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Sample records for aequorea victoria gfp

  1. Sound scattering by the gelatinous zooplankters Aequorea victoria and Pleurobrachia bachei

    NASA Astrophysics Data System (ADS)

    Monger, B. C.; Chinniah-Chandy, S.; Meir, E.; Billings, S.; Greene, C. H.; Wiebe, P. H.

    Target strengths were determined for individual Aequorea victoria and Pleurobrachia bachei using separate 200, 420 and 1,000 kHz transducers in an enclosure deployed off the dock of the Friday Harbor Laboratories in northern Puget Sound, Washington, USA. The target strength for Aequorea, when averaged over all animal sizes and all three trnasducer frequencies, was -72.6 dB. The target strength for Pleurobrachia was -75.9 dB when similarly averaged. Average reduced target strengths for Aequorea and Pleurobrachia were -43.3 and -33.1 dB, respectively. Differences in the sound scattering contrasts for Pleurobrachia and Aequorea are proposed to explain the larger reduced target strength of Pleurobrachia, relative to that of Aequorea. A new sound scattering model was developed to predict the backscattering cross-section of Aequorea. The model takes into account expected variations in target strength due to changes in shape of the jellyfish as it propels itself through the water. The scattering model was tuned specifically for Aequorea in the present study, but has a general formulation that is widely applicable to any medusa-shaped gelatinous zooplankter.

  2. 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

  3. A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

    PubMed

    Wang, Y; Wang, G; O'Kane, D J; Szalay, A A

    2001-01-01

    We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time. PMID:11212912

  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. Chemical nature of the light emitter of the Aequorea green fluorescent protein

    PubMed Central

    Niwa, Haruki; Inouye, Satoshi; Hirano, Takashi; Matsuno, Tatsuki; Kojima, Satoshi; Kubota, Masayuki; Ohashi, Mamoru; Tsuji, Frederick I.

    1996-01-01

    The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (λmax = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission. PMID:8942983

  6. Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene.

    PubMed

    Izadi, Manizheh; Abiri, Maryam; Keramatipour, Mohammad

    2009-04-01

    The green fluorescent protein (GFP) was originally isolated from the Jellyfish Aequorea Victoria that fluoresces green when exposed to blue light. GFP protein is composed of 238 amino acids with the molecular mass of 26.9 kD. The GFP gene is frequently used in cellular and molecular biology as a reporter gene. To date, many bacterial, yeast, fungal, plants, fly and mammalian cells, including human, have been created which express GFP. Martin Chalfie, Osamu Shimomura, and Roger Tsien were awarded the 2008 noble prize in chemistry for their discovery and development of GFP. In many studies on mammalian cells, GFP gene is introduced into cells using vector-based systems or a recombinant virus to track the location of a target protein or to study the expression level of the gene of interest, but in these studies there is no selection marker to normalize transfection. According to the importance of neomycin gene as a selection marker in mammalian cells, we aimed to produce a GFP expression vector that contains neomycin gene. GFP gene was separated from pEGFP-N1 vector and was inserted in the back-bone of pCDNA3.1/His/LacZ vector that contained the neomycin gene. The resulted vector contained GFP beside neomycin gene. PMID:23407141

  7. 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.

  8. 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

  9. 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.

  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. Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

    PubMed Central

    Nishiuchi, Yuji; Inui, Tatsuya; Nishio, Hideki; Bódi, József; Kimura, Terutoshi; Tsuji, Frederick I.; Sakakibara, Shumpei

    1998-01-01

    The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor. PMID:9811837

  12. 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.

  13. 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.

  14. 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…

  15. Partway to 'Victoria'

    NASA Technical Reports Server (NTRS)

    2005-01-01

    This image shows the route that NASA's Mars Exploration Rover Opportunity had driven through its 659th Martian day, or sol, (Dec. 1, 2005) relative to the potential destination of 'Victoria Crater' farther south. The base image is a portion of a mosaic (previously released as PIA07506) combining images from the Mars Observer Camera on NASA's Mars Global Surveyor orbiter, the Thermal Emission Imaging System on NASA's Mars Odyssey orbiter, and Opportunity's own Descent Image Motion Estimation System. The scale bar at lower right is 800 meters (one-half mile).

  16. 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,...

  17. 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,...

  18. 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,...

  19. 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,...

  20. 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,...

  1. 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

  2. Hepatitis E Infections, Victoria, Australia

    PubMed Central

    Adamopoulos, Jim; Carter, Karen; Kelly, Heath

    2005-01-01

    In the first half of 2004, acute hepatitis E virus infections diagnosed in Victoria, Australia, increased 7-fold. Of the interviewed patients with highly reactive serologic results, 90% reported recent clinically compatible illness and overseas travel. The increase is compared with a background of exposure in countries in which hepatitis E is endemic. PMID:15757573

  3. 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…

  4. Green Fluorescent Protein as a Reporter To Monitor Gene Expression and Food Colonization by Aspergillus flavus

    PubMed Central

    Du, Wanglei; Huang, Zhengyu; Flaherty, Joseph E.; Wells, Kevin; Payne, Gary A.

    1999-01-01

    Transformants of Aspergillus flavus containing the Aequorea victoria gfp gene fused to a viral promoter or the promoter region and 483 bp of the coding region of A. flavus aflR expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants. PMID:9925624

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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…

  10. Degradation of Victoria Crater, Mars

    NASA Technical Reports Server (NTRS)

    Wilson, Sharon A.; Grant, John A.; Cohen, Barbara A.; Golombek, Mathew P.; Geissler, Paul E.; Sullivan, Robert J.; Kirk, Randolph L.; Parker, Timothy J.

    2008-01-01

    The $\\sim$750 m diameter and $\\sim$75 m deep Victoria crater in Meridiani Planum, Mars, presents evidence for significant degradation including a low, serrated, raised rim characterized by alternating alcoves and promontories, a surrounding low relief annulus, and a floor partially covered by dunes. The amount and processes of degradation responsible for the modified appearance of Victoria crater were evaluated using images obtained in situ by the Mars Exploration Rover Opportunity in concert with a digital elevation model created using orbital HiRISE images. Opportunity traversed along the north and northwest rim and annulus, but sufficiently characterized features visible in the DEM to enable detailed measurements of rim relief, ejecta thickness, and wall slopes around the entire degraded, primary impact structure. Victoria retains 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 $\\sim$120 to 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 to 750 m across indicate the original, pristine crater was close to 600 m in diameter. Hence, the crater has been erosionally widened by approximately 150 m and infilled by about 50 m of sediments. Eolian processes are responsible for modification at Victoria, but lesser contributions from mass wasting or other processes cannot be ruled out. Erosion by prevailing winds is most significant along the exposed rim and upper walls and accounts for $\\sim$50 m widening across a WNW-ESE diameter. The volume of material eroded from the crater walls and rim is $\\sim$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 $\\sim$1 m deflation of the ejecta created a lag of more resistant hematite spherules that trapped darker, regional

  11. Overview of Approach to 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

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

    This image from the Mars Orbiter Camera aboard NASA's Mars Global Surveyor spacecraft shows an overview of 'Victoria Crater' and a portion of the area NASA's Mars Exploration Rover Opportunity has covered to reach the enormous depression.

    Images such as this one from the Mars Orbiter Camera on NASA's Mars Global Surveyor are helping scientists and engineers decide the best path for NASA's Mars Exploration Rover Opportunity as it approaches 'Victoria Crater.'

    In figure 1, a blue dot indicates 'Cape Verde' and a red dot 'Cabo Frio.' These two points mark the extent of the crater visible from the rover's position on its 945th Martian day, or sol (Sept. 20, 2006), a location it had reached two sols earlier and from which much of this monster depression was still out of sight. The green annotations indicate 'Duck Bay,' a location expected to allow a view to the other side of the crater. A dune, or ripple, is to the left of the crater, right in front of the green dot location. This is where the team initially talked about sending Opportunity for the rover's first view down into the crater. After further consideration, the team opted for a drive to the right of that ripple (south of the green dot) near the rim.

    The yellow lines that surround and intersect Victoria Crater are used to measure the crater and the distance to the far 'bays.' North is up. Victoria Crater is about 800 meters (half a mile) in diameter.

  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. Sensitivity of Superfolder GFP to Ionic Agents

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2014-01-01

    Superfolder variant of the green fluorescent protein (sfGFP) became a favorite probe for examination of the unfolding–refolding processes of fluorescent proteins with beta-barrel structure owing to its reversible unfolding in comparison with other fluorescent proteins. Its benefit is the proper folding even in fusion constructions with poorly folded polypeptides. We noticed that guanidine thiocyanate affects not only the structure of protein but its chromophore directly. Therefore we studied the influence of ionic denaturants and salts including guanidine thiocyanate, guanidine hydrochloride, sodium chloride and sodium thiocyanate on spectral features of sfGFP. It was shown that moderate amounts of the studied agents do not disrupt sfGFP structure but provoke pronounced alteration of its spectral characteristics. Changes in absorption and CD spectra in visible spectral range indicate the specific binding of SCN− and Cl− anions in the sfGFP chromophore vicinity. The anion binding results in the redistribution of sfGFP molecules with neutral and anionic chromophores. This also hinders the proton transfer in the chromophore excited state, considerably decreasing the fluorescence intensity of sfGFP. Our results indicate that when ionic denaturants are used in the studies of fluorescent protein folding their effect on fluorophore charge state should be taken into account. PMID:25347822

  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. '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).

