to 1.08) compared with the comparison arm, no difference at 3 or 6 months, nor in any LGA in breastfeeding before and after the intervention. Some issues were experienced with intervention protocol fidelity. Conclusions Early home-based and community-based support proved difficult to implement. Interventions to increase breastfeeding in complex community settings require sufficient time and partnership building for successful implementation. We cannot conclude that additional community-based support is ineffective in improving breastfeeding

  4. Inhibitory Interneurons That Express GFP in the PrP-GFP Mouse Spinal Cord Are Morphologically Heterogeneous, Innervated by Several Classes of Primary Afferent and Include Lamina I Projection Neurons among Their Postsynaptic Targets.

    PubMed

    Ganley, Robert P; Iwagaki, Noboru; del Rio, Patricia; Baseer, Najma; Dickie, Allen C; Boyle, Kieran A; Polgár, Erika; Watanabe, Masahiko; Abraira, Victoria E; Zimmerman, Amanda; Riddell, John S; Todd, Andrew J

    2015-05-13

    The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to "unclassified" cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2006-05-01

    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 ACV-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

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

    PubMed Central

    Paul, Anna-Lisa; Bamsey, Matthew; Berinstain, Alain; Braham, Stephen; Neron, Philip; Murdoch, Trevor; Graham, Thomas; Ferl, Robert J.

    2008-01-01

    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.

  8. Dose-dependent fate of GFP-expressing Escherichia coli in the alimentary canal of adult house flies.

    PubMed

    Kumar, N H V; Nayduch, D

    2016-06-01

    The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe-rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose-dependent survival of bacteria in house flies. Flies were fed four different 'doses' of green fluorescent protein (GFP)-expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post-ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time-points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time-points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose-fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune-mediated. Understanding dose-dependent bacterial survival in flies can help in predicting bacteria transmission potential.

  9. Temporospatial fate of bacteria and immune effector expression in house flies fed GFP-Escherichia coli O157:H7.

    PubMed

    Fleming, A; Kumar, H V; Joyner, C; Reynolds, A; Nayduch, D

    2014-12-01

    The house fly Musca domestica L. (Diptera: Muscidae) harbours and transmits a variety of human enteropathogens including Escherichia coli (Enterobacteriales: Enterobacteriaceae) O157:H7. Interactions between ingested bacteria and the fly gut directly impact bacterial persistence, survival and ultimately fly vector competence. We assessed the temporospatial fate of green fluorescent protein (GFP)-expressing E. coli O157:H7 (GFP-ECO157) in house flies along with fly antimicrobial responses up to 12 h post-ingestion. In flies fed GFP-ECO157, culture and microscopy revealed a steady decrease in bacterial load over 12 h, which is likely to be attributable to the combined effects of immobilization within the peritrophic matrix, lysis and peristaltic excretion. However, flies can putatively transmit this pathogen in excreta because intact bacteria were observed in the crop and rectum. Quantitative reverse-transcriptase polymerase chain reaction analysis of antimicrobial peptides (AMPs) and lysozyme gene expression showed minimal upregulation in both the gut and carcass of house flies fed GFP-ECO157. However, these genes were upregulated in fly heads and salivary glands, and effector proteins were detected in the gut in some flies. Collectively, these data indicate that house flies can serve as reservoirs of E. coli O157:H7 for up to 12 h, and factors in addition to AMPs and lysozyme may contribute to bacteria destruction in the gut.

  10. Dose-dependent fate of GFP-expressing Escherichia coli in the alimentary canal of adult house flies.

    PubMed

    Kumar, N H V; Nayduch, D

    2016-06-01

    The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe-rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose-dependent survival of bacteria in house flies. Flies were fed four different 'doses' of green fluorescent protein (GFP)-expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post-ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time-points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time-points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose-fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune-mediated. Understanding dose-dependent bacterial survival in flies can help in predicting bacteria transmission potential. PMID:26843509

  11. Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluation of resistance or susceptibility of corn inbreds to infection by Aspergillus flavus was evaluated by a kernel screening assay. A GFP-expressing strain of A. flavus was used to accomplish this study to measure fungal spread and aflatoxin levels in real time. Among the four inbreds tested, ...

  12. Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

    SciTech Connect

    Yang Yongmin; Barankiewicz, Teresa J.; He Mingyue; Taussig, Michael J.; Chen, Swey-Shen . E-mail: alexchen.IGE.com@gmail.com

    2007-07-27

    Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.

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

    ERIC Educational Resources Information Center

    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…

  14. Tracking CO2 Geosequestration Using Downhole Gravity Gradiometry, Otway Basin, Victoria, Australia.

    NASA Astrophysics Data System (ADS)

    Matthews, S.; O'Neill, C.

    2015-12-01

    Carbon dioxide sequestration is a vital technique for the mitigation of greenhouse gas emissions and anthropogenic global warming. The viability of CO2 geosequestration, and the ability to prevent any leakage or migration depends on the capacity to monitor, and model, the CO2 reservoir. Downhole gravity gradiometry offers an additional capability to CO2 monitoring, and here we demonstrate the downhole gradient effect of CO2 injection at the Otway Basin, Victoria, by the CO2CRC corporation. Data from reservoir modelling has been simulated to calculate gravity gradients to gain an understanding of the behaviour of CO2 once injected into geological storage. Monte Carlo simulations were utilised to determine the uncertainties inherent in the deep subsurface. Presently; models have been created to demonstrate the response caused by the injection of CO2, and we present a statistical analysis to constrain the uncertainties posed by the results.

  15. World without borders: integrating clinical perspectives into the coronial jurisdiction in Victoria, Australia.

    PubMed

    Bohensky, M; Ibrahim, J E; O'Brien, A J; Emmett, S L; Newman, E; Charles, A; Young, C; Ranson, D L

    2006-03-01

    A professional world without borders would allow for more appropriate collaboration between the Coroner's office and the healthcare sector in their endeavours to improve patient safety. In Victoria (Australia), the Clinical Liaison Service draws on the distinct experiences and expertise of medical, nursing and research personnel to evaluate clinical evidence for the investigation of healthcare deaths reported to the State Coroner's Office. This approach allows for greater intersectoral collaboration between the Coroner's office and healthcare sector than a traditional English-style coronial system that relies on the expertise of coroners, forensic pathologists and police officers to investigate unexpected deaths. Encouraging collaboration between these two sectors may have averted or at least mitigated the atrocities caused by Harold Shipman or the events at Bristol's Royal Infirmary. This paper describes the work processes employed by the Clinical Liaison Service (CLS) to investigate healthcare deaths in a coronial setting. To describe how this work has fostered the advancement of patient care.

  16. Companion rabbit and companion bird management practices among a representative sample of guardians in Victoria, Australia.

    PubMed

    Howell, Tiffani J; Mornement, Kate; Bennett, Pauleen C

    2015-01-01

    Although companion animal management practices used by caregivers can influence the welfare of the companion animals, there is little existing information about the ways in which people attempt to meet their companion animals' needs. A representative sample of rabbit guardians (n = 93, representing 63,000 people) and bird guardians (n = 203, representing 157,000 people) in Victoria, Australia, completed an online survey. Items were related to the environmental, diet/exercise, behavioral, social, and health management practices used by guardians. Guardians sometimes meet their companion animals' welfare needs, but they do not always engage in best practices. Most (79%) bird guardians reported that they interacted with their birds daily, but only 68% of rabbit guardians did the same. Likewise, 32% of rabbit guardians and 55% of bird guardians never had their companion animals vaccinated. These results may be used for educational campaigns for improving companion animal welfare.

  17. Seismicity and arrival-time residuals from the Victoria Earthquake of June 9, 1980

    SciTech Connect

    Wong, V.; Frez, J.

    1981-01-01

    Hypocenter distribution in space and time of the aftershock activity from the Victoria Earthquake of June 9, 1980 was studied. It was concluded that the main event excited aftershocks in several pre-existing nests at the northwest end of the Cerro Prieto Fault, but no significant activity occurred at the immediate neighborhood of the main event. The depth of the aftershocks increases with the distance from the northwest end of the fault and this feature might be related with the higher temperatures and the spreading center located between the ends of the Imperial and Cerro Prieto Faults. The significance of the arrival-times residuals for local and regional stations is discussed both for P and S-waves and the importance of obtaining station corrections is emphasized. The non-uniqueness in determining a structure which minimizes the residuals is illustrated. Two different structures which satisfy the local data are presented.

  18. Suffering by comparison: Twitter users' reactions to the Victoria's Secret Fashion Show.

    PubMed

    Chrisler, Joan C; Fung, Kaitlin T; Lopez, Alexandra M; Gorman, Jennifer A

    2013-09-01

    Social comparison theory suggests that evaluating the self in comparison with others (e.g., peers, celebrities, models) can influence body image. Experimental studies that have tested effects of viewing idealized images in the media often show that women feel worse about themselves after seeing images that illustrate the beauty ideal. Twitter presents a naturally occurring opportunity to study viewers' reactions. An analysis was conducted of 977 tweets sent immediately before and during the 2011 Victoria's Secret Fashion Show that reference the show. Although the majority were idiosyncratic remarks, many tweets contain evidence of upward social comparisons to the fashion models. There were tweets about body image, eating disorders, weight, desires for food or alcohol, and thoughts about self-harm. The results support social comparison theory, and suggest that vulnerable viewers could experience negative affect, or even engage in harmful behaviors, during or after viewing the show or others like it. PMID:23769929

  19. Steranes and triterpanes in the Beacon Supergroup samples from southern Victoria Land in Antarctica

    SciTech Connect

    Matsumoto, Genki I.; Watanuki, Kunihiko ); Machihara, Tsutomu ); Suzuki, Noriyuki ); Funaki, Minoru )

    1987-10-01

    Steranes and triterpanes in Beacon Supergroup samples (sedimentary rock and silicified wood) from Allan Hills and Carapace Nunatak of southern Victoria Land in Antarctica were studied to elucidate sources of organic materials, sedimentary paleoenvironment and thermal history after deposition. Relative abundances of C{sub 27}, C{sub 28} and C{sub 29} steranes and visual kerogen results of Beacon Supergroup samples from Allan Hills imply that organic materials in the sedimentary paleoenvironments are contributed mainly by vascular plants with some influence of microorganisms, while those of the Carapace Nunatak sample may be largely due to fern spores. The pristane/phytane and pristane/heptadecane ratios of the samples were generally close to unity and between 0.50 and 0.99, respectively, suggesting that the sedimentary paleoenvironment was shallow lacustrine with alternating oxic and anoxic conditions.

  20. Was Queen Victoria depressed? 1. Natural history and differential diagnosis of presenting problem.

    PubMed

    Powles, W E; Alexander, M G

    1987-02-01

    For some years we have speculated as to whether Queen Victoria suffered a definable psychiatric illness in her notorious and prolonged seclusion after the Prince Consort's death. We here summarize criteria for grief and depression from three authorities. Against these, we examine the natural history of the Queen's bereavement and restitution. We find that her suffering and her portrayal of the role of widow were related to her personal style and were culturally accepted. Her self-esteem, ego functions, and object relatedness were preserved. While some clinicians might favour a diagnosis of Dysthymic Disorder, we find the evidence strongly in favour of an intense, prolonged, normal human grief (Uncomplicated Bereavement of DSM III) coloured by a romantic and histrionic personal style. Intensity and duration do not, in this case, establish a diagnosis of depression.

  1. A pharyngeal jaw evolutionary innovation facilitated extinction in Lake Victoria cichlids.

    PubMed

    McGee, Matthew D; Borstein, Samuel R; Neches, Russell Y; Buescher, Heinz H; Seehausen, Ole; Wainwright, Peter C

    2015-11-27

    Evolutionary innovations, traits that give species access to previously unoccupied niches, may promote speciation and adaptive radiation. Here, we show that such innovations can also result in competitive inferiority and extinction. We present evidence that the modified pharyngeal jaws of cichlid fishes and several marine fish lineages, a classic example of evolutionary innovation, are not universally beneficial. A large-scale analysis of dietary evolution across marine fish lineages reveals that the innovation compromises access to energy-rich predator niches. We show that this competitive inferiority shaped the adaptive radiation of cichlids in Lake Tanganyika and played a pivotal and previously unrecognized role in the mass extinction of cichlid fishes in Lake Victoria after Nile perch invasion.

  2. A new resonance Rayleigh scattering spectral method for determination of O3 with victoria blue B

    NASA Astrophysics Data System (ADS)

    Wen, Guiqing; Yang, Duo; Jiang, Zhiliang

    2014-01-01

    Ozone (O3) could be absorbed by boric acid-potassium iodide (BKI) absorbent solution to produce tri-iodine ion (I3-) that react with victoria blue B (VBB) to form the associated particle (VBB-I3)n and exhibited a strong resonance Rayleigh scattering (RRS) peak at 722 nm. Under the chosen conditions, the RRS peak intensity was linear with O3 concentration in the range of 0.2-50 μmol/L, with a linear regression equation of ΔI722 = 17.9c - 45.4 and detection limit of 0.057 μmol/L. Accordingly, a simple, rapid and sensitive RRS spectral method was set up for determination of trace O3 in air, with satisfactory results.

  3. Chemostratigraphy and Fe Mineralogy of the Victoria Crater Duck Bay Section: Opportunity APXS and Moessbauer Results

    NASA Technical Reports Server (NTRS)

    Mittlefehldt, D. W.; Schroeder, C.; Gellert, R.; Klingelhoefer, G.; Jolliff, B. L.; Morris, R. V.

    2008-01-01

    Meridiani Planum is a vast plain of approximately horizontally bedded sedimentary rocks composed of mixed and reworked basaltic and evaporitic sands containing secondary, diagenetic minerals [e.g., 1-5]. Because bedding planes are subparallel to topography, investigation of contiguous stratigraphy requires examining exposures in impact craters. Early in the mission (sols 130-317), Opportunity was commanded to do detailed study of exposed outcrops in Endurance crater, including the contiguous Karatepe section at the point of ingress. Just over 1000 sols later and roughly 7 km to the south, the rover is being commanded to do a similar study of the Duck Bay section of Victoria crater. Here we report on the preliminary results from the Alpha Particle X-ray Spectrometer (APXS) and Moessbauer instruments.

  4. Protecting genetic materials and genetic information: a case study of Guthrie Cards in Victoria.

    PubMed

    Lawson, C; Smith, R

    2001-11-01

    The authors are privileged to have been provided with correspondence about a dispute over the ongoing storage of genetic material (as Guthrie Cards) in Victoria. The correspondence details confusion over the roles of government and the private sector service provider in accounting for the storage, use and destruction of these stored genetic materials collected as part of a government public health program. The purpose in publishing this account is to highlight the present inadequacies in current practices and the ongoing potential for a crisis in the management of collected genetic materials through a lack of appropriate regulation, transparency and accountability. The article suggests measures to remedy some of the existing inadequacies in contractual arrangements and recommends that the government retain ownership and control of both the genetic materials and the derived information to ensure some accountability in the present legal environment.

  5. Basal Adare volcanics, Robertson Bay, North Victoria Land, Antarctica: Late Miocene intraplate basalts of subaqueous origin

    USGS Publications Warehouse

    Mortimer, N.; Dunlap, W.J.; Isaac, M.J.; Sutherland, R.P.; Faure, K.

    2007-01-01

    Late Cenozoic lavas and associated hyaloclastite breccias of the Adare volcanics (Hallett volcanic province) in Robertson Bay, North Victoria Land rest unconformably on Paleozoic greywackes. Abundant hyaloclastite breccias are confined to a paleovalley; their primary geological features, and the stable isotope ratios of secondary minerals, are consistent with eruption in a subaqueous environment with calcite formation probably involving seawater. In contrast, the lavas which stratigraphically overlie the hyaloclastites on Mayr Spur probably were erupted subaerially. K-Ar dating of eight samples from this basal sequence confirms the known older age limit (Late Miocene) of the Hallett volcanic province. Geochemical data reveal an ocean island basalt-like affinity, similar to other Cenozoic igneous rocks of the Hallett volcanic province. If a submarine eruptive paleoenvironment is accepted then there has been net tectonic or isostatic post-Late Miocene uplift of a few hundred metres in the Robertson Bay-Adare Peninsula area

  6. A new Holocene relative sea-level curve for Terra Nova Bay, Victoria Land, Antarctica

    NASA Astrophysics Data System (ADS)

    Baroni, Carlo; Hall, Brenda L.

    2004-05-01

    More than 100 radiocarbon dates of penguin guano and remains, shells and seal skin afford ages for raised beaches adjacent to Terra Nova Bay, Antarctica. These dates permit construction of a new relative sea-level curve that bears on the timing of deglaciation. Recession of the Ross Sea ice-sheet grounding line from Terra Nova Bay occurred no earlier than 7200 14C yr (8000 cal. yr) BP. Retreat along the Victoria Land coast may have been rapid, possibly contributing to eustatic sea-level rise centred at ca. 7600 cal. yr BP. The presence of a significant amount of ice remaining in the Ross Sea Embayment in Holocene time lessens the chance that Antarctica contributed significantly to meltwater pulse 1A several thousand years earlier. Copyright

  7. Writing history: case study of the university of Victoria School of Nursing.

    PubMed

    Scaia, Margaret R; Young, Lynne

    2013-04-23

    A historical examination of a nursing curriculum is a bridge between past and present from which insights to guide curriculum development can be gleaned. In this paper, we use the case study method to examine how the University of Victoria School of Nursing (UVic SON), which was heavily influenced by the ideology of second wave feminism, contributed to a change in the direction of nursing education from task-orientation to a content and process orientation. This case study, informed by a feminist lens, enabled us to critically examine the introduction of a "revolutionary" caring curriculum at the UVic SON. Our research demonstrates the fault lines and current debates within which a feminist informed curriculum continues to struggle for legitimacy and cohesion. More work is needed to illuminate the historical basis of these debates and to understand more fully the complex landscape that has constructed the social and historical position of women and nursing in Canadian society today.

  8. Genetic diversity of the endangered endemic Agave victoriae-reginae (Agavaceae) in the Chihuahuan Desert.

    PubMed

    Martínez-Palacios, A; Eguiarte, L E; Furnier, G R

    1999-08-01

    Long-lived perennials are a species-rich, ecologically important component of the North American deserts, yet we know little about their genetic structure, information important for their conservation. Agave victoriae-reginae is an endemic of the Chihuahuan Desert of northern Mexico that is endangered by collection for the ornamental trade. We examined levels and patterns of variation at ten polymorphic allozyme loci in ten populations representing the range of the species. Levels of genetic variation (mean H(e)= 0.335) and differentiation (mean F(ST) = 0.236) were high. Phenetic clustering suggested the existence of at least three distinct groups of populations. If this pattern of variation is representative of other long-lived desert perennials, it may explain the species richness of this group and will pose a real challenge to gene conservation efforts.

  9. A pharyngeal jaw evolutionary innovation facilitated extinction in Lake Victoria cichlids.

    PubMed

    McGee, Matthew D; Borstein, Samuel R; Neches, Russell Y; Buescher, Heinz H; Seehausen, Ole; Wainwright, Peter C

    2015-11-27

    Evolutionary innovations, traits that give species access to previously unoccupied niches, may promote speciation and adaptive radiation. Here, we show that such innovations can also result in competitive inferiority and extinction. We present evidence that the modified pharyngeal jaws of cichlid fishes and several marine fish lineages, a classic example of evolutionary innovation, are not universally beneficial. A large-scale analysis of dietary evolution across marine fish lineages reveals that the innovation compromises access to energy-rich predator niches. We show that this competitive inferiority shaped the adaptive radiation of cichlids in Lake Tanganyika and played a pivotal and previously unrecognized role in the mass extinction of cichlid fishes in Lake Victoria after Nile perch invasion. PMID:26612951

  10. Perceptions of prescribed burning in a local forest community in Victoria, Australia.

    PubMed

    Bell, Tina; Oliveras, Immaculada

    2006-11-01

    The general perceptions of prescribed burning were elicited from forest users for an area that has been subject to this form of land management for at least 20 years. The largest group consisted of local residents living in and around the Wombat State Forest with two smaller groups of students from a nearby university campus and local professional land managers. A questionnaire was given to each participant in order to explore how the forest was used, to determine the level of knowledge of burning in the targeted forest and Victoria and the perception of the appearance, effectiveness of protection, and accessibility to the forest after prescribed burning. Generally all groups had similar responses with community members having stronger views on the effectiveness and practicalities of prescribed burning, whereas students were more neutral in their opinions. All participants claimed knowledge of prescribed burning activities within Victoria, but fewer had experience of planned fires in the Wombat State Forest. All groups agreed that areas that had not been recently burned had a better appearance than those that had, but this result may have included a range of value judgments. Land managers had a greater understanding of the ecological importance of season and timing of burning; however, some students and community members were equally knowledgeable. Prescribed burning did not impede access to the forest, nor did smoke from prescribed burns pose any great problem. The majority of the participants felt that the amount of prescribed burning done in the forest was adequate for engendering a feeling of protection to life and property, yet many were still suspicious of this management practice. These initial findings indicate several areas in which further research would be useful including the efficacy of education programs for community members and improved communication of burn plans by land managers.