  16. 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

  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

    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.

  20. 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.

  1. Circular permutant GFP insertion folding reporters

    DOEpatents

    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.

  2. Circular permutant GFP insertion folding reporters

    DOEpatents

    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.

  3. 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.'

  4. Looking Forward: Community Gateways at Victoria University

    ERIC Educational Resources Information Center

    Mountford, Christine

    2011-01-01

    The mission and values of Victoria University provide the underlying criteria for the development and implementation of the Community Gateways initiative, which aims to transform the lives of those living in the west of Melbourne through the power of further education. Community Gateways takes the university into the community by providing career…

  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. 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

  7. On the GFP-based analysis of dynamic concentration profiles.

    PubMed

    Berezhkovskii, Alexander M; Shvartsman, Stanislav Y

    2014-02-01

    Studies with GFP-tagged proteins can be used to investigate the dynamics of concentration profiles of regulatory proteins in cells and tissues. Analysis of these experiments must account for the finite rate with which the GFP-tagged proteins mature to the fluorescent state. Toward this end, we present an analytical framework that provides an explicit connection between the apparent kinetics of concentration gradients and the rates of GFP maturation. PMID:24507618

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

    PubMed

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

    2016-01-25

    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

  9. 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

  10. Differential GFP expression patterns induced by different heavy metals in Tg(hsp70:gfp) transgenic medaka (Oryzias latipes).

    PubMed

    Ng, Grace Hwee Boon; Xu, Hongyan; Pi, Na; Kelly, Barry C; Gong, Zhiyuan

    2015-06-01

    Heat shock protein 70 (Hsp70) is one of the most widely used biomarker for monitoring environment perturbations in biological systems. To facilitate the analysis of hsp70 expression as a biomarker, we generated a Tg(hsp70:gfp) transgenic medaka line in which green fluorescence protein (GFP) reporter gene was driven by the medaka hsp70 promoter. Here, we characterized Tg(hsp70:gfp) medaka for inducible GFP expression by seven environment-relevant heavy metals, including mercury, arsenic, lead, cadmium, copper, chromium, and zinc. We found that four of them (mercury, arsenic, lead, and cadmium) induced GFP expression in multiple and different organs. In general, the liver, kidney, gut, and skin are among the most frequent organs to show induced GFP expression. In contrast, no detectable GFP induction was observed to copper, chromium, or zinc, indicating that the transgenic line was not responsive to all heavy metals. RT-qPCR determination of hsp70 mRNA showed similar induction and non-induction by these metals, which also correlated with the levels of metal uptake in medaka exposed to these metals. Our observations suggested that these heavy metals have different mechanisms of toxicity and/or differential bioaccumulation in various organs; different patterns of GFP expression induced by different metals may be used to determine or exclude metals in water samples tested. Furthermore, we also tested several non-metal toxicants such as bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 4-introphenol, and lindane; none of them induced significant GFP expression in Tg(hsp70:gfp) medaka, further suggesting that the inducibility of Tg(hsp70:gfp) for GFP expression is specific to a subset of heavy metals. PMID:25652692

  11. '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.

  12. '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.

  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. Factors influencing birth control habits in Victoria.

    PubMed

    Selwood, T; Leeton, J

    1981-02-01

    A survey was conducted in March 1978 for the purpose of comparing the contraceptive habits of Melbourne and ex-Melbourne populations in Victoria. Of the 1312 records of sexually active persons collected in 1978, 1254 of those persons were between the ages of 15 and 49. Recorded information included age, sex, marital status, and place of domicile (Melbourne or ex-Melbourne, Victoria). When the Melbourne population was compared with the rest of Victoria, the proportion using each method of contraception was much the same for each group. A marked difference was the proportion using withdrawal in Melbourne; this was over 3 times the proportion among the ex-Melbourne population. Over 35% of persons under age 30 used oral contraceptives (OCs), compared with 24.2 in the over 30 age range. Barrier methods and natural family planning methods and withdrawal were commonly used by the older age groups. A large proportion of the over age 30 group used sterilization and hysterectomy as forms of birth control. Individuals in "de facto" relationships showed a tendency towards more reliable forms of contraception than did other groups. Only 20.3% were not using some form of contraception. Single persons were frequent OC users and users of barrier methods. 28.1% used no form of contraception. The sex of the person interviewed in the survey revealed a bias. Males tended to report male birth control methods and female reported female methods rather than those used by partners of the opposite sex. Marital status influenced birth control choice. The choice of birth control for each marital status was influenced by the confounding factor of mean age of each group. PMID:7247847

  15. 'Victoria' After Sol 950 Drive (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Left-eye view of a stereo pair for PIA08778

    [figure removed for brevity, see original site] Right-eye view of a stereo pair for PIA08778 [figure removed for brevity, see original site] Cylindrical view for PIA08778

    A drive of about 30 meters (about 100 feet) on the 950th Martian day, or sol, of Opportunity's exploration of Mars' Meridiani Planum region (Sept. 25, 2006) brought the NASA rover to within about 20 meters (about 66 feet) of the rim of 'Victoria Crater.' From that position, the rover's navigation camera took the exposures combined into this stereo anaglyph, which appears three-dimensional when viewed through red-green glasses. The scalloped shape of the crater is visible on the left edge. Due to a small dune or ripple close to the nearest part of the rim, the scientists and engineers on the rover team planned on sol 951 to drive to the right of the ripple, but not quite all the way to the rim, then to proceed to the rim the following sol. The image is presented in cylindrical projection with geometric seam correction.

    Victoria Crater is about 800 meters (one-half mile) in diameter, 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.

  16. 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.

  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. Degradation of Victoria Crater, Meridiani Planum, Mars

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

    Victoria crater (2.05N, 354.51E) is ~750 m in diameter and the largest crater on Mars observed in situ. The Mars Exploration Rover Opportunity traversed NW to SE across a broad annulus dominated by dark sand that at least partially surrounds the crater before navigating the northern crater rim. Rover observations of the crater and ejecta deposits are complemented by images with 26-52 cm/pixel scales from the High Resolution Imaging Science Experiment (HiRISE) on Mars Reconnaissance Orbiter and enable assessment of degradation state. The present depth/diameter ratio for Victoria is 0.1, less than the 0.2 expected for a pristine primary impact structure. Together with the eroded, serrated rim, this implies an originally smaller crater diameter and/or considerable infilling consistent with occurrence of a large dune field and few exposed rocks on the crater floor. The height and width of the raised rim is generally 4-5 m and 150-225 m, respectively, less than the 30 m and 500-600 m, respectively, expected for a pristine 750 m diameter crater. Ejecta thicknesses around the rim were derived using rover-based and HiRISE images and yield consistent estimates averaging ~3 m. The serrated rim plan creates a series of promontories extending up to 50 m into the crater and generally fronted by 30-60 degree slopes that are locally vertical and are separated by bays whose floors typically slope 15-25 degrees. A crater originally on order of 600-650 m in diameter and subsequently enlarged by mass wasting and aeolian erosion may yield a structure resembling Victoria today. The steep expression of the promontories and local outcroppings of rocks in the ejecta blanket points to some ongoing mass wasting, but the relative paucity of associated flanking talus indicates derived blocks of sulfate sandstone are not resistant to saltating sand and are rapidly broken down by the wind or are completely covered/filled in by aeolian drift. At Cape St. Vincent, the promontory appears undercut

  1. Specific cell surface labeling of GPCRs using split GFP.

    PubMed

    Jiang, Wen-Xue; Dong, Xu; Jiang, Jing; Yang, Yu-Hong; Yang, Ju; Lu, Yun-Bi; Fang, San-Hua; Wei, Er-Qing; Tang, Chun; Zhang, Wei-Ping

    2016-01-01

    Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications. PMID:26857153

  2. 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.…

  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. 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.

  5. 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.

  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. 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.

  10. GFP Loss-of-Function Mutations in Arabidopsis thaliana

    PubMed Central

    Fu, Jason L.; Kanno, Tatsuo; Liang, Shih-Chieh; Matzke, Antonius J. M.; Matzke, Marjori

    2015-01-01

    Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate–induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability. PMID:26153075

  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. PMID:11685538

  12. 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

  13. 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

  14. 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.

  15. '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.

  16. 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

  17. Opportunity's First Dip into Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity entered Victoria Crater during the rover's 1,291st Martian day, or sol, (Sept. 11, 2007). The rover team commanded Opportunity to drive just far enough into the crater to get all six wheels onto the inner slope, and then to back out again and assess how much the wheels slipped on the slope. The driving commands for the day included a precaution for the rover to stop driving if the wheels were slipping more than 40 percent. Slippage exceeded that amount on the last step of the drive, so Opportunity stopped with its front pair of wheels still inside the crater. The rover team planned to assess results of the drive, then start Opportunity on an extended exploration inside the crater.

    This wide-angle view taken by Opportunity's front hazard-identification camera at the end of the day's driving shows the wheel tracks created by the short dip into the crater. The left half of the image looks across an alcove informally named 'Duck Bay' toward a promontory called 'Cape Verde' clockwise around the crater wall. The right half of the image looks across the main body of the crater, which is 800 meters (half a mile) in diameter.