  11. Impact of an outright ban on the availability of commercial tanning services in Victoria, Australia.

    PubMed

    Sinclair, C; Cleaves, N; Dunstone, K; Makin, J; Zouzounis, S

    2016-08-01

    Australian states have introduced an outright ban on commercial artificial tanning sunbeds. This was based on evidence from systematic reviews affirming a strong association between tanning bed use and increased risk of melanoma. The implementation of the ban provides an opportunity to assess the effectiveness of the legislation in preventing access to artificial UV radiation. Compliance with the ban has been enforced and monitored by government departments. We additionally monitored online tanning bed advertisements before and after the ban, showing a decline over time in the number of sunbeds advertised for sale on gumtree.com.au and ebay.com.au, from a peak of 115 advertisements in May 2014 to 50 or fewer per month from February 2015. There was also an increase in advertised price, with the percentage of sunbeds offered in Victoria on gumtree.com.au at higher than $2 000 increasing from around 25% between January and June 2014 to 65% in the quarter following the introduction of the ban. Advertisements on gumtree.com.au from individuals wishing to access a sunbed in a private home in Victoria increased from only isolated instances in the months immediately prior to the ban, to between 25 and 77 advertisements per month in the quarter following the ban. In summary, the introduction of an outright ban of commercial sunbeds has achieved a dramatic reduction in the availability of harmful artificial UV radiation sources in Australia. Long-term benefits to the health of the population and a reduction in costs to the health system are expected to result.

  12. Mantle metasomatism beneath western Victoria, Australia: I. Metasomatic processes in Cr-diopside lherzolites

    NASA Astrophysics Data System (ADS)

    O'Reilly, Suzanne Y.; Griffin, W. L.

    1988-02-01

    Most Cr-diopside spinel lherzolite xenoliths from Bullenmerri and Gnotuk Maars, western Victoria, show modal metasomatism involving the growth of amphibole ± mica ± apatite at the expense of primary pyroxenes + spinel. The metasomatism is attributed to CO 2-rich fluids, observed in fluid inclusions. Median values of Ni, Cr, V, Sc, Y, Ti, K and Na are similar to those in depleted mantle xenoliths and the source regions of N-type MORB. Median concentrations of Ba, Th, U, Ta, Nb, Sr and REE range from 1-10 times "primordial" values. REE patterns of anhydrous lherzolites range from LREE-depleted ( (La/Yb) n ≈ 0.3 ) to LREE-enriched ( (La/Yb) n = 30-60 ), and show an inverse correlation of Nd/Sm with CaO. Amphibole-rich peridotites are enriched in LREE ( (La/Yb) n = 10-30 ), Zr and Ta, with high K/Rb. Mica-rich rocks are enriched in K, Rb, Ba, Ta and Ti, with low K/ Rb. Introduction of apatite leads to high σREE (with (La/Yb) n = 40-100 ), Sr, U and Th contents. The distribution of trace and minor elements in the Iherzolites is thus controlled by the crystal chemistry of the primary and metasomatic phases. Micaceous xenoliths may be derived from thin selvedges on pyroxenite veins. Abundant amphibole lherzolites may form a matrix enclosing relict volumes of anhydrous lherzolites showing varying degrees of cryptic metasomatism. The overall pattern of trace-element enrichment in the mantle beneath Victoria will depend on the volumetric proportions of these rock types.

  13. Simulation of Lake Victoria Circulation Patterns Using the Regional Ocean Modeling System (ROMS).

    PubMed

    Nyamweya, Chrispine; Desjardins, Christopher; Sigurdsson, Sven; Tomasson, Tumi; Taabu-Munyaho, Anthony; Sitoki, Lewis; Stefansson, Gunnar

    2016-01-01

    Lake Victoria provides important ecosystem services including transport, water for domestic and industrial uses and fisheries to about 33 million inhabitants in three East African countries. The lake plays an important role in modulating regional climate. Its thermodynamics and hydrodynamics are also influenced by prevailing climatic and weather conditions on diel, seasonal and annual scales. However, information on water temperature and circulation in the lake is limited in space and time. We use a Regional Oceanographic Model System (ROMS) to simulate these processes from 1st January 2000 to 31st December 2014. The model is based on real bathymetry, river runoff and atmospheric forcing data using the bulk flux algorithm. Simulations show that the water column exhibits annual cycles of thermo-stratification (September-May) and mixing (June-August). Surface water currents take different patterns ranging from a lake-wide northward flow to gyres that vary in size and number. An under flow exists that leads to the formation of upwelling and downwelling regions. Current velocities are highest at the center of the lake and on the western inshore waters indicating enhanced water circulation in those areas. However, there is little exchange of water between the major gulfs (especially Nyanza) and the open lake, a factor that could be responsible for the different water quality reported in those regions. Findings of the present study enhance understanding of the physical processes (temperature and currents) that have an effect on diel, seasonal, and annual variations in stratification, vertical mixing, inshore-offshore exchanges and fluxes of nutrients that ultimately influence the biotic distribution and trophic structure. For instance information on areas/timing of upwelling and vertical mixing obtained from this study will help predict locations/seasons of high primary production and ultimately fisheries productivity in Lake Victoria. PMID:27030983

  14. Long-term groundwater storage change in Victoria, Australia from satellite gravity and in situ observations

    NASA Astrophysics Data System (ADS)

    Chen, J. L.; Wilson, C. R.; Tapley, B. D.; Scanlon, Bridget; Güntner, Andreas

    2016-04-01

    Analysis based on satellite gravity measurements from the Gravity Recovery and Climate Experiment (GRACE) and land surface models indicates that groundwater storage in Victoria, Australia had been declining steadily, until a trend reversal around early 2010, attributed to two wetter seasons in 2010 and 2011. In situ groundwater level measurements (from a network of 1395 bores in Victoria) also indicate a steady groundwater depletion since the early 1990's, and show remarkable agreement with GRACE estimates for the 10-year period (2003-2012) in common with the GRACE mission. Groundwater depletion rates for 2005 to 2009 are relatively large as indicated by both GRACE estimates (8.0 ± 1.7 km3/yr) and in situ measurements (8.3 ± 3.4 km3/yr). Over the same period (2005-2009), GRACE measurements capture significant groundwater depletion in a wider region covering much of the southern Murray-Darling Basin, and the total groundwater depletion rate in this region is about 17.2 ± 4.7 km3/yr. Annual groundwater storage changes are strongly correlated with precipitation anomalies, but only about one-fifth of anomalous precipitation contributes to groundwater recharge. The strong correlation suggests that this groundwater depletion is primarily related to drought with related groundwater pumping for agricultural and domestic consumption. The remarkable agreement between GRACE estimates and in situ measurements demonstrates the great potential of satellite gravity observations in combination with land surface model estimates to quantify changes in regional groundwater resources, especially when in situ measurements are limited or unavailable. This study shows the importance of reducing leakage bias in GRACE observations and the effectiveness of the forward modeling iterative method used.

  15. A Paramedic-staffed Helicopter Emergency Medical Service's Response to Winch Missions in Victoria, Australia.

    PubMed

    Meadley, Ben; Heschl, Stefan; Andrew, Emily; de Wit, Anthony; Bernard, Stephen A; Smith, Karen

    2016-01-01

    Winching emergency medical care providers from a helicopter to the scene enables treatment of patients in otherwise inaccessible locations, but is not without risks. The objective of this study was to define characteristics of winch missions undertaken by Intensive Care Flight Paramedics (ICFP) in Victoria, Australia with a focus on extraction methods and clinical care delivered at the scene. A retrospective data analysis was performed to identify all winch missions between November 2010 and March 2014. Demographic data, winch characteristics, physiological parameters, and interventions undertaken on scene by the ICFP were extracted. Out of 5,003 missions in the study period, 125 were identified as winch operations. Winter missions were significantly less frequent than those of any other season. Patients were predominantly male (78.4%) and had a mean age of 38 years (±17.6). A total of 109 (87.2%) patients were identified as experiencing trauma with a mean Revised Trauma Score of 7.5288, and isolated limb fractures were the most frequently encountered injury. Falls and vehicle-related trauma were the most common mechanisms of injury. The total median scene duration was 49 minutes (IQR 23-91). Sixty-three patients (50.4%) were extracted using a stretcher, 45 (36.0%) using a hypothermic strop, and 6 (4.8%) via normal rescue strop. Eleven patients (8.8%) were not winched to the helicopter. Vascular access (38.4%), analgesia (44.0%), and anti-emetic administration (28.8%) were the most frequent clinical interventions. Forty-nine patients (39.2%) did not receive any clinical intervention prior to winch extraction. Winch operations in Victoria, Australia consisted predominantly of patients with minor to moderate traumatic injuries. A significant proportion of patients did not require any clinical treatment prior to winching, and among those who did, analgesia was the most frequent intervention. Advanced medical procedures were rarely required prior to winch extraction.

  16. Impact of an outright ban on the availability of commercial tanning services in Victoria, Australia.

    PubMed

    Sinclair, C; Cleaves, N; Dunstone, K; Makin, J; Zouzounis, S

    2016-08-01

    Australian states have introduced an outright ban on commercial artificial tanning sunbeds. This was based on evidence from systematic reviews affirming a strong association between tanning bed use and increased risk of melanoma. The implementation of the ban provides an opportunity to assess the effectiveness of the legislation in preventing access to artificial UV radiation. Compliance with the ban has been enforced and monitored by government departments. We additionally monitored online tanning bed advertisements before and after the ban, showing a decline over time in the number of sunbeds advertised for sale on gumtree.com.au and ebay.com.au, from a peak of 115 advertisements in May 2014 to 50 or fewer per month from February 2015. There was also an increase in advertised price, with the percentage of sunbeds offered in Victoria on gumtree.com.au at higher than $2 000 increasing from around 25% between January and June 2014 to 65% in the quarter following the introduction of the ban. Advertisements on gumtree.com.au from individuals wishing to access a sunbed in a private home in Victoria increased from only isolated instances in the months immediately prior to the ban, to between 25 and 77 advertisements per month in the quarter following the ban. In summary, the introduction of an outright ban of commercial sunbeds has achieved a dramatic reduction in the availability of harmful artificial UV radiation sources in Australia. Long-term benefits to the health of the population and a reduction in costs to the health system are expected to result. PMID:27535604

  17. Simulation of Lake Victoria Circulation Patterns Using the Regional Ocean Modeling System (ROMS)

    PubMed Central

    Sigurdsson, Sven; Tomasson, Tumi; Taabu-Munyaho, Anthony; Sitoki, Lewis; Stefansson, Gunnar

    2016-01-01

    Lake Victoria provides important ecosystem services including transport, water for domestic and industrial uses and fisheries to about 33 million inhabitants in three East African countries. The lake plays an important role in modulating regional climate. Its thermodynamics and hydrodynamics are also influenced by prevailing climatic and weather conditions on diel, seasonal and annual scales. However, information on water temperature and circulation in the lake is limited in space and time. We use a Regional Oceanographic Model System (ROMS) to simulate these processes from 1st January 2000 to 31st December 2014. The model is based on real bathymetry, river runoff and atmospheric forcing data using the bulk flux algorithm. Simulations show that the water column exhibits annual cycles of thermo-stratification (September–May) and mixing (June–August). Surface water currents take different patterns ranging from a lake-wide northward flow to gyres that vary in size and number. An under flow exists that leads to the formation of upwelling and downwelling regions. Current velocities are highest at the center of the lake and on the western inshore waters indicating enhanced water circulation in those areas. However, there is little exchange of water between the major gulfs (especially Nyanza) and the open lake, a factor that could be responsible for the different water quality reported in those regions. Findings of the present study enhance understanding of the physical processes (temperature and currents) that have an effect on diel, seasonal, and annual variations in stratification, vertical mixing, inshore—offshore exchanges and fluxes of nutrients that ultimately influence the biotic distribution and trophic structure. For instance information on areas/timing of upwelling and vertical mixing obtained from this study will help predict locations/seasons of high primary production and ultimately fisheries productivity in Lake Victoria. PMID:27030983

  18. Meteoritic and bedrock constraints on the glacial history of Frontier Mountain in northern Victoria Land, Antarctica

    NASA Astrophysics Data System (ADS)

    Welten, K. C.; Folco, L.; Nishiizumi, K.; Caffee, M. W.; Grimberg, A.; Meier, M. M. M.; Kober, F.