  18. A suite of engineered GFP molecules for oligomeric scaffolding

    DOE PAGESBeta

    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

  19. 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.

  20. GFP: from jellyfish to the Nobel prize and beyond.

    PubMed

    Zimmer, Marc

    2009-10-01

    On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for "the discovery and development of the green fluorescent protein, GFP". The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications. PMID:19771329

  1. Neurochemical characterisation of lamina II inhibitory interneurons that express GFP in the PrP-GFP mouse

    PubMed Central

    2013-01-01

    Background Inhibitory interneurons in the superficial dorsal horn play important roles in modulating sensory transmission, and these roles are thought to be performed by distinct functional populations. We have identified 4 non-overlapping classes among the inhibitory interneurons in the rat, defined by the presence of galanin, neuropeptide Y, neuronal nitric oxide synthase (nNOS) and parvalbumin. The somatostatin receptor sst2A is expressed by ~50% of the inhibitory interneurons in this region, and is particularly associated with nNOS- and galanin-expressing cells. The main aim of the present study was to test whether a genetically-defined population of inhibitory interneurons, those expressing green fluorescent protein (GFP) in the PrP-GFP mouse, belonged to one or more of the neurochemical classes identified in the rat. Results The expression of sst2A and its relation to other neurochemical markers in the mouse was similar to that in the rat, except that a significant number of cells co-expressed nNOS and galanin. The PrP-GFP cells were entirely contained within the set of inhibitory interneurons that possessed sst2A receptors, and virtually all expressed nNOS and/or galanin. GFP was present in ~3-4% of neurons in the superficial dorsal horn, corresponding to ~16% of the inhibitory interneurons in this region. Consistent with their sst2A-immunoreactivity, all of the GFP cells were hyperpolarised by somatostatin, and this was prevented by administration of a selective sst2 receptor antagonist or a blocker of G-protein-coupled inwardly rectifying K+ channels. Conclusions These findings support the view that neurochemistry provides a valuable way of classifying inhibitory interneurons in the superficial laminae. Together with previous evidence that the PrP-GFP cells form a relatively homogeneous population in terms of their physiological properties, they suggest that these neurons have specific roles in processing sensory information in the dorsal horn. PMID

  2. Age- and Light-Dependent Development of Localised Retinal Atrophy in CCL2−/−CX3CR1GFP/GFP Mice

    PubMed Central

    Chen, Mei; Hombrebueno, Jose R.; Luo, Chang; Penalva, Rosana; Zhao, Jiawu; Colhoun, Liza; Pandi, Sudha Pirya Soundara; Forrester, John V.; Xu, Heping

    2013-01-01

    Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2−/−CX3CR1GFP/GFP mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2−/−CX3CR1GFP/GFP mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17∼60% of 12-month, and 30∼100% of 18-month CCL2−/−CX3CR1GFP/GFP mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2−/−CX3CR1GFP/GFP mice respectively, but not in wild-type mice. All CCL2−/−CX3CR1GFP/GFP mice exposed to extra-light (∼800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20–25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2−/−CX3CR1GFP/GFP mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2−/−CX3CR1GFP/GFP mice. GABA expression was reduced in the inner retina of aged CCL2−/−CX3CR1GFP/GFP mice. Significantly increased Müller glial and microglial activation was observed in CCL2−/−CX3CR1GFP/GFP mice compared to age-matched WT mice. Macrophages from CCL2−/−CX3CR1GFP/GFP mice were less phagocytic, but expressed higher levels of iNOS, IL-1β, IL-12 and TNF-α under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy. PMID:23637822

  3. Geologic map and structural analysis of the Victoria quadrangle, Mercury

    NASA Astrophysics Data System (ADS)

    Galluzzi, Valentina; Di Achille, Gaetano; Ferranti, Luigi; Rothery, David A.; Palumbo, Pasquale

    2015-04-01

    In this work we present a new geologic map and structural analysis of the Victoria quadrangle (H2) of Mercury, along with a reconnaissance study of the geometry and kinematics of lobate scarps in this area. To this end, we 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

  4. 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

  5. Selection for intrabody solubility in mammalian cells using GFP fusions.

    PubMed

    Guglielmi, Laurence; Denis, Vincent; Vezzio-Vié, Nadia; Bec, Nicole; Dariavach, Piona; Larroque, Christian; Martineau, Pierre

    2011-12-01

    Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay. PMID:21997307

  6. 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…

  7. Mapping eGFP Oligomer Mobility in Living Cell Nuclei

    PubMed Central

    Zwerger, Monika; Müller, Gabriele; Waldeck, Waldemar; Langowski, Jörg

    2009-01-01

    Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers. PMID:19347038

  8. 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

  9. Aboriginal Students in Victoria. ACER Research Monograph No. 3.

    ERIC Educational Resources Information Center

    de Lemos, Marion M.

    An estimated 80%-90% of all Aboriginal students enrolled in the primary and secondary schools of Victoria, Australia, were tested and surveyed to determine their numbers, distribution, attendance, achievement, attitudes, and school leaving patterns. Most of the 1244 Aboriginals surveyed attended state schools and 75% were schooled in rural areas.…

  10. 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…

  11. Reaching Year 12 in Victoria, Australia: Student and School Influences

    ERIC Educational Resources Information Center

    Marks, Gary

    2014-01-01

    This paper examines student and school influences on reaching Year 12, the final year of schooling in Victoria, Australia. It analyses data from the population of students who were in Year 9 in 2008. Male, English-speaking background, government school, and especially Indigenous students were less likely to reach Year 12 than comparison groups.…

  12. 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…

  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. Catalogue Use at the State Library of Victoria

    ERIC Educational Resources Information Center

    Hider, Philip

    2008-01-01

    A questionnaire survey conducted at the State Library of Victoria indicates that users in general, and not just scholars, value the standard elements of bibliographic data found in the Library's catalogues, and consider all these elements useful for finding, identifying and selecting items. Rather than making do with less, users wanted more…

  15. 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.

  16. Model for heat-up of structures in VICTORIA

    SciTech Connect

    Bixler, N.E.

    1993-12-01

    VICTORIA is a mechanistic computer code that treats fission product behavior in the reactor coolant system during a severe accident. During an accident, fission products that deposit on structural surfaces produce heat loads that can cause fission products to revaporize and possibly cause structures, such as a pipe, to fail. This mechanism had been lacking from the VICTORIA model. This report describes the structural heat-up model that has recently been implemented in the code. A sample problem shows that revaporization of fission products can occur as structures heat up due to radioactive decay. In the sample problem, the mass of deposited fission products reaches a maximum, then diminishes. Similarly, temperatures of the deposited film and adjoining structure reach a maximum, then diminish.

  17. 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).

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro.

    PubMed

    Luo, Jun; Zhao, Jing; Tian, Qin; Mo, Weiyu; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-06-01

    Several studies have demonstrated the rabies virus to be a perfect potential vaccine vector to insert foreign genes into the target genome. For this study, a green fluorescent protein (GFP) gene was cloned into the rabies virus (RABV) genome between the N and P gene. CT dinucleotide was inserted as intergenic region. The recombinant high egg passage Flury strain (HEP-Flury) of RABV, carrying GFP (rHEP-NP-GFP), was generated in BHK-21 cells using reverse genetics. According to the viral growth kinetics assay, the addition of GFP between N and P gene has little effect on the viral growth compared to the parental strain HEP-Flury. Quantitative real-time PCR (qPCR) indicated that rHEP-NP-GFP showed different viral gene transcription, especially for G gene, compared to HEP-Flury. The same is true for one other recombinant RABV carrying GFP between G and L gene in NA cells. In addition, parent HEP-Flury showed more expression of innate immune-related molecules in NA cells. Compared to HEP-Flury, Western blotting (WB) indicated that insertion of a foreign gene following N gene enhanced the expression of M and G proteins. According to the qPCR and WB, GFP expression levels of rHEP-NP-GFP were significantly higher than rHEP-GFP. This study indicates HEP-Flury as valid vector to express exogenous genes between N and P. PMID:26957093

  8. 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.

  9. 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., III; Calvin, W.M.; Christensen, P.R.; Clark, B. C.; Cohen, B. A.; De Souza, P.A., Jr.; 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., Jr.; 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.

  10. 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.

  11. 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

  12. Injury surveillance in Victoria, Australia: developing comprehensive injury incidence estimates.

    PubMed

    Watson, W L; Ozanne-Smith, J

    2000-03-01

    This study aimed to develop an estimate of the incidence of all medically-treated injury by level of severity and to broadly describe the epidemiology of injury in the Australian State of Victoria in a given year. Victoria has developed a relatively comprehensive injury surveillance system. Data is currently collected by various agencies on injury deaths, hospitalisations and emergency department attendances. The method used to establish the incidence of both unintentional and intentional injury is described. Incidence figures were directly derived, or estimated from, the available Victorian health sector and Coronial data bases for three level of severity (deaths, hospitalisations and medical treatment only) and for causes of injury, age and gender groups, location of the injury event and activity at the time of injury. In 1993/1994, injuries resulted in at least 1487 deaths, 67,402 persons hospitalised and an estimated 397,160 medically-treated, non-hospitalised injured persons in Victoria. In total, over 466,000 people were injured or 10.5 persons per year for every 100 residents. Males sustain 62% of all injuries yet represent 49.5% of the population. Almost three-quarters of injury fatalities and over 60% of non-fatal injuries occur among males. Young people aged 15-24 years account for 22% of all injuries yet represent only about 16% of the Victorian population. Children (0-14 years) also suffer relatively high injury rates, although mainly less severe, while the elderly are at risk of more severe injuries. The leading cause of injury death in Victoria is suicide, followed by motor vehicle accidents, whereas falls are the leading cause of all non-fatal injury. Most injuries occur in the home (36%), areas of sport and recreation (12.5%) and transport (11.7%). They are mainly associated with leisure activities (33.1%), work (11%) and transportation (10.8%). This study demonstrates a method for the development of comprehensive injury incidence estimates. The

  13. Exploration of Victoria Crater by the Mars Rover Opportunity

    NASA Astrophysics Data System (ADS)

    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.

    2009-05-01

    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.

  14. 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…

  15. 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…

  16. 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…

  17. 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.

  18. Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells

    PubMed Central

    Noda, Asao; Suemori, Hirofumi; Hirai, Yuko; Hamasaki, Kanya; Kodama, Yoshiaki; Mitani, Hiroshi; Landes, Reid D.; Nakamura, Nori

    2015-01-01

    It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation

  19. 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

    2015-01-01

    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). PMID:26782515

  20. Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression.

    PubMed

    Blokpoel, Marian C J; O'Toole, Ronan; Smeulders, Marjan J; Williams, Huw D

    2003-08-01

    Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria. PMID:12782376

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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...

  6. 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

  7. Expression of nestin-GFP transgene marks oval cells in the adult liver

    PubMed Central

    Gleiberman, Anatoli S.; Encinas, Juan M.; Mignone, John L.; Michurina, Tatyana; Rosenfeld, Michael G.; Enikolopov, Grigori

    2009-01-01

    Oval cells which become apparent in the liver after chronic injury, serve as bi-potent progenitors for differentiated hepatocytes and cholangiocytes. We found that in the liver of adult transgenic mice in which expression of green fluorescent protein (GFP) is driven by regulatory elements of the nestin gene, the GFP signal marks a subpopulation of small epithelial cells which meet the criteria for oval cells, including morphology, localization, antigenic profile, and reactivity in response to injury. In the regenerating and developing liver we also found nestin-GFP-positive cells which express hepatocyte markers; such cells may correspond to transiently appearing differentiating progeny of oval cells. During development, GFP-expressing cells in the liver emerge relatively late, after the appearance of differentiated hepatocytes and cholangiocytes. Our results suggest that nestin-GFP cells in the liver correspond to a specialized cell type whose primary function may be to serve as a reserve for adult liver epithelial cell types. PMID:16127706

  8. Cationic Triarylmethane Photosensitizers For Selective Photochemotherapy: Victoria Blue-Bo, Victoria Blue-R And Malachite Green

    NASA Astrophysics Data System (ADS)

    Wadwa, K.; Smith, S.; Oseroff, A. R.

    1989-03-01

    We have investigated the malignant cell selective phototoxicity of some triarylmethane dyes including Victoria Blue BO (VB-BO), Victoria Blue R (VB-R), and Malachite Green (MG). Phototoxicity was studied in human squamous (FaDu) and colon (CX-1) carcinoma, and in human and murine melanoma (NEL, B-16) cell lines, as well as in non-malignant monkey kidney cells (CV-1). Cultured cells were exposed to varying concentrations of the dyes for 60 min, washed with PBS, irradiated after an efflux time of 90 min in culture media, and placed in a colony forming assay. VB-BO was the most effective photosensitizer, giving 90% killing of malignant cells such as B-16 when treated with 5x10-8 M dye and 13 J/cm2 light. CV-1 cells were unaffected under these conditions. VB-R was about 10 fold less effective, while MG had minimal phototoxicity in this assay. VB-BO was studied in vivo, using subcutaneous FaDu tumors in nude mice. At a dose of 3 mg/kg followed 4 hrs later by dye laser irradiation under conditions which did not produce hyperthermia, there was an 80% complete remission rate without significant phototoxicity to overlying or adjacent normal skin. The triarylmethanes are a novel class of photosensitizers which may have promise in vitro and in vivo.

  9. Anti-Fading Media for Live Cell GFP Imaging

    PubMed Central

    Bogdanov, Alexey M.; Kudryavtseva, Elena I.; Lukyanov, Konstantin A.

    2012-01-01

    Photostability is one of the most important characteristic of a dye for fluorescence microscopy. Recently we demonstrated that vitamins present in imaging media dramatically accelerate photobleaching of Enhanced Green Fluorescent Protein (EGFP) and many other green fluorescent and photoactivatable proteins. Here we tested all vitamins of commonly used media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and found that only two vitamins, riboflavin and pyridoxal, decrease photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can be used as an imaging medium, which ensures high photostability of GFPs at the expense of minimal biochemical disturbance. Then, we tested some antioxidants and found that a plant flavonoid rutin greatly enhances photostability of EGFP during live cell microscopy. In complete DMEM, rutin increased EGFP photostability up to the level of vitamin-depleted DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to further suppress EGFP photobleaching. Potentially, new medium formulations can be widely used for fluorescence microscopy of GFP-expressing cells and model multicellular organisms in a variety of imaging applications, where photostability represents a challenge. PMID:23285248

  10. 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

  11. Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER

    PubMed Central

    Hoseki, Jun; Oishi, Asami; Fujimura, Takaaki; Sakai, Yasuyoshi

    2016-01-01

    The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases. The ER redox state, which is an oxidative environment suitable for disulfide-bond formation, is essential for ER protein quality control. Hence, detection of the ER redox state, especially in living cells, is essential to understand the mechanism by which the redox state of the ER is maintained. However, methods to detect the redox state of the ER have not been well-established because of inefficient folding and stability of roGFP variants with oxidative redox potential like roGFP-iL. Here we have improved the folding efficiency of ER-targeted roGFP-iL (ERroGFP-iL) in cells by introducing superfolder GFP (sfGFP) mutations. Four specific amino acid substitutions (S30R, Y39N, T105N and I171V) greatly improved folding efficiency in Escherichia coli and in the ER of HeLa cells, as well as the thermostability of the purified proteins. Introduction of these mutations also enhanced the dynamic range for redox change both in vitro and in the ER of living cells. ER-targeted roGFP-S4 (ERroGFP-S4) possessing these four mutations could detect physiological redox changes within the ER. ERroGFP-S4 is therefore a novel probe suitable for monitoring redox change in the ER. ERroGFP-S4 can be applied to detect aberrant ER redox states associated with various pathological conditions and to identify the mechanisms used to maintain the redox state of the ER. PMID:26934978

  12. VENUS - The Victoria Experimental Network Under the Sea: First Light

    NASA Astrophysics Data System (ADS)

    Dewey, R. K.; Tunnicliffe, V.; Round, A.

    2006-05-01

    The Victoria Experimental Network Under the Sea (VENUS) is a recently installed cabled ocean observatory representing the next generation of infrastructure technology. VENUS is scheduled to consist of two major segments, one in Saanich Inlet (installed in February 2006), and a second in the Strait of Georgia (planned for October 2006). The technologies associated with both the hardware and software that will deliver data from instruments to scientists is being developed in collaboration with both the NEPTUNE Canada and MARS observatories. The presentation will provide an overview of the science projects planned for stage one of the installed instruments, the engineering design of the infrastructure, the preliminary design of the Data Management and Archive System (DMAS), lessons learned so far, and preliminary results from the first few months of data from Saanich Inlet.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing

    PubMed Central

    Hughes, Alex J.; Tentori, Augusto M.; Herr, Amy E.

    2012-01-01

    We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the E222G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF were employed to monitor dark (non-fluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3–4 formal charges distinguish the bright and dark populations of avGFP, compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events. PMID:23017083

  1. 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.

  2. Color variations on Victoria quadrangle: support for the geological mapping

    NASA Astrophysics Data System (ADS)

    Zambon, F.; Galluzzi, V.; Carli, C.; Giacomini, L.; Massironi, M.; Palumbo, P.; Guzzetta, L.; Mancinelli, P.; Vivaldi, V.; Ferranti, L.; Pauselli, C.; Frigeri, A.; Zusi, M.; Pozzobon, R.; Cremonese, G.; Ferrari, S.; Capaccioni, F.