    2008-06-01

    In 2001, a small H4 chondrite, Frontier Mountain (FRO) 01149, was found on a glacially eroded surface near the top of Frontier Mountain, Antarctica, about 600 m above the present ice level. The metal and sulphides are almost completely oxidized due to terrestrial weathering. We used a chemical leaching procedure to remove weathering products, which contained atmospheric 10Be and 36Cl in a ratio similar to that found in Antarctic ice. The FRO 01149 meteorite has a terrestrial age of 3.0 ± 0.3 Myr based on the concentrations of the cosmogenic radionuclides 10Be, 26Al and 36Cl. This age implies that FRO 01149 is the oldest stony meteorite (fossil meteorites excluded) discovered on Earth. The noble gas cosmic ray exposure age of FRO 01149 is ~ 30 Myr. The meteorite thus belongs to the 33 Myr exposure age peak of H-chondrites. The bedrock surface on which FRO 01149 was found has wet-based glacial erosional features recording a former high-stand of the East Antarctic ice sheet. This ice sheet evidently overrode the highest peaks (> 2800 m a.s.l.) of the inland sector of the Transantarctic Mountains in northern Victoria Land. We argue that FRO 01149 was a local fall and that its survival on a glacially eroded bedrock surface constrains the age of the last overriding event to be older than ~ 3 Myr. The concentrations of in-situ produced cosmogenic 10Be, 26Al and 21Ne in a glacially eroded bedrock sample taken from near the summit of Frontier Mountain yield a surface exposure age of 4.4 Myr and indicate that the bedrock was covered by several meters of snow. The exposure age is also consistent with bedrock exposure ages of other summit plateaus in northern Victoria Land.

  19. Prevalence of Schistosomes and Soil-Transmitted Helminths among Schoolchildren in Lake Victoria Basin, Tanzania.

    PubMed

    Siza, Julius E; Kaatano, Godfrey M; Chai, Jong-Yil; Eom, Keeseon S; Rim, Han-Jong; Yong, Tai-Soon; Min, Duk-Young; Chang, Su Young; Ko, Yunsuk; Changalucha, John M

    2015-10-01

    The objectives of this study was to conduct a survey on schistosomiasis and soil-transmitted helminth (STH) infections in order to come up with feasible control strategies in Lake Victoria basin, Tanzania. Depending on the size of the school, 150-200 schoolchildren were recruited for the study. Duplicate Kato-Katz stool smears were prepared from each child and microscopically examined for Schistosoma mansoni and STHs. Urine specimens were examined for Schistosoma haematobium eggs using the filtration technique. After the survey, mass drug administration was done using praziquantel and albendazole for schistosomiasis and STHs infections, respectively. A total of 5,952 schoolchildren from 36 schools were recruited for the study and had their stool and urine specimens examined. Out of 5,952 schoolchildren, 898 (15.1%) were positive for S. mansoni, 754 (12.6%) for hookworms, 188 (3.2%) for Ascaris lumblicoides, and 5 (0.008%) for Trichuris trichiura. Out of 5,826 schoolchildren who provided urine samples, 519 (8.9%) were positive for S. haematobium eggs. The results revealed that intestinal schistosomiasis, urogenital schistosomiasis, and STH infections are highly prevalent throughought the lake basin. The high prevalence of intestinal and urogenital schistosomisiasis in the study area was a function of the distance from Lake Victoria, the former being more prevalent at localities close to the lake, whilst the latter is more so away from it. Control of schistosomiasis and STHs in the study area requires an integrated strategy that involves provision of health education to communities, regular treatments, and provision of adequate safe water supply and sanitation facilities.

  20. Detailed glaciochemical investigations in southern Victoria Land - a proxy climatic record

    SciTech Connect

    Mayewski, P.A.

    1987-09-01

    Local accumulation-basins in the Transantarctic Mountains possess sites suitable for recovering ice-core records that are valuable for studying climate change. These sites are also unique, because they are close to the sites of other ice-core studies and to areas with established terrestrial records. The objective is to study a snowpit in detail and collect ice cores in southern Victoria Land; this work will be similar to the investigations that the authors has conducted in south Greenland and in the Dominion Range near the Beardmore Glacier. The proposed sites are in Convoy, Asgard, and Royal Society ranges. The authors will select one site at which he will recover two ice cores, each approximately 200 to 300 meters in depth. Samples will be analyzed for major anions (chloride, sulfate, nitrate, fluoride) and cations (sodium, potassium, magnesium, ammonium, silicate), total acidity, conductivity, density, and core stratigraphy with dating provided by cross-calibration of all of the preceding plus total beta-activity, lead-210, oxygen isotopes, and microparticles. This investigation will yield a detailed record of several thousand years of glacial history, climate change, and volcanic activity for southern Victoria Land. This record will be compared to existing terrestrial records to add necessary detail and to other global ice-core records to assess global climatic change. It will also help to document volcanic activity for Mount Erebus as well as other volcanos in the Southern Hemisphere and possibly some in the Northern Hemisphere. With this record, the author will be able to evaluate the influence of volcanic and solar activity on climate as well as add greatly to the understanding of the chemistry of the global atmosphere.

  1. Prevalence of Schistosomes and Soil-Transmitted Helminths among Schoolchildren in Lake Victoria Basin, Tanzania

    PubMed Central

    Siza, Julius E.; Kaatano, Godfrey M.; Chai, Jong-Yil; Eom, Keeseon S.; Rim, Han-Jong; Yong, Tai-Soon; Min, Duk-Young; Chang, Su Young; Ko, Yunsuk; Changalucha, John M.

    2015-01-01

    The objectives of this study was to conduct a survey on schistosomiasis and soil-transmitted helminth (STH) infections in order to come up with feasible control strategies in Lake Victoria basin, Tanzania. Depending on the size of the school, 150-200 schoolchildren were recruited for the study. Duplicate Kato-Katz stool smears were prepared from each child and microscopically examined for Schistosoma mansoni and STHs. Urine specimens were examined for Schistosoma haematobium eggs using the filtration technique. After the survey, mass drug administration was done using praziquantel and albendazole for schistosomiasis and STHs infections, respectively. A total of 5,952 schoolchildren from 36 schools were recruited for the study and had their stool and urine specimens examined. Out of 5,952 schoolchildren, 898 (15.1%) were positive for S. mansoni, 754 (12.6%) for hookworms, 188 (3.2%) for Ascaris lumblicoides, and 5 (0.008%) for Trichuris trichiura. Out of 5,826 schoolchildren who provided urine samples, 519 (8.9%) were positive for S. haematobium eggs. The results revealed that intestinal schistosomiasis, urogenital schistosomiasis, and STH infections are highly prevalent throughought the lake basin. The high prevalence of intestinal and urogenital schistosomisiasis in the study area was a function of the distance from Lake Victoria, the former being more prevalent at localities close to the lake, whilst the latter is more so away from it. Control of schistosomiasis and STHs in the study area requires an integrated strategy that involves provision of health education to communities, regular treatments, and provision of adequate safe water supply and sanitation facilities. PMID:26537030

  2. Tsetse Fly (G.f. fuscipes) Distribution in the Lake Victoria Basin of Uganda

    PubMed Central

    Albert, Mugenyi; Wardrop, Nicola A; Atkinson, Peter M; Torr, Steve J; Welburn, Susan C

    2015-01-01

    Tsetse flies transmit trypanosomes, the causative agent of human and animal African trypanosomiasis. The tsetse vector is extensively distributed across sub-Saharan Africa. Trypanosomiasis maintenance is determined by the interrelationship of three elements: vertebrate host, parasite and the vector responsible for transmission. Mapping the distribution and abundance of tsetse flies assists in predicting trypanosomiasis distributions and developing rational strategies for disease and vector control. Given scarce resources to carry out regular full scale field tsetse surveys to up-date existing tsetse maps, there is a need to devise inexpensive means for regularly obtaining dependable area-wide tsetse data to guide control activities. In this study we used spatial epidemiological modelling techniques (logistic regression) involving 5000 field-based tsetse-data (G. f. fuscipes) points over an area of 40,000 km2, with satellite-derived environmental surrogates composed of precipitation, temperature, land cover, normalised difference vegetation index (NDVI) and elevation at the sub-national level. We used these extensive tsetse data to analyse the relationships between presence of tsetse (G. f. fuscipes) and environmental variables. The strength of the results was enhanced through the application of a spatial autologistic regression model (SARM). Using the SARM we showed that the probability of tsetse presence increased with proportion of forest cover and riverine vegetation. The key outputs are a predictive tsetse distribution map for the Lake Victoria basin of Uganda and an improved understanding of the association between tsetse presence and environmental variables. The predicted spatial distribution of tsetse in the Lake Victoria basin of Uganda will provide significant new information to assist with the spatial targeting of tsetse and trypanosomiasis control. PMID:25875201

  3. Geology and petrogenesis of the Kirkpatrick Basalt, Pain Mesa and Solo Nunatak, northern Victoria Land, Antarctica

    SciTech Connect

    Mensing, T.M.

    1987-01-01

    The flows on Pain Mesa were divided into a Lower Suite and an Upper Suite based on differences in their chemical compositions. The Upper Suite has higher concentrations of SiO/sub 2/, alkalies, total Fe, MnO, TiO/sub 2/, and P/sub 2/O/sub 5/ than the Lower Suite. In addition, the flows of the Upper Suite are homogeneous in chemical composition compared to the Lower Suite which is differentiated. The isotope compositions of Sr and O of the Lower Suite are positively correlated which indicates that the magma was contaminated by crustal material. The isotopic and chemical compositions of the flows of the Lower Suite were explained by fractional crystallization and simultaneous assimilation of a crustal contaminant. The initial Sr isotope ratios of the Upper Suite, calculated for an assumed age of 175 Ma, were lower than those of the Lower Suite, and are not constant. However, by lowering the assumed age of the Upper Suite to 92 Ma the range of the initial Sr-isotope ratios was greatly reduced. Paleomagnetic results and K-Ar dating of Ferrar rocks at the Litell Rocks Nunatak in northern Victoria Land also support the hypothesis that the Upper Suite is Cretaceous in age. The magma source of both suites has elevated /sup 87/Sr//sup 86/Sr ratios compared to normal mantle values because of prior enrichment in Rb. The magma source was enriched in Rb about 475 Ma years ago. This data coincides with the Ross Orogeny which may reflect a causal relationship between these events. The occurrence of igneous activity in northern Victoria Land and Marie Byrd Land were juxtaposed during the Mesozoic Era but were later separated as a result of major right lateral displacement along the Transantarctic rift system.

  4. Mineralogy and Geochemistry of Granitic rocks within Lichen Hills, Outback Nunatak, Northern Victoria Land, Antarctica

    NASA Astrophysics Data System (ADS)

    KIM, T.; KIM, Y.; Lee, I.; Lee, J.; Woo, J.

    2015-12-01

    The study areas, Lichen Hills and Outback Nunatak are located in the Northern Victoria Land which is close to Pacific Ocean side of Transantarctic Mountain (TAM), Antarctica. According to the study of Zeller and Dreschoff (1990), the radioactivity values of Lichen hills and Frontier Mt. area in the Victoria Land were very high. To identify the geochemical characteristics of granitic rocks in these areas, 13 samples of Lichen Hills rocks and 4 samples of Outback Nunatak rocks are analyzed. For mineralogical study, samples were observed in macroscale as well as microscale including microscope electron probe analysis. Rock samples of Lichen Hills, Outback Nunatak are mainly leucogranite and granitic pegmatite. These rock samples are composed of quartz, k-feldspar, plagioclase, muscovite, garnet, tourmaline like granite. In SEM-EDS analysis, the observed light colored minerals show relatively high Th, U, Dy, Ce, Nb concentration. This suggests that rock samples may contain minerals such as fergusonite, monazite, thorite, allanite, karnasurtite which are considered to be REE-bearing minerals. Samples of related rocks have been analyzed in terms of major, trace and rare earth element (REE) concentrations using X-ray fluorescence (XRF) spectrometer and Inductively Coupled Plasma Mass Spectrometer (ICP-MS). As concentration of SiO2 increase, Al2O3, TiO2, Fe2O3, MgO, P2O5 concentration decrease and Na2O, K2O, MnO concentration increase. Analyzed trace elements and REE are normalized using CI Chondrite, Primitive mantle. The normalized data show that LREE are enriched compared to HREE. The distinct negative anomalies of Eu, Sr are observed, indicating that rock-forming melts are fairly processed state of fractional crystallization. It means that Th, U, Nb, Ta are much enriched in the melts.

  5. Impact of Urbanization on Precipitation Distribution and Intensity over Lake Victoria Basin

    NASA Astrophysics Data System (ADS)

    Gudoshava, M.; Semazzi, F. H. M.

    2014-12-01

    In this study, sensitivity simulations on the impact of rapid urbanization over Lake Victoria Basin in East Africa were done using a Regional Climate Model (RegCM4.4-rc29) with the Hostetler lake model activated. The simulations were done for the rainy seasons that is the long rains (March-April-May) and short rains (October-November-December). Africa is projected to have a surge in urbanization with an approximate rate of 590% in 2030 over their 2000 levels. The Northern part of Lake Victoria Basin and some parts of Rwanda and Burundi are amongst the regions with high urbanization projections. Simulations were done with the land cover for 2000 and the projected 2030 urbanization levels. The results showed that increasing the urban fraction over the northern part of the basin modified the physical parameters such as albedo, moisture and surface energy fluxes, aerodynamic roughness and surface emissivity, thereby altering the precipitation distribution, intensity and frequency in the region. The change in the physical parameters gave a response of an average increase in temperature of approximately 2oC over the urbanized region. A strong convergence zone was formed over the urbanized region and thereby accelerating the lake-breeze front towards the urbanized region center. Precipitation in the urbanized region and regions immediate to the area increased by approximately 4mm/day, while drying up the southern (non-urbanized) side of the basin. The drying up of the southern side of the basin could be a result of divergent flow and subsidence that suppresses vertical development of storms.

  6. Tsetse fly (G. f. fuscipes) distribution in the Lake Victoria basin of Uganda.

    PubMed

    Albert, Mugenyi; Wardrop, Nicola A; Atkinson, Peter M; Torr, Steve J; Welburn, Susan C

    2015-04-01

    Tsetse flies transmit trypanosomes, the causative agent of human and animal African trypanosomiasis. The tsetse vector is extensively distributed across sub-Saharan Africa. Trypanosomiasis maintenance is determined by the interrelationship of three elements: vertebrate host, parasite and the vector responsible for transmission. Mapping the distribution and abundance of tsetse flies assists in predicting trypanosomiasis distributions and developing rational strategies for disease and vector control. Given scarce resources to carry out regular full scale field tsetse surveys to up-date existing tsetse maps, there is a need to devise inexpensive means for regularly obtaining dependable area-wide tsetse data to guide control activities. In this study we used spatial epidemiological modelling techniques (logistic regression) involving 5000 field-based tsetse-data (G. f. fuscipes) points over an area of 40,000 km2, with satellite-derived environmental surrogates composed of precipitation, temperature, land cover, normalised difference vegetation index (NDVI) and elevation at the sub-national level. We used these extensive tsetse data to analyse the relationships between presence of tsetse (G. f. fuscipes) and environmental variables. The strength of the results was enhanced through the application of a spatial autologistic regression model (SARM). Using the SARM we showed that the probability of tsetse presence increased with proportion of forest cover and riverine vegetation. The key outputs are a predictive tsetse distribution map for the Lake Victoria basin of Uganda and an improved understanding of the association between tsetse presence and environmental variables. The predicted spatial distribution of tsetse in the Lake Victoria basin of Uganda will provide significant new information to assist with the spatial targeting of tsetse and trypanosomiasis control.

  7. Simulation of Lake Victoria Circulation Patterns Using the Regional Ocean Modeling System (ROMS).

    PubMed

    Nyamweya, Chrispine; Desjardins, Christopher; Sigurdsson, Sven; Tomasson, Tumi; Taabu-Munyaho, Anthony; Sitoki, Lewis; Stefansson, Gunnar

    2016-01-01

    Lake Victoria provides important ecosystem services including transport, water for domestic and industrial uses and fisheries to about 33 million inhabitants in three East African countries. The lake plays an important role in modulating regional climate. Its thermodynamics and hydrodynamics are also influenced by prevailing climatic and weather conditions on diel, seasonal and annual scales. However, information on water temperature and circulation in the lake is limited in space and time. We use a Regional Oceanographic Model System (ROMS) to simulate these processes from 1st January 2000 to 31st December 2014. The model is based on real bathymetry, river runoff and atmospheric forcing data using the bulk flux algorithm. Simulations show that the water column exhibits annual cycles of thermo-stratification (September-May) and mixing (June-August). Surface water currents take different patterns ranging from a lake-wide northward flow to gyres that vary in size and number. An under flow exists that leads to the formation of upwelling and downwelling regions. Current velocities are highest at the center of the lake and on the western inshore waters indicating enhanced water circulation in those areas. However, there is little exchange of water between the major gulfs (especially Nyanza) and the open lake, a factor that could be responsible for the different water quality reported in those regions. Findings of the present study enhance understanding of the physical processes (temperature and currents) that have an effect on diel, seasonal, and annual variations in stratification, vertical mixing, inshore-offshore exchanges and fluxes of nutrients that ultimately influence the biotic distribution and trophic structure. For instance information on areas/timing of upwelling and vertical mixing obtained from this study will help predict locations/seasons of high primary production and ultimately fisheries productivity in Lake Victoria.

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

    PubMed

    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

  9. Excited-State Proton-Transfer-Induced Trapping Enhances the Fluorescence Emission of a Locked GFP Chromophore

    PubMed Central

    2016-01-01

    The chemical locking of the central single bond in core chromophores of green fluorescent proteins (GFPs) influences their excited-state behavior in a distinct manner. Experimentally, it significantly enhances the fluorescence quantum yield of GFP chromophores with an ortho-hydroxyl group, while it has almost no effect on the photophysics of GFP chromophores with a para-hydroxyl group. To unravel the underlying physical reasons for this different behavior, we report static electronic structure calculations and nonadiabatic dynamics simulations on excited-state intramolecular proton transfer, cis–trans isomerization, and excited-state deactivation in a locked ortho-substituted GFP model chromophore (o-LHBI). On the basis of our previous and present results, we find that the S1 keto species is responsible for the fluorescence emission of the unlocked o-HBI and the locked o-LHBI species. Chemical locking does not change the parts of the S1 and S0 potential energy surfaces relevant to enol–keto tautomerization; hence, in both chromophores, there is an ultrafast excited-state intramolecular proton transfer that takes only 35 fs on average. However, the locking effectively hinders the S1 keto species from approaching the keto S1/S0 conical intersections so that most of trajectories are trapped in the S1 keto region for the entire 2 ps simulation time. Therefore, the fluorescence quantum yield of o-LHBI is enhanced compared with that of unlocked o-HBI, in which the S1 excited-state decay is efficient and ultrafast. In the case of the para-substituted GFP model chromophores p-HBI and p-LHBI, chemical locking hardly affects their efficient excited-state deactivation via cis–trans isomerization; thus, the fluorescence quantum yields in these chromophores remain very low. The insights gained from the present work may help to guide the design of new GFP chromophores with improved fluorescence emission and brightness. PMID:26744782

  10. HSP70 kinetics study by continuous observation of HSP-GFP fusion protein expression on a perfusion heating stage.

    PubMed

    Wang, Sihong; Xie, Weijun; Rylander, Marissa Nichole; Tucker, Philip W; Aggarwal, Shanti; Diller, Kenneth R

    2008-01-01

    The direct correlation between levels of heat shock protein expression and efficiency of its tissue protection function motivates this study of how thermal doses can be used for an optimal stress protocol design. Heat shock protein 70 (HSP70) expression kinetics were visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a microscope heating stage using green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP(p)-HSP70-GFP which expresses an HSP70-GFP fusion protein under control of the HSP70 promoter. Expression levels were validated by western blot analysis. Transfected cells were heated on a controlled temperature microscope stage at 42 degrees C for a defined period, then shifted to 37 degrees C for varied post-heating times. The expression of HSP70-GFP and its sub-cellular localization were visualized via fluorescence microscopy. The progressive expression kinetics were measured by quantitative analysis of serial fluorescence images captured during heating protocols from 1 to 2 h and post-heating times from 0 to 20 h. The results show two sequential peaks in HSP70 expression at approximately 3 and 12 h post-heat shock. A progressive translocation of HSP70 from the cytoplasm to the nucleus was observed from 6 to 16 h. We conclude that we have successfully combined molecular cloning and optical imaging to study HSP70 expression kinetics. The kinetic profile for HSP70-GFP fusion protein is consistent with the endogenous HSP70. Furthermore, information on dynamic intracellular translocation of HSP70 was extracted from the same experimental data.

  11. Variation in Growth, Colonization of Maize, and Metabolic Parameters of GFP- and DsRed-Labeled Fusarium verticillioides Strains.