    2015-10-01

    Mercury is the closest planet to the Sun. Its extreme thermal environment makes it difficult to explore onsite. In 1974, Mariner 10, the first mission dedicated to Mercury, covered 45% of the surface during of the three Hermean flybys [1]. For about 30 years after Mariner 10, no other mission has flownto Mercury. Many unresolved issues need an answer, and in recent years the interest about Mercury has increased. MESSENGER mission contributed to understand Mercury's origin, its surface structure, and the nature of its magnetic field, exosphere, and magnetosphere [1]. The Mercury Dual Imaging System (MDIS) provided a global coverage of Mercury surface with variable spatial resolution. MDIS is equipped with a narrow angle camera (NAC), dedicated to the study of the geology and a wide angle camera (WAC) with 12 filters useful to investigate the surface composition[2]. Mercury has been divided into 15 quadrangles for mapping purposes [3]. The mapping process permits integration of different geological surface information to better understand the planet crust formation and evolution. Merging spectroscopically data is a poorly followed approach in planetary mapping, but it gives additional information about lithological composition, contributing to the construction of a more complete geological map [e.g. 4]. Recently, [5] proposed a first detailed map of all the Victoria quadrangle (H2). Victoria quadrangle is located in a longitude range between 270°E and 360°E and a latitude range of 22.5°N and 65°N,and itwas only partially mapped by Mariner 10 data[3]. Here we investigate the lithological variation by using the MDIS-WAC data to produce a set of color map products which could be asupport to the geological mapping [5]. The future ESA-JAXA mission to Mercury, BepiColombo, will soon contribute to improve the knowledge of Mercury surface composition and geology thanks to the Spectrometer and Imagers for MPO BepiColombo-Integrated Observatory SYStem (SIMBIO-SYS)[6].

  3. 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.

  4. 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

  5. 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.

  6. Specific in vivo labeling with GFP retroviruses, lentiviruses, and adenoviruses for imaging

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.; Kishimoto, Hiroyuki; Fujiwara, Toshiyoshi

    2008-02-01

    Fluorescent proteins have revolutionized the field of imaging. Our laboratory pioneered in vivo imaging with fluorescent proteins. Fluorescent proteins have enabled imaging at the subcellular level in mice. We review here the use of different vectors carrying fluorescent proteins to selectively label normal and tumor tissue in vivo. We show that a GFP retrovirus and telomerase-driven GFP adenovirus can selectively label tumors in mice. We also show that a GFP lentivirus can selectively label the liver in mice. The practical application of these results are discussed.

  7. 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

  8. 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.

  9. Origin of the superflock of cichlid fishes from Lake Victoria, East Africa.

    PubMed

    Verheyen, Erik; Salzburger, Walter; Snoeks, Jos; Meyer, Axel

    2003-04-11

    Lake Victoria harbors a unique species-rich flock of more than 500 endemic haplochromine cichlid fishes. The origin, age, and mechanism of diversification of this extraordinary radiation are still debated. Geological evidence suggests that the lake dried out completely about 14,700 years ago. On the basis of phylogenetic analyses of almost 300 DNA sequences of the mitochondrial control region of East African cichlids, we find that the Lake Victoria cichlid flock is derived from the geologically older Lake Kivu. We suggest that the two seeding lineages may have already been lake-adapted when they colonized Lake Victoria. A haplotype analysis further shows that the most recent desiccation of Lake Victoria did not lead to a complete extinction of its endemic cichlid fauna and that the major lineage diversification took place about 100,000 years ago. PMID:12649486

  10. Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation.

    PubMed

    Liu, Guangpeng; Ye, Xinhai; Zhu, Yuchang; Li, Yulin; Sun, Jian; Cui, Lei; Cao, Yilin

    2011-10-01

    The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering. PMID:21684270

  11. Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization.

    PubMed

    Eshaghi, Majid; Sun, Guangyu; Grüter, Andreas; Lim, Chiew Ling; Chee, Yuemin Celina; Jung, Gregor; Jauch, Ralf; Wohland, Thorsten; Chen, Swaine L

    2015-11-16

    Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the "vGFP strategy" based on structural analysis of GFP bound to a single-domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50%), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single-molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single-step immunofluorescence in vitro (3× brighter due to dimerization); expression in bacteria and human cells in vivo (1.5× brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general. PMID:26447926

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  13. Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed (Conyza canadensis) hybrids.

    PubMed

    Halfhill, Matthew D; Good, Laura L; Basu, Chhandak; Burris, Jason; Main, Christopher L; Mueller, Thomas C; Stewart, C Neal

    2007-03-01

    The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha(-1) of glyphosate (i.e. Roundup) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 microM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F(1) hybrids between GR and GS horseweed. Thirty GSxGR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GSxGFP/GR F(1) hybrids produced F(2) seeds, and F(2) plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype. PMID:17024451

  14. 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

  15. 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

  16. 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

  17. Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

    PubMed

    Soler-Jover, Alex; Blasi, Juan; Gómez de Aranda, Inma; Navarro, Piedad; Gibert, Maryse; Popoff, Michel R; Martín-Satué, Mireia

    2004-07-01

    Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human. PMID:15208360

  18. 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

  19. Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Shcherbakova, Daria M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2012-01-01

    Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a β-barrel topology. Here, we studied the unfolding–refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations of GTC (from 0.7 to 1.7 M), induce a disruption of the sfGFP structure, that is manifested in simultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in which inner alpha-helix and hence green chromophore and Trp57 are still not tuned up to (properly integrated into) the already formed β-barrel scaffold of protein. Incorporation of the chromophore in the β-barrel in the pathway of refolding and restoration of its ability to fluoresce occur in a narrow range of GTC concentrations from 1.0 to 0.7 M, and a correct insertion of Trp 57 occurs at concentrations ranging from 0.7 to 0 M GTC. These two processes determine the hysteresis of protein unfolding and refolding. PMID:23144981

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Novel fluorescent protein from Hydnophora rigida possess cyano emission.

    PubMed

    Bokhari, H; Smith, C; Veerendra, K; Sivaraman, J; Sikaroodi, M; Gillevet, P

    2010-06-01

    Currently, a broad diversity of fluorescent proteins among marine organisms range from cyano-red emissions. Fluorescent proteins differ in their DNA sequences from green fluorescent protein (GFP). We identified cDNA encoding the gene of a new protein from the reef coral Hydnophora rigida of the Merulinidae family. Both the spectral properties and putative primary sequence of the protein has been determined. The cloned cDNA encode peptide we call HriCFP is comprised of 134 amino acids. It has characteristics of a cyano fluorescent protein (HriCFP) and its sequence is markedly different from known GFP from the hydroid jellyfish Aequorea victoria. HriCFP was cloned, expressed, purified and exist as monomer. The peptide mass finger print on the purified protein confirmed identity of HriCFP. PMID:20435020

  5. 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

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

    PubMed Central

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

    2009-01-01

    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 D9k 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 Ca2+-dependent. PMID:19472338

  7. 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

  8. 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

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

    PubMed

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

    2012-01-01

    Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals. PMID:23056388

  10. 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

  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. 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

  13. 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.

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

    PubMed

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

    2013-07-01

    This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant. PMID:23695435

  15. A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization

    PubMed Central

    LeSauter, Joseph; Yan, Lily; Vishnubhotla, Bhavana; Quintero, Jorge E.; Kuhlman, Sandra J.; McMahon, Douglas G.; Silver, Rae

    2012-01-01

    Period1 (Per1) is one of several clock genes driving the oscillatory mechanisms that mediate circadian rhythmicity. Per1 mRNA and protein are highly expressed in the suprachiasmatic nuclei, which contain oscillator cells that drive circadian rhythmicity in physiological and behavioral responses. We examined a transgenic mouse in which degradable green fluorescent protein (GFP) is driven by the mPer1 gene promoter. This mouse expresses precise free-running rhythms and characteristic light induced phase shifts. GFP protein (reporting Per1 mRNA) is expressed rhythmically as measured by either fluorescence or immunocytochemistry. In addition the animals show predicted rhythms of Per1 mRNA, PER1 and PER2 proteins. The localization of GFP overlaps with that of Per1 mRNA, PER1 and PER2 proteins. Together, these results suggest that GFP reports rhythmic Per1 expression. A surprising finding is that, at their peak expression time GFP, Per1 mRNA, PER1 and PER2 proteins are absent or not detectable in a subpopulation of SCN cells located in the core region of the nucleus. PMID:12576188

  16. 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

  17. Characterising groundwater dynamics in Western Victoria, Australia using Menyanthes software

    NASA Astrophysics Data System (ADS)

    Woldeyohannes, Yohannes; Webb, John

    2010-05-01

    Water table across much of the western Victoria, Australia have been declining for at least the last 10-15 years, and this is attributed to the consistently low rainfall for these years, but over the same period of time there has been substantial change in land use, with grazing land replaced by cropping and tree plantations appearing in some areas. Hence, it is important to determine the relative effect the climate and land use factors on the water table changes. Monitoring changes in groundwater levels to climate variables and/or land use change is helpful in indicating the degree of threat faced to agricultural and public assets. The dynamics of the groundwater system in the western Victoria, mainly on the basalt plain, have been modelled to determine the climatic influence in water table fluctuations. In this study, a standardized computer package Menyanthes was used for quantifying the influence of climatic variables on the groundwater level, statistically estimating trends in groundwater levels and identify the properties that determine the dynamics of groundwater system. This method is optimized for use on hydrological problems and is based on the use of continuous time transfer function noise model, which estimates the Impulse response function of the system from the temporal correlation between time series of groundwater level and precipitation surplus. In this approach, the spatial differences in the groundwater system are determined by the system properties, while temporal variation is driven by the dynamics of the input into the system. 80 time series models are analysed and the model output parameter values characterized by their moments. The zero-order moment Mo of a distribution function is its area and M1 is related to the mean of the impulse response function. The relation is M1/Mo. It is a measure of the system's memory. It takes approximately 3 times the mean time (M1/Mo) for the effect of a shower to disappear completely from the system. Overall