    PubMed

    Wu, Lei; Conner, R L; Wang, Xiaoming; Xu, Rongqi; Li, Hongjie

    2016-08-01

    Autofluorescent proteins are frequently applied as visual markers in the labeling of filamentous fungi. Genes gfp and DsRed were transformed into the genome of Fusarium verticillioides via the Agrobacterium tumefaciens-mediated transformation method. The selected transformants displayed a bright green or red fluorescence in all the organelles of the growing fungal mycelia and spores (except for the vacuoles) both in cultures and in the maize (Zea mays) roots they colonized. The results of gene-specific polymerase chain reaction (PCR) analysis and the thermal asymmetrical interlaced (TAIL)-PCR analysis demonstrated that gfp and DsRed were integrated on different chromosomes of the fungus. Reductions in the colony growth on the plates at pH 4.0 and 5.5 was observed for the green fluorescent protein (GFP)-transformant G3 and the DsRed-transformant R4, but transformants G4 and R1 grew as well as the wild-type strain at pH 4.0. The speed of growth of all the transformants was similar to the wild-type strain at pH ≥ 7. The insertion of gfp and DsRed did not alter the production of extracellular enzymes and fumonisin B by F. verticillioides. The transformants expressing GFP and DsRed proteins were able to colonize maize roots. However, the four transformants examined produced fewer CFU in the root samples than the wild-type strain during a sampling period of 7 to 28 days after inoculation. PMID:27088391

  12. Excited-State Proton-Transfer-Induced Trapping Enhances the Fluorescence Emission of a Locked GFP Chromophore.

    PubMed

    Liu, Xiang-Yang; Chang, Xue-Ping; Xia, Shu-Hua; Cui, Ganglong; Thiel, Walter

    2016-02-01

    The chemical locking of the central single bond in core chromophores of green fluorescent proteins (GFPs) influences their excited-state behavior in a distinct manner. Experimentally, it significantly enhances the fluorescence quantum yield of GFP chromophores with an ortho-hydroxyl group, while it has almost no effect on the photophysics of GFP chromophores with a para-hydroxyl group. To unravel the underlying physical reasons for this different behavior, we report static electronic structure calculations and nonadiabatic dynamics simulations on excited-state intramolecular proton transfer, cis-trans isomerization, and excited-state deactivation in a locked ortho-substituted GFP model chromophore (o-LHBI). On the basis of our previous and present results, we find that the S1 keto species is responsible for the fluorescence emission of the unlocked o-HBI and the locked o-LHBI species. Chemical locking does not change the parts of the S1 and S0 potential energy surfaces relevant to enol-keto tautomerization; hence, in both chromophores, there is an ultrafast excited-state intramolecular proton transfer that takes only 35 fs on average. However, the locking effectively hinders the S1 keto species from approaching the keto S1/S0 conical intersections so that most of trajectories are trapped in the S1 keto region for the entire 2 ps simulation time. Therefore, the fluorescence quantum yield of o-LHBI is enhanced compared with that of unlocked o-HBI, in which the S1 excited-state decay is efficient and ultrafast. In the case of the para-substituted GFP model chromophores p-HBI and p-LHBI, chemical locking hardly affects their efficient excited-state deactivation via cis-trans isomerization; thus, the fluorescence quantum yields in these chromophores remain very low. The insights gained from the present work may help to guide the design of new GFP chromophores with improved fluorescence emission and brightness. PMID:26744782

  13. Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro.

    PubMed

    van Vollenstee, Fiona A; Jackson, Carlo; Hoffmann, Danie; Potgieter, Marnie; Durandt, Chrisna; Pepper, Michael S

    2016-10-01

    Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

  14. Excited-State Proton-Transfer-Induced Trapping Enhances the Fluorescence Emission of a Locked GFP Chromophore.

    PubMed

    Liu, Xiang-Yang; Chang, Xue-Ping; Xia, Shu-Hua; Cui, Ganglong; Thiel, Walter

    2016-02-01

    The chemical locking of the central single bond in core chromophores of green fluorescent proteins (GFPs) influences their excited-state behavior in a distinct manner. Experimentally, it significantly enhances the fluorescence quantum yield of GFP chromophores with an ortho-hydroxyl group, while it has almost no effect on the photophysics of GFP chromophores with a para-hydroxyl group. To unravel the underlying physical reasons for this different behavior, we report static electronic structure calculations and nonadiabatic dynamics simulations on excited-state intramolecular proton transfer, cis-trans isomerization, and excited-state deactivation in a locked ortho-substituted GFP model chromophore (o-LHBI). On the basis of our previous and present results, we find that the S1 keto species is responsible for the fluorescence emission of the unlocked o-HBI and the locked o-LHBI species. Chemical locking does not change the parts of the S1 and S0 potential energy surfaces relevant to enol-keto tautomerization; hence, in both chromophores, there is an ultrafast excited-state intramolecular proton transfer that takes only 35 fs on average. However, the locking effectively hinders the S1 keto species from approaching the keto S1/S0 conical intersections so that most of trajectories are trapped in the S1 keto region for the entire 2 ps simulation time. Therefore, the fluorescence quantum yield of o-LHBI is enhanced compared with that of unlocked o-HBI, in which the S1 excited-state decay is efficient and ultrafast. In the case of the para-substituted GFP model chromophores p-HBI and p-LHBI, chemical locking hardly affects their efficient excited-state deactivation via cis-trans isomerization; thus, the fluorescence quantum yields in these chromophores remain very low. The insights gained from the present work may help to guide the design of new GFP chromophores with improved fluorescence emission and brightness.

  15. Superparamagnetic iron oxide nanoparticle-labeled cells as an effective vehicle for tracking the GFP gene marker using magnetic resonance imaging

    PubMed Central

    Zhang, Z; Mascheri, N; Dharmakumar, R; Fan, Z; Paunesku, T; Woloschak, G; Li, D

    2010-01-01

    Background Detection of a gene using magnetic resonance imaging (MRI) is hindered by the magnetic resonance (MR) targeting gene technique. Therefore it may be advantageous to image gene-expressing cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles by MRI. Methods The GFP-R3230Ac (GFP) cell line was incubated for 24 h using SPIO nanoparticles at a concentration of 20 μg Fe/mL. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using fluorescent microscopy and MRI. Results SPIO was used to label GFP cells effectively, with no effects on cell function and GFP expression. Iron-loaded GFP cells were successfully imaged with both fluorescent microscopy and T2*-weighted MRI. Prussian blue staining showed intracellular iron accumulation in the cells. All cells were labeled (100% labeling efficiency). The average iron content per cell was 4.75±0.11 pg Fe/cell (P<0.05 versus control). Discussion This study demonstrates that the GFP expression of cells is not altered by the SPIO labeling process. SPIO-labeled GFP cells can be visualized by MRI; therefore, GFP, a gene marker, was tracked indirectly with the SPIO-loaded cells using MRI. The technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker and enhancing the understanding of cell- and gene-based therapeutic strategies. PMID:18956269

  16. A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae.

    PubMed

    Frey, Simone; Sott, Kristin; Smedh, Maria; Millat, Thomas; Dahl, Peter; Wolkenhauer, Olaf; Goksör, Mattias

    2011-01-01

    Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration. Our analysis separates the different bleaching rates of Mig1-GFP and background, and the background-to-Mig1-GFP ratio. By applying our model to experimental data, we can thus extract the Mig1-GFP signal from the overall acquired signal and investigate the influence of kinase and phosphatase on Mig1. We found a stronger regulation of Mig1 through its kinase than through its phosphatase when controlled by the glucose concentration, with a constant (de)phosphorylation rate independent of the glucose concentration. By replacing the term for decreasing excited Mig1-GFP concentration with a constant, we were able to reconstruct the dynamics of Mig1-GFP, as it would occur without bleaching and background noise. Our model effectively demonstrates how data, acquired with an optical microscope, can be processed and used for a systems biology analysis of signal transduction pathways.

  17. Development of infectious transcripts from full-length and GFP-tagged cDNA clones of Pepper mottle virus and stable systemic expression of GFP in tobacco and pepper.

    PubMed

    Lee, Mi Yeon; Song, Yeon Sook; Ryu, Ki Hyun

    2011-02-01

    A full-length cDNA clone (pSP6PepMoV-Vb1) of the genomic RNA of a Korean isolate of Pepper mottle virus (PepMoV-Vb1) was constructed downstream of a bacteriophage SP6 RNA polymerase promoter in the plasmid. In vitro RNA transcripts generated from pSP6PepMoV-Vb1 corresponded to PepMoV-Vb1 RNA (9641nt) with an extra guanosine residue at the 5' terminus and a 15-nt, poly (A) tract at the 3' end. The RNAs synthesized from the pSP6PepMoV-Vb1 clone, by in vitro run-off transcription in the presence of the 5' cap analog m(7)GpppG, were highly infectious in Nicotiana benthamiana and Capsicum annuum cv. Early Calwonder. Visible symptoms appeared at 4-5 days post-inoculation, at essentially the same time as occurred on these host plant species inoculated with wild-type PepMoV-Vb. Symptoms induced by progeny virus of the transcripts were indistinguishable from wild-type PepMoV-Vb on their experimental and natural hosts. The gene encoding the green fluorescent protein (GFP), turboGFP, was inserted between the coding regions for NIb and CP in the pSP6PepMoV-Vb1 clone. RNA transcripts of the resulting GFP-tagged clone, designated SP6PepMoV-Vb1/GFP, were highly infectious and symptoms were not different from those induced by either transcripts of pSP6PepMoV-Vb1 or wild-type PepMoV-Vb. However, GFP expression could be detected earlier than virus-induced symptom in plants infected by SP6PepMoV-Vb1/GFP. This study is the first report of the construction of a biologically active, full-length cDNA copy of the Pepper mottle virus RNA genome and the stable expression of a foreign gene within the modified virus.

  18. Dual Fluorescence in GFP Chromophore Analogues: Chemical Modulation of Charge Transfer and Proton Transfer Bands.

    PubMed

    Chatterjee, Tanmay; Mandal, Mrinal; Das, Ananya; Bhattacharyya, Kalishankar; Datta, Ayan; Mandal, Prasun K

    2016-04-14

    Dual fluorescence of GFP chromophore analogues has been observed for the first time. OHIM (o-hydroxy imidazolidinone) shows only a charge transfer (CT) band, CHBDI (p-cyclicamino o-hydroxy benzimidazolidinone) shows a comparable intensity CT and PT (proton transfer) band, and MHBDI (p-methoxy o-hydroxy benzimidazolidinone) shows a higher intensity PT band. It could be shown that the differential optical behavior is not due to conformational variation in the solid or solution phase. Rather, control of the excited state electronic energy level and excited state acidity constant by functional group modification could be shown to be responsible for the differential optical behavior. Chemical modification-induced electronic control over the relative intensity of the charge transfer and proton transfer bands could thus be evidenced. Support from single-crystal X-ray structure, NMR, femtosecond to nanosecond fluorescence decay analysis, and TDDFT-based calculation provided important information and thus helped us understand the photophysics better. PMID:26998908

  19. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

    PubMed

    Marc; Granger; Brincat; Fisher; Kao; McCubbin; Cyr

    1998-11-01

    Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation. PMID:9811799

  20. Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase.

    PubMed

    Graham, David J L; Tseng, Shu-Fen; Hsieh, Jer-Tsong; Chen, David J; Alexandrakis, George

    2015-11-01

    Photobleaching is a key limitation in two-photon imaging of fluorescent proteins with femtosecond pulsed excitation. We present measurements of the dependence of eGFP photobleaching on the spectral amplitude and phase of the pulses used. A strong dependence on the excitation wavelength was confirmed and measured over a 800-950 nm range. A fiber continuum light source and pulse shaping techniques were used to investigate photobleaching with broadband, 15 fs transform limited, pulses with differing spectral amplitude and phase. Narrow band pulses, >150 fs transform limited, typical of femtosecond laser sources used in two-photon imaging applications, were also investigated for their photobleaching dependence on pulse dispersion and bandwidth. The bleach rate for broadband pulses was found to be primarily determined by the second harmonic spectrum of the excitation light. On the other hand, for narrow band excitation pulses with similar center wavelengths improvement in bleach rate was found to be mostly dependent on reducing the pulse length. A simple model to predict the relative bleach rates for broadband pulses is presented and compared to the experimental data.

  1. Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

    PubMed Central

    Botstein, David; Noyes, Marcus B.

    2013-01-01

    Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided. PMID:24300440

  2. In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen.

    PubMed

    Fernando, Danilo D; Richards, Javonna L; Kikkert, Julie R

    2006-05-01

    The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4 degrees C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at -20 degrees C or -80 degrees C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4 degrees C for a couple of weeks and then at -80 degrees C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).

  3. The hole in the barrel: water exchange at the GFP chromophore.

    PubMed

    Shinobu, Ai; Agmon, Noam

    2015-02-26

    Internal water molecules in proteins are conceivably part of the protein structure, not exchanging easily with the bulk. We present a detailed molecular dynamics study of the water molecule bound to the green fluorescent protein (GFP) chromophore that conducts its proton following photoexcitation. It readily exchanges above 310 K through a hole that forms between strands 7 and 10, due to fluctuations in the 6-7 loop. As the hole widens, rapid succession of water exchange events occur. The exiting water molecule passes three layers of atoms, constituting the binding, internal, and surface sites. Along this pathway, hydrogen bonding protein residues are replaced with water molecules. The mean squared displacement along this pathway is initially subdiffusive, becomes superdiffusive as the water traverses the protein wall in a flip-flop motion, and reverts to normal diffusion in the bulk. The residence correlation function for the bound state decays biexponentially, supporting this three-site scenario. For a favorable orientation of the Thr203 side-chain, the hole often fills with a single file of water molecules that could indeed rapidly conduct the photodissociated proton outside the protein. The activation enthalpy for its formation, 26 kJ/mol, agrees with the experimental value for a protein conformation change suggested to gate proton escape.

  4. Real Time Metastatic Route Tracking of Orthotopic PC-3-GFP Human Prostate Cancer Using Intravital Imaging.

    PubMed

    Zhang, Yong; Wang, Xiaoen; Hoffman, Robert M; Seki, Naohiko

    2016-04-01

    The cellular basis of metastasis is poorly understood. An important step to understanding this process is to be able to visualize the routes by which cancer cells migrate from the primary tumor to various distant sites to eventually form metastasis. Our laboratory previously developed single-cell in vivo imaging using fluorescent proteins to label cancer cells. In the present study, using PC-3 human prostate cancer cells labeled with green fluorescent protein (GFP) and orthotopic tumor transplantation, we have imaged in live mice various highly diverse routes by which PC-3 cells metastasize superiorly and inferiorly to distant sites, including in the portal area, stomach area, and urogenital system. Imaging began at day 9, at which time distant metastasis had already occurred, and increased at each imaging point at days 10, 13, 14, and 16. Metastatic cells were observed migrating superiorly and inferiorly from the primary tumor as well as in lymphatic channels and trafficking in various organ systems demonstrating that PC-3 has multiple metastatic routes similar to hormone-independent advanced-stage prostate cancer in the clinic. PMID:26515240

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Estrogenic activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a GFP expression system.

    PubMed

    Kuruto-Niwa, Ryoko; Nozawa, Ryushi; Miyakoshi, Takashi; Shiozawa, Tatsushi; Terao, Yoshiyasu

    2005-01-01

    Alkylphenol ethoxylates, widely used non-ionic surfactants, are biodegraded into alkylphenols such as nonylphenol (NP) and t-octylphenol (OP), short-chain ethoxylates such as NP-monoethoxylate (NP1EO) and NP-diethoxylate (NP2EO), and alkylphenoxy carboxylic acids such as 4-t-octylphenoxyacetic acid (OP1EC). Bisphenol S (BPS) is more heat-stable and photo-resistant than bisphenol A (BPA), and therefore replaces BPA. These chemicals could be chlorinated during wastewater treatment. We synthesized these compounds and their chlorinated derivatives to estimate their estrogenic activities using a GFP expression system. The EC(50) ranking of NP-related compounds was NP > ClNP > diClNP > NP1EO > ClNP1EO > NP2EO. The estrogenic activity of OP1EC was 10 times less potent than parent OP. Furthermore, BPS showed comparable estrogenic activity with BPA. The EC(50) ranking of BPS-related compounds was BPA ≥ BPS > triClBPS > diClBPS > ClBPS. Other tested BPS derivatives had no estrogenic activity. Chlorination of the tested chemicals did not enhance their estrogenic activity, in contrast to certain chlorinated BPAs. Thus, our results demonstrated that chlorinated derivatives of NP, OP, and BPS, even if artificially produced during wastewater processing, were less estrogenic than their parent chemicals, known as endocrine disruptors.

  7. Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP.

    PubMed

    Masullo, Tiziana; Puccio, Riccardo; Di Pierro, Mariarosaria; Tagliavia, Marcello; Censi, Paolo; Vetri, Valeria; Militello, Valeria; Cuttitta, Angela; Colombo, Paolo

    2014-02-01

    Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu(2+), Cd(2+), Pb(2+) and Ni(2+) concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 μM). Moreover, starting from these data, using the combination of molecular biology techniques and optical setup, we developed a device for the detection of Cu(2+) in water solutions. The quenching effect detected with the device showed that the relative attenuation of the signal (0.46 ± 0.02 AU) was slightly larger than the data measured by fluorescence spectra (0.65 ± 0.03 AU). The good sensitivity in the span of two orders of the magnitude of Cu(2+) concentration, the fact that the instrument is made up of low-cost and sturdy parts and the selective quenching of rAsGFP to copper ions make this setup suited as a low cost, on-the-field, copper ion-specific biosensor. PMID:24338300

  8. Estrogenic activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a GFP expression system.

    PubMed

    Kuruto-Niwa, Ryoko; Nozawa, Ryushi; Miyakoshi, Takashi; Shiozawa, Tatsushi; Terao, Yoshiyasu

    2005-01-01

    Alkylphenol ethoxylates, widely used non-ionic surfactants, are biodegraded into alkylphenols such as nonylphenol (NP) and t-octylphenol (OP), short-chain ethoxylates such as NP-monoethoxylate (NP1EO) and NP-diethoxylate (NP2EO), and alkylphenoxy carboxylic acids such as 4-t-octylphenoxyacetic acid (OP1EC). Bisphenol S (BPS) is more heat-stable and photo-resistant than bisphenol A (BPA), and therefore replaces BPA. These chemicals could be chlorinated during wastewater treatment. We synthesized these compounds and their chlorinated derivatives to estimate their estrogenic activities using a GFP expression system. The EC(50) ranking of NP-related compounds was NP > ClNP > diClNP > NP1EO > ClNP1EO > NP2EO. The estrogenic activity of OP1EC was 10 times less potent than parent OP. Furthermore, BPS showed comparable estrogenic activity with BPA. The EC(50) ranking of BPS-related compounds was BPA ≥ BPS > triClBPS > diClBPS > ClBPS. Other tested BPS derivatives had no estrogenic activity. Chlorination of the tested chemicals did not enhance their estrogenic activity, in contrast to certain chlorinated BPAs. Thus, our results demonstrated that chlorinated derivatives of NP, OP, and BPS, even if artificially produced during wastewater processing, were less estrogenic than their parent chemicals, known as endocrine disruptors. PMID:21783468

  9. Generation of Tg(cyp1a:gfp) Transgenic Zebrafish for Development of a Convenient and Sensitive In Vivo Assay for Aryl Hydrocarbon Receptor Activity.

    PubMed

    Xu, Hongyan; Li, Caixia; Li, Yan; Ng, Grace Hwee Boon; Liu, Chunsheng; Zhang, Xiaoyan; Gong, Zhiyuan

    2015-12-01

    Both dioxins/dioxin-like compounds and polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants and cause multiple adverse health effects on human and wildlife. Cyp1a is the most commonly used biomarker induced by these pollutants through activation of the aryl hydrocarbon receptor (AhR) pathway. Here we generated Tg(cyp1a:gfp) transgenic zebrafish for establishing a convenient in vivo assay for analysing these xenobiotic compounds. The Tg(cyp1a:gfp) larvae at 4 day post-fertilization were tested with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and GFP induction was observed mainly in the kidney, liver and gut. Similar GFP expression was also induced strongly by two dioxin-like chemicals, co-planar polychlorinated biphenyl (PCB126) and polychlorinated dibenzo-p-furan (PeCDF) and relatively weakly by two PAHs, 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BAP). The lowest observed effective concentration (LOEC) of TCDD was estimated to be ∼1 pM and the EC50 (effective concentration to induce GFP in 50 % of Tg(cyp1a:gfp) larvae) was ∼10 pM. PCB126 and PeCDF had ∼10× lower potencies in GFP induction than TCDD, while the potencies for 3-MC and BAP were at least 1000× lower. The sensitivity of Tg(cyp1a:gfp) larvae to respond TCDD was also favourable compared to that of ethoxyresorufin-O-deethylase (EROD) assay in both zebrafish larvae and adult livers. As GFP-based assay in transgenic zebrafish can be easily accommodated in multi-well dishes, the Tg(cyp1a:gfp) zebrafish should provide not only a valuable biomonitoring tool for aquatic contaminants but also a potential high-throughput chemical screening platform for identification of new AhR agonists.

  10. Fusion of mature HIV-1 particles leads to complete release of a gag-GFP-based content marker and raises the intraviral pH.