  18. Evaluation of vehicle side airbag effectiveness in Victoria, Australia.

    PubMed

    D'Elia, Angelo; Newstead, Stuart; Scully, Jim

    2013-05-01

    Side airbag systems were first introduced into vehicles around 1995 to help protect occupants from injury in side impact crashes. International studies have shown that side airbags are effective in reducing the risk of death and injury, however, serious injuries can still occur even when side airbags deploy. The objective of this study was to use detailed injury information from insurance injury compensation claims data linked to Police reported crash data to determine the effectiveness of side airbags in reducing the risk of death or injury for occupants involved in side impact crashes in Victoria, Australia based on the specific body regions that side airbag systems are designed to protect. It was found that head and torso-protecting dual airbag systems designed to protect the head, neck, face, chest and abdomen are highly effective in reducing driver death or injury due to near side crashes. They were associated with a statistically significant reduction of 41.1% (25.9%, 53.2%) in the odds of death or injury across all body regions; and a 48.0% (28.0%, 62.4%) reduction in the odds of death or injury to the head, neck, face, chest and abdomen. The study did not find any evidence that torso-protecting airbags alone are effective in reducing death or injury. Analysis results indicate that head and torso-protecting side airbag systems in vehicles are a highly effective technology for reducing the risk of death or injury to vehicle occupants in near side crashes. The magnitude of the injury reduction benefits estimated indicate that fitment of this technology to all vehicles should be a high priority and will yield significant savings in overall road trauma. PMID:23499979

  19. 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.

  20. 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. PMID:27447051

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

    NASA Astrophysics Data System (ADS)

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

    2008-08-01

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

  2. Photophysics of GFP-related chromophores imposed by a scaffold design.

    PubMed

    Dolgopolova, E A; Moore, T M; Fellows, W B; Smith, M D; Shustova, N B

    2016-06-14

    In this paper, a rigid scaffold imposes the photophysics of chromophores with a benzylidene imidazolidinone core by mimicking the β-barrel structure of the green fluorescent protein (GFP) and its analogs. The designed artificial frameworks maintain fluorescence responses and, therefore, conformational rigidity of typically non-emissive GFP-related chromophores. To replicate a small weight percent of the chromophore inside the natural GFP, two synthetic approaches were utilized: coordinative immobilization and non-coordinative inclusion. Despite low chromophore loading in the rigid matrix, both approaches resulted in formation of photoluminescent hybrid materials. Furthermore, the rigid scaffold dictates chromophore fluorescence by replicating its behavior in solution or the solid state. The presented results open an avenue for utilization of rigid scaffolds in the engineering of materials with tunable photoluminescence profiles for a variety of practical applications. PMID:26927627

  3. 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.

  4. Off-target effects of siRNA specific for GFP

    PubMed Central

    Tschuch, Cordula; Schulz, Angela; Pscherer, Armin; Werft, Wiebke; Benner, Axel; Hotz-Wagenblatt, Agnes; Barrionuevo, Leticia Serra; Lichter, Peter; Mertens, Daniel

    2008-01-01

    Background Gene knock down by RNAi is a highly effective approach to silence gene expression in experimental as well as therapeutic settings. However, this widely used methodology entails serious pitfalls, especially concerning specificity of the RNAi molecules. Results We tested the most widely used control siRNA directed against GFP for off-target effects and found that it deregulates in addition to GFP a set of endogenous target genes. The off-target effects were dependent on the amount of GFP siRNA transfected and were detected in a variety of cell lines. Since the respective siRNA molecule specific for GFP is widely used as negative control for RNAi experiments, we studied the complete set of off-target genes of this molecule by genome-wide expression profiling. The detected modulated mRNAs had target sequences homologous to the siRNA as small as 8 basepairs in size. However, we found no restriction of sequence homology to 3'UTR of target genes. Conclusion We can show that even siRNAs without a physiological target have sequence-specific off-target effects in mammalian cells. Furthermore, our analysis defines the off-target genes affected by the siRNA that is commonly used as negative control and directed against GFP. Since off-target effects can hardly be avoided, the best strategy is to identify false positives and exclude them from the results. To this end, we provide the set of false positive genes deregulated by the commonly used GFP siRNA as a reference resource for future siRNA experiments. PMID:18577207

  5. 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.

  6. Institutional responses to communicable diseases at Victoria College, University of Toronto, 1900-1940.

    PubMed

    Gidney, Catherine

    2007-01-01

    Between 1900 and 1940 students and staff risked their lives to attend, and teach at, Victoria College. Not only did Victoria experience three major epidemics--diphtheria in 1911, influenza from 1918 to 1920, and smallpox in 1927--but almost yearly one or two students contracted diseases such as scarlet fever, measles, and mumps. Yet at a time when there was no health insurance and few hospital facilities, how did the university cope with the problem? This paper examines the care provided to Victoria's residential students. In the process the paper illustrates not only the upheaval endured by individual students but also the enormous financial and emotional toll paid by the institution, especially by members of its female staff. PMID:18447307

  7. 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..., at 7300 Lone Tree Road, Victoria, Texas. The facility is used for the production of excavators...

  8. Reduction of photo bleaching and long term archiving of chemically cleared GFP-expressing mouse brains.

    PubMed

    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

  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. Attachment of Anti-GFP Antibodies to Microspheres for Optical Trapping Experiments

    PubMed Central

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

    2016-01-01

    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. PMID:22046049

  11. 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. PMID:22046049

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

    PubMed Central

    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

  13. 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

  14. 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

  15. Differential age susceptibility to influenza B/Victoria lineage viruses in the 2015 Australian influenza season.

    PubMed

    Barr, Ian G; Vijaykrishna, Dhanasekaran; Sullivan, Sheena G

    2016-01-01

    Influenza B viruses make up an important part of the burden from seasonal influenza globally. The 2015 season in Australia saw an unusual predominance of influenza B with a distinctive switch during the season from B/Yamagata/16/88 lineage viruses to B/Victoria/2/87 lineage viruses. We also noted significant differences in the age groups infected by the different B lineages, with B/Victoria infecting a younger population than B/Yamagata, that could not be explained by potential prior exposure. PMID:26848118

  16. 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

  17. Characterization of the Ugandan inshore waters of Lake Victoria based on temperature-conductivity diagrams

    NASA Astrophysics Data System (ADS)

    Cózar, AndréS.; Bracchini, Luca; Dattilo, Arduino; Loiselle, Steven; Azza, Nicholas

    2004-12-01

    Temperature-conductivity diagrams are shown as a valid instrument to analyze the hydrographic structure of freshwater ecosystems, even along the surface waters. We put this method in practice in the Ugandan inshore waters of Lake Victoria. A complementary parameter (T-C anomaly) was used to differentiate between upland water intrusions. The relative value of the T-C anomaly provided information about the nature of the water intrusions and showed a considerable correlation with the biological characteristics of the water masses. The results indicated that the connections between catchment attributes, water characteristics, and biological community are quite direct in the inshore waters of Lake Victoria.

  18. 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

  19. A simple and sensitive high-throughput GFP screening in woody and herbaceous plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Green fluorescent protein (GFP) has been used widely as a powerful bioilluminant reporter, but its visualization by existing methods in tissues or whole plants remains challenging in most species. Here, we report for the first time that a fluorescence scanner can be used as a sensitive means to det...

  20. It's cheap to be colorful. Anthozoans show a slow turnover of GFP-like proteins.

    PubMed

    Leutenegger, Alexandra; D'Angelo, Cecilia; Matz, Mikhail V; Denzel, Andrea; Oswald, Franz; Salih, Anya; Nienhaus, G Ulrich; Wiedenmann, Jörg

    2007-05-01

    Pigments homologous to the green fluorescent protein (GFP) contribute up to approximately 14% of the soluble protein content of many anthozoans. Maintenance of such high tissue levels poses a severe energetic penalty to the animals if protein turnover is fast. To address this as yet unexplored issue, we established that the irreversible green-to-red conversion of the GFP-like pigments from the reef corals Montastrea cavernosa (mcavRFP) and Lobophyllia hemprichii (EosFP) is driven by violet-blue radiation in vivo and in situ. In the absence of photoconverting light, we subsequently tracked degradation of the red-converted forms of the two proteins in coral tissue using in vivo spectroscopy and immunochemical detection of the post-translational peptide backbone modification. The pigments displayed surprisingly slow decay rates, characterized by half-lives of approximately 20 days. The slow turnover of GFP-like proteins implies that the associated energetic costs for being colorful are comparatively low. Moreover, high in vivo stability makes GFP-like proteins suitable for functions requiring high pigment concentrations, such as photoprotection. PMID:17419724

  1. CopA:GFP localizes to putative Golgi equivalents in Aspergillus nidulans.

    PubMed

    Breakspear, Andrew; Langford, Kelly J; Momany, Michelle; Assinder, Susan J

    2007-12-01

    The Golgi complex is a main component of the eukaryotic secretory system and functions to modify nascent proteins sent from the endoplasmic reticulum. Ultrastructural studies of filamentous fungi have shown Golgi to be individual smooth membrane cisternae that are referred to as Golgi equivalents or dictyosomes. The Aspergillus nidulans copA gene encodes a homolog of mammalian coat protein (alpha-COP), a constituent of the Golgi-localized COPI vesicle coat. Here, the localization of A. nidulansalpha-COP was examined in live cells using the reporter green fluorescent protein (GFP). CopA:GFP localized to putative Golgi equivalents that were concentrated at hyphal tips. The localization was disrupted by the fungal metabolite brefeldin A. To investigate the significance of the microtubule cytoskeleton in the localization of putative Golgi equivalents, the copA:gfp fusion was expressed in a temperature-sensitive dynein mutant. In addition, a wild-type strain expressing copA:gfp was treated with the microtubule-disrupting drug nocodazole. The results suggest that the microtubule cytoskeleton is not the primary mechanism of localizing putative Golgi equivalents in A. nidulans. PMID:17986089

  2. 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.

  3. A functional Bucky ball-GFP transgene visualizes germ plasm in living zebrafish.

    PubMed

    Riemer, Stephan; Bontems, Franck; Krishnakumar, Pritesh; Gömann, Jasmin; Dosch, Roland

    2015-01-01

    In many animals, the germline is specified by maternal RNA-granules termed germ plasm. The correct localization of germ plasm during embryogenesis is therefore crucial for the specification of germ cells. In zebrafish, we previously identified Bucky ball (Buc) as a key regulator of germ plasm formation. Here, we used a Buc antibody to describe its continuous germ plasm localization. Moreover, we generated a transgenic Buc-GFP line for live imaging, which visualizes germ plasm from its assembly during oogenesis up to the larval stages. Live imaging of Buc-GFP generated stunning movies, as they highlighted the dynamic details of germ plasm movements. Moreover, we discovered that Buc was still detected in primordial germ cells 2 days after fertilization. Interestingly, the transgene rescued buc mutants demonstrating genetically that the Buc-GFP fusion protein is functional. These results show that Buc-GFP exerts all biochemical interactions essential for germline development and highlight the potential of this line to analyze the molecular regulation of germ plasm formation. PMID:26143227

  4. 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

  5. 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...

  6. 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.

  7. Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons.

    PubMed

    Meera, Pratap; Dodson, Paul D; Karakossian, Movses H; Otis, Thomas S

    2005-11-01

    Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ. PMID:16212990

  8. Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore.

    PubMed

    Frizler, Maxim; Yampolsky, Ilia V; Baranov, Mikhail S; Stirnberg, Marit; Gütschow, Michael

    2013-09-21

    An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE. PMID:23912233

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

    PubMed

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

    2016-01-01

    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. PMID:26779599

  10. Effect of GFP expression on the sensitivity of glioma cell lines to photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Khatami, S.; Rendon, A.; Yoshimitsu, M.; Medin, J.; Lilge, L.

    2005-09-01

    Enhanced green fluorescent protein (EGFP)-expressing cells are customarily used in a variety of in vitro and in vivo studies and assays to ease visualization and localization. Nonetheless, the effects of EGFP expression on cellular responsivity to Photodynamic therapy (PDT), a combination therapy combining a photoactive drug and light, have yet to be characterized. To address this effect, rat astrocytoma cells (CNS-1), a lentivirus-transduced EGFP variant (CNS-1 GFP), human glioblastoma (U-87), and the transfected EGFP variant (U-87 GFP) are analyzed in terms of cell survival following PDT mediated by two different photoactive drugs. Cell survival is quantified via colony forming assays and Alamar blue assays, as a function of light dose, using the photosensitizers Photofrin (1ug ml-1 for 24h) and ALA (200ug ml-1 for 5h). Furthermore, effect of GFP expression on the responsivity to Cisplatin, a DNA-binding chemotherapeutic agent is determined for these cell lines. Our results show that EGFP expression does not affect the responsivity of Photofrin-PDT in comparison to parental cell lines (non GFP expressing cells), but does alter that of ALA-PDT. No change in responsivity is observed for Cisplatin treatment for either cell line. These results can be explained by oxidative stress induced by EGFP expression. This work will establish under which circumstances it is appropriate to use EGFP-expressing cell lines in the context of PDT preclinical research in vivo and in vitro.

  11. Mice expressing GFP and CreER in osteochondro progenitor cells in the periosteum

    PubMed Central

    Kawanami, Aya; Matsushita, Takehiko; Chan, Yuk Yu; Murakami, Shunichi

    2009-01-01

    We generated Prx1CreER-GFP transgenic mice that express tamoxifen-inducible Cre recombinase and GFP under the control of a 2.4 kb Prx1 promoter. The transgene is expressed in osteochondro progenitor cells in the developing limb buds and in a subpopulation of periosteal cells that is closely associated with the cortical bone. GFP-expressing cells isolated from the diaphyses of long bones by cell sorting express multiple markers of periosteal cells, including Prx-1, Fgf18, Tenascin-W, Periostin, and Thrombospondin 2. In addition, these cells undergo chondrogenic and osteogenic differentiation in culture upon induction. Cell fate analysis using the Rosa26 LacZ reporter indicated that transgene-expressing cells give rise to some of the chondrocytes and osteoblasts in the fracture callus. Collectively, these observations strongly suggest that the transgene-expressing cells are osteochondro progenitor cells in the periosteum. The established Prx1CreER-GFP mice would offer novel approaches for analyzing the functions of periosteal cells in vitro and in vivo. PMID:19538944

  12. 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.

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

    PubMed

    Talman, Arthur M; Blagborough, Andrew M; Sinden, Robert E

    2010-01-01

    The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito. PMID:20161781

  14. 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

  15. NMR-derived Topology of a GFP-photoprotein Energy Transfer Complex*

    PubMed Central

    Titushin, Maxim S.; Feng, Yingang; Stepanyuk, Galina A.; Li, Yang; Markova, Svetlana V.; Golz, Stefan; Wang, Bi-Cheng; Lee, John; Wang, Jinfeng; Vysotski, Eugene S.; Liu, Zhi-Jie

    2010-01-01

    Förster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca2+-regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously 15N,13C,2H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain 1H-15N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (KD = 0.9 mm). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase. PMID:20926380

  16. Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus

    PubMed Central

    Manicassamy, Balaji; Manicassamy, Santhakumar; Belicha-Villanueva, Alan; Pisanelli, Giuseppe; Pulendran, Bali; García-Sastre, Adolfo

    2010-01-01

    Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b+CD11c−, CD11b−CD11c+, and CD11b+CD11c+, as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type–specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field. PMID:20534532

  17. 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

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

    PubMed

    Sukup-Jackson, Michelle R; Kiraly, Orsolya; 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

    2014-06-01

    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

  19. Generation of Recombinant Rabies Virus CVS-11 Expressing eGFP Applied to the Rapid Virus Neutralization Test

    PubMed Central

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

    2014-01-01

    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. PMID:24714411

  20. 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. PMID:24714411

  1. Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 a...

  2. 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.

  3. Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding module.

    PubMed

    Ojima, Yoshihiro; Suparman, Asep; Nguyen, Minh Hong; Sakka, Makiko; Sakka, Kazuo; Taya, Masahito

    2015-07-01

    A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs. PMID:25978970

  4. 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

    ... Quality Standards (NAAQS) for 1- hour ozone (43 FR 8962). As required by the CAA, the state of Texas... in March 25, 1980 (45 FR 19231) and another in August 13, 1984 (49 FR 32180). An additional SIP..., 1995 (60 FR 12438). On July 27, 1994, Texas submitted a request to redesignate Victoria County...

  5. Supervision and Satisfaction among School Psychologists: An Empirical Study of Professionals in Victoria, Australia

    ERIC Educational Resources Information Center

    Thielking, Monica; Moore, Susan; Jimerson, Shane R.

    2006-01-01

    This study examined the supervision arrangements and job satisfaction among school psychologists in Victoria, Australia. Participation in professional supervision was explored in relation to the type of employment and job satisfaction. The results revealed that the frequency of participation in supervision activities was less than optimal, with…

  6. 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…

  7. Communicating Risk with Parents: Exploring the Methods and Beliefs of Outdoor Education Coordinators in Victoria, Australia

    ERIC Educational Resources Information Center

    Dallat, Clare

    2009-01-01

    This paper examines the risk communication strategies currently being employed by seven outdoor education co-ordinators in Government schools in Victoria, Australia. Of particular interest are the beliefs and assumptions held by these co-ordinators in relation to communicating risk with parents. Current policy stipulates that parents must be…

  8. 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...

  9. Rhetoric, Reality and Resources: The New 3R's for Victoria's "Schools of the Future."

    ERIC Educational Resources Information Center

    Townsend, Tony

    The new government of the state of Victoria (Australia) announced its "Schools of the Future" program in 1993. The emphasis on the role of the community in education and self-management by schools seemed promising for the quality of education in the state, but after 3 years of implementation, the reality does not match the rhetoric of this reform.…

  10. 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…

  11. 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

  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. 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…

  14. 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,…

  15. 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…

  16. 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…

  17. 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…

  18. 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…

  19. Establishing an Online Community of Inquiry at the Distance Education Centre, Victoria

    ERIC Educational Resources Information Center

    Jackson, Luke C.; Jackson, Alun C.; Chambers, Dianne

    2013-01-01

    This pilot intervention focused on three courses that were redesigned to utilize the online environment to establish an online community of inquiry (CoI). The setting for this research study was the Distance Education Centre, Victoria (DECV), an Australian co-educational school with approximately 3000 students who, for a variety of reasons, are…

  20. What Do the Public Search for on the Catalogue of the State Library of Victoria?

    ERIC Educational Resources Information Center

    Waller, Vivienne

    2009-01-01

    This study examines what the public search for in the catalogue of the State Library of Victoria (SLV). As well as indicating the type of content being accessed, this gives an indication of what catalogue users expect of the State Library collection. A content analysis was undertaken of a random, stratified sample of 4,000 search queries typed in…

  1. 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…

  2. 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)…

  3. 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,…

  4. 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.