    PubMed

    Padilla-Parra, Sergi; Marin, Mariana; Gahlaut, Nivriti; Suter, Rolf; Kondo, Naoyuki; Melikyan, Gregory B

    2013-01-01

    By imaging the release of a GFP-based viral content marker produced upon virus maturation, we have previously found that HIV-1 fuses with endosomes. In contrast, fusion at the cell surface did not progress beyond a lipid mixing stage (hemifusion). However, recent evidence suggesting that free GFP can be trapped within the mature HIV-1 capsid raises concerns that this content marker may not be released immediately after the formation of a fusion pore. To determine whether a significant portion of GFP is trapped in the mature capsid, we first permeabilized the viral membrane with saponin. The overwhelming majority of pseudoviruses fully released GFP while the remaining particles exhibited partial loss or no loss of content. The extent of GFP release correlated with HIV-1 maturation, implying that incomplete Gag processing, but not GFP entrapment by mature capsids, causes partial content release. Next, we designed a complementary assay for visualizing pore formation by monitoring the intraviral pH with an additional pH-sensitive fluorescent marker. The loss of GFP through saponin-mediated pores was associated with a concomitant increase in the intraviral pH due to equilibration with the pH of an external buffer. We next imaged single HIV-cell fusion and found that these events were manifested in a highly correlated loss of content and increase in the intraviral pH, as it equilibrated with the cytosolic pH. Fused or saponin-permeabilized pseudoviruses that partially lost GFP did not release the remaining content marker under conditions expected to promote the capsid dissociation. We were thus unable to detect significant entrapment of GFP by the mature HIV-1 capsid. Together, our results validate the use of the GFP-based content marker for imaging single virus fusion and inferring the sites of HIV-1 entry.

  11. Comparing women pharmacy consumers’ experiences with weight loss treatment in Victoria and Nottingham: a cross-sectional study

    PubMed Central

    2014-01-01

    Background There has been a recent increase in weight management services available in pharmacies across Australia and England. The aim of this study was to determine the following between women in Victoria and Nottingham: similarities and differences of what weight management options are preferred by women pharmacy consumers; how they feel about pharmacists providing advice in this area; and what they desire in a weight management program. Method Women pharmacy consumers were randomly approached by a researcher in community pharmacies in Victoria and Nottingham and asked to complete a questionnaire regarding their own weight management experiences. The questionnaire was self-completed or researcher-administered and was comprised of four main sections that focused on the participant’s general health, previous weight loss experiences, their ideal weight management program and their demographics. Data was entered in SPSS 19 and logistic regression was used to identify any differences in weight loss experiences between women. Results The participant rates were high: 86% (n = 395/460) in Victoria and 98% in Nottingham (n = 215/220). Overall, women in Victoria and Nottingham were similar with comparable demographics. Approximately 50% (250/507) of women were in the overweight or obese body mass index category, with over 70% (n = 436/610) of women having attempted to lose weight in the past. The majority of women (n = 334/436) felt comfortable receiving advice from pharmacists. In the logistic regression analysis women in Nottingham were found to be significantly less likely to have utilised a pharmacy weight management program in the last five years (OR: 0.23 CI: 0.08, 0.63) and were significantly less likely to want an ideal weight management program located in a pharmacy (OR: 0.49 CI: 0.30, 0.82) compared to women in Victoria. No significant associations between location and feeling comfortable with a pharmacist advising on weight loss or wanting a pharmacist in an

  12. Engraftment of retrovirally transduced Bet v 1-GFP expressing bone marrow cells leads to allergen-specific tolerance.

    PubMed

    Gattringer, Martina; Baranyi, Ulrike; Pilat, Nina; Hock, Karin; Klaus, Christoph; Buchberger, Elisabeth; Ramsey, Haley; Iacomini, John; Valenta, Rudolf; Wekerle, Thomas

    2013-09-01

    Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5. Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells. Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays. Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays. These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

  13. Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

    NASA Technical Reports Server (NTRS)

    Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

  14. Medicine of senescence or managing a hospital system: the resistible rise of geriatric medicine in the State of Victoria.

    PubMed

    Hunter, Cecily

    2006-04-01

    This paper investigates the conditions in which geriatric medicine emerged in the state of Victoria between the late 1950s and the late 1970s. It shows how, in transforming a charity model of care into a medical model of service, nascent geriatricians were caught up in the shifting balance of responsibilities between state and Commonwealth governments in funding health services and care for the aged, and in the efforts of a small segment of the Australian medical profession to establish an organisational setting and professional context for social medicine. In Victoria, the potential for doctors to circumvent bureaucratic and professional limitations on their work lay in their relationship with their institutional committees of management whose members were drawn from the general community. The paper argues that pioneer 'geriatricians' failed to establish a field of practice that met all the needs they discerned in their patient group because they could not change community ideas about appropriate responses to old age infirmity.

  15. Abrupt out-of-plane edge folding of a circular thin plate: Implication for a mature Victoria regia leaf.

    PubMed

    Yang, Pengfei; Zhang, Chen; Dang, Fei; Yan, Yuan; Liu, Yilun; Chen, Xi

    2016-09-01

    Inspired by the observation of the configurations of Victoria regia leaves, we establish a phenomenological buckling model for the abrupt out-of-plane edge folding of a circular thin sheet. A reduced model is first developed, and further refined by a more sophisticated growth strain field so that the resulting buckling morphology resembles that of a mature Victoria regia leaf. Parametric studies are carried out to investigate the effects of geometric, material, and strain field parameters on the buckling morphology. Several main characteristics discovered through numerical studies are verified by theoretical analysis of a simple geometry-based model. Besides, the roles of the thickness variation and cracks are examined. This work may not only shed some light on the morphogenesis of certain plants, but also provide some useful insights on three-dimensional fabrications using mechanical self-assembly. PMID:27628696

  16. Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat.

    PubMed

    Duisit, Ghislaine; Conrath, Hervé; Saleun, Sylvie; Folliot, Sebastien; Provost, Nathalie; Cosset, François-Loïc; Sandrin, Virginie; Moullier, Philippe; Rolling, Fabienne

    2002-10-01

    The purpose of our study was to evaluate lentiviral vector-mediated rat retinal transduction using simian immunodeficiency virus (SIV) pseudotyped with envelope proteins from vesicular stomatitis virus G glycoprotein (VSV-G), Mokola virus G protein (MK-G), amphotropic murine leukemia virus envelope (4070A-Env), influenza A virus hemagglutinin (HA), lymphocytic choriomeningitis virus G protein (LCMV-G), and RD114 retrovirus envelope (RD114-Env). The six pseudotyped lentivirus vectors carried CMV-driven green fluorescent protein (GFP) or beta-galactosidase (beta-gal) reporter genes. Intravitreal and subretinal injections of each pseudotyped recombinant SIV were performed in cohorts of Wistar rats. Our results showed that no transgene expression was detected after intravitreal injection of each pseudotyped SIV vector. Also, no transduction could be detected following subretinal injection of RD114 pseudotyped SIV vectors. However, selective transduction of retinal pigment epithelium (RPE) cells was repeatedly obtained after subretinal delivery of VSV-G, MK-G, 4070A-Env, HA, and LCMV-G pseudotyped SIV. GFP expression was maximum as soon as 4 days postadministration for VSV-G, MK-G, 4070A-Env, and HA pseudotypes, with no evidence of pseudotransduction for VSV-G. Maximum transgene expression was observed 3 weeks postinjection for LCMV-6. Importantly, HA and VSV-G pseudotyped SIV lead to such a high level of transgene expression that GFP-related toxicity occurred. Therefore, when a high level of GFP synthesis is achieved, replacement of enhanced GFP (egfp, Aequorea victoria) by a low-toxicity GFP (Renilla reniformis) cDNA is necessary to allow long-term expression.

  17. Scenario-based water resources planning for utilities in the Lake Victoria region

    NASA Astrophysics Data System (ADS)

    Mehta, V. K.; Aslam, O.; Dale, L.; Miller, N.; Purkey, D.

    2010-12-01

    Cities in the Lake Victoria (LV) region are experiencing the highest growth rates in Africa, at the same time that their water resource is threatened by domestic waste and industrial pollution. Urban centers use local springs, wetlands and Lake Victoria as source waters. As efforts to meet increasing demand accelerate, integrated water resources management (IWRM) tools provide opportunities for utilities and other stakeholders to develop a planning framework comprehensive enough to include short term (e.g. landuse change), as well as longer term (e.g. climate change) scenarios. This paper presents IWRM models built using the Water Evaluation And Planning (WEAP) decision support system, for three pilot towns in the LV region - Bukoba (Tanzania), Masaka (Uganda), and Kisii (Kenya). Their current populations are 100,000, 70,000 and 200,000 respectively. Demand coverage is ~70% in Masaka and Bukoba, and less than 50% in Kisii. IWRM models for each town were calibrated under current system performance based on site visits, utility reporting and interviews. Projected water supply, demand, revenues and costs were then evaluated against a combination of climate, demographic and infrastructure scenarios upto 2050. In Masaka, flow and climate data were available to calibrate a runoff model to simulate streamflow at water intake. In Masaka, without considering climate change, the system is infrastructure-limited and not water availability (hydrology) limited until 2035, under projected population growth of 2.17%. Under a wet climate scenario as projected by GCM’s for the LV region, the current wetland source could supply all expected demands until 2050. Even under a drought scenario, the wetland could supply all demand until 2032, if the supply infrastructure is updated at an estimated cost of USD 10.8 million. However, demand targets can only be met at the expense of almost no water returning to the wetland downstream of the intake by 2035, unless substantial investments

  18. Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System.

    PubMed

    Beretta, Carlo A; Dross, Nicolas; Engel, Ulrike; Carl, Matthias

    2016-01-01

    The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

  19. Pdx1 (GFP/w) mice for isolation, characterization, and differentiation of pancreatic progenitor cells.

    PubMed

    Williams, Michael D; Wong, Wilson; Rixon, Amanda; Satoor, Sarang N; Hardikar, Anandwardhan A; Joglekar, Mugdha V

    2014-01-01

    It is well known that human cells are diverse with respect to their epigenome, transcriptome, and proteome. In the context of regenerative medicine, it is important for the transplanted cells or tissues to faithfully recapitulate their intended tissue type in each of these respects. Whether the cells chosen for such an application are embryonic, postnatal, or induced pluripotent stem cells, the transplanted product must behave in a predictable and reliable manner to be a safe and effective treatment option. Irrespective of the choice of cells used in such an application, the characterization and understanding of the developmental cues responsible for establishing and maintaining the desired cell phenotype are essential.Animal models are extremely important in understanding the development of a specific tissue, which can then be subsequently extrapolated to human studies. Generation of transgenic animal models with whole-body gene knockout, conditional knockout, constitutive fluorescent gene reporters, and Cre-Lox-based conditional and lineage reporters has revolutionized the field of developmental biology. An intrinsically complex network of the actions and interactions of the multitude of different signalling cascades is required for development. A thorough understanding of such networks, gained through studies on transgenic animal models, is essential for the development of the techniques necessary to reliably differentiate a given stem or progenitor cell population into a specific cell type, such as an islet-like, insulin-producing cell aggregate.In this chapter, we describe the use of GFP (green fluorescent protein)-based reporter mice for isolation of cells of choice, analyzing gene expression in those cells as well as their use for screening signalling molecules to understand their effect on differentiation.

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

    NASA Astrophysics Data System (ADS)

    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.