  5. 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

  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. Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line.

    PubMed

    Mangravite, L M; Lipschutz, J H; Mostov, K E; Giacomini, K M

    2001-05-01

    Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK(1) cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway. PMID:11292631

  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. 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

  10. 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%.

  11. 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.

  12. 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.

  13. 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.

  14. Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum.

    PubMed

    Nikonov, Andrei V; Snapp, Erik; Lippincott-Schwartz, Jennifer; Kreibich, Gert

    2002-08-01

    In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP-Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP-Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP-Dad1 in the ER membranes, but to a level that is still lower than that of free GFP-Dad1. This suggests that GFP-Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains. PMID:12163472

  15. Subpopulations of GFP-Marked Mouse Pancreatic β-Cells Differ in Size, Granularity, and Insulin Secretion

    PubMed Central

    Katsuta, Hitoshi; Aguayo-Mazzucato, Cristina; Katsuta, Rimiko; Akashi, Tomoyuki; Hollister-Lock, Jennifer; Sharma, Arun J.; Bonner-Weir, Susan

    2012-01-01

    There is growing information about the heterogeneity of pancreatic β-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze β-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of β-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of β-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1–9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about β-cell heterogeneity. PMID:22919061

  16. 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.

  17. Live-Cell Imaging of the Association of STAT6-GFP with Mitochondria

    PubMed Central

    Khan, Rasel; Lee, Jason E.; Yang, Yang-Ming; Liang, Feng-Xia; Sehgal, Pravin B.

    2013-01-01

    The transcription factor STAT3 has been previously reported to be associated with mitochondria. However, we have been unable to visualize an association of STAT3-GFP, STAT3-DsRed or STAT3-Flag with mitochondria in human Hep3B hepatocytes thus far even though an association of these molecules with other cytoplasmic organelles (endosomes) was readily demonstrable. We then addressed the broader question of a possible association of other STAT-family of proteins with mitochondria by first using immunolocalization assays in Hep3B and human pulmonary arterial endothelial and smooth muscle cells. Strong anti-STAT6-immunolocalization with mitochondria was apparent in fluorescence and electron microscopy assays of cells first washed with a digitonin-sucrose buffer to remove bulk soluble STAT proteins. In live-cell imaging studies, STAT6-GFP, but not N1-GFP, was observed to constitutively colocalize with MitoTracker- and tetramethylrhodamine ethyl ester (TMRE)-positive mitochondria, and with mitochondrial F1-ATPase when assayed by immunofluorescence after fixation. This association was Tyr-phosphorylation independent in that a STAT6 truncated protein (STAT61-459-GFP) which lacked the SH2 domain (517–632) and the cytokine-activated Y641 phosphorylation site also accumulated in MitoTracker-positive mitochondria. This was consistent with the unexpected discovery that anti-STAT6-immunofluoresence also associated with mitochondria in mouse embryo fibroblasts (MEFs) from both wild-type and the STAT6SH2-/SH2- mouse. MEFs from the latter mouse, which had been engineered in 1996 to be deleted in the STAT6 SH2 domain (amino acids 505–584) expressed an immune-specific ∼50 kDa protein detectable in whole cell and mitochondria-enriched fractions. Taken together, the present data provide the first definitive evidence of the association of any STAT-protein family member with mitochondria - that of STAT6. PMID:23383189

  18. 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

  19. GFP-SCFV: expression and possible applications as a tool for experimental investigations of atherosclerosis.

    PubMed

    Faulin, Tanize do Espirito Santo; Guilherme, Daniel Ferreira; Silva, Aldacilene Souza; Abdalla, Dulcineia Saes Parra; Hering, Vitor Renaux; Politi, Mario José; Maranhão, Andrea Queiroz

    2014-01-01

    Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti-LDL(-) scFv, previously obtained from a hybridoma secreting an anti-LDL(-) monoclonal antibody, was inserted into the bacterial vector pET-28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP-scFv was assessed by ELISA, which determined its affinity for LDL(-) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(-). In conclusion, our data suggest that the anti-LDL(-) GFP-scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis. PMID:24911875

  20. GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

    PubMed Central

    Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

    2014-01-01

    Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

  1. Strain-Specific Regulation of Striatal Phenotype in Drd2-eGFP BAC Transgenic Mice

    PubMed Central

    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

    2012-01-01

    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 D2 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 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 which are hemizygous for the Drd2 BAC transgene provide a reliable tool for studying basal ganglia function. PMID:22764222

  2. Mechanistic Insights into Reversible Photoactivation in Proteins of the GFP Family

    PubMed Central

    Gayda, Susan; Nienhaus, Karin; Nienhaus, G. Ulrich

    2012-01-01

    Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore’s protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pKa. In mIrisGFP, the cis chromophore protonates in two steps, with pKcis of 5.3 and 6, which is much lower than pKtrans (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes. PMID:23260054

  3. 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

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

    PubMed

    Bello-Morales, Raquel; Pérez-Hernández, Marta; Rejas, María Teresa; Matesanz, Fuencisla; Alcina, Antonio; López-Guerrero, José Antonio

    2011-01-01

    The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP), the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP)-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested. PMID:21573057

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  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. PMID:17753283

  11. Modes of climate variability and heat waves in Victoria, southeastern Australia

    NASA Astrophysics Data System (ADS)

    Parker, Teresa J.; Berry, Gareth J.; Reeder, Michael J.; Nicholls, Neville

    2014-10-01

    Summertime heat waves in the southeastern state of Victoria, Australia, are associated with broad anticyclonic upper level potential vorticity (PV) anomalies. The current research establishes the relationship between heat waves, precipitation, and three modes of climate variability of importance for rainfall in Australia: the El Niño-Southern Oscillation (ENSO), Madden-Julian Oscillation (MJO), and Southern Annular Mode (SAM). Heat waves in Victoria in summer are more common during phases 3-6 of the MJO (when convection is enhanced over the eastern Indian Ocean, Maritime Continent, and western Pacific Ocean) and La Niña phases of ENSO. The PV-Theta (Θ) structure of the heat wave and pattern of convection varies with the phase of each mode of variability. Enhanced tropical convection results in a heat wave characterized by a monopole of anticyclonic PV, whereas suppressed convection is associated with a dipole of anticyclonic and cyclonic PV.

  12. 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

  13. Episodic rifting of phanerozoic rocks in the Victoria Land basin, Western Ross Sea, Antarctica

    USGS Publications Warehouse

    Cooper, A. K.; Davey, F.J.

    1985-01-01

    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.

  14. Environmental impacts of cage culture in Lake Victoria: the case of Shirati Bay-Sota, Tanzania.

    PubMed

    Kashindye, Benedicto Boniphace; Nsinda, P; Kayanda, R; Ngupula, G W; Mashafi, C A; Ezekiel, C N

    2015-01-01

    The experimental cage culture was conducted at Shirati bay, Lake Victoria from February to August 2013, to investigate the impacts of the small scale cage culture on the environment. Three locations along the cages, at the intermediate and one in the offshore (control) were sampled for water quality parameters, phytoplankton and macro invertebrates. A notable increase in nutrient concentration was observed after the set of cages among the stations. However DO, pH, and water transparency showed no major changes and was within the recommended ranges. Cyanophytes an indicator of inorganic pollution dominated before and after the set of cages, an increase in phytoplankton numerical abundance was observed after stocking of fish in cages. In addition there was an increase in the invertebrate community especially bivalves and gastropods. In conclusion we found no consistent environmental change caused by cage culture, and therefore it can be allowed in Lake Victoria, Tanzania part, with close monitoring of its impacts. PMID:26361576

  15. 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

  16. 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

  17. 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.

  18. A novel subset of enteric neurons revealed by ptf1a:GFP in the developing zebrafish enteric nervous system.

    PubMed

    Uribe, Rosa A; Gu, Tiffany; Bronner, Marianne E

    2016-03-01

    The enteric nervous system, the largest division of the peripheral nervous system, is derived from vagal neural crest cells that invade and populate the entire length of the gut to form diverse neuronal subtypes. Here, we identify a novel population of neurons within the enteric nervous system of zebrafish larvae that express the transgenic marker ptf1a:GFP within the midgut. Genetic lineage analysis reveals that enteric ptf1a:GFP(+) cells are derived from the neural crest and that most ptf1a:GFP(+) neurons express the neurotransmitter 5HT, demonstrating that they are serotonergic. This transgenic line, Tg(ptf1a:GFP), provides a novel neuronal marker for a subpopulation of neurons within the enteric nervous system, and highlights the possibility that Ptf1a may act as an important transcription factor for enteric neuron development. PMID:26865080

  19. 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

  20. 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

  1. Simulation of pollution buffering capacity of wetlands fringing the Lake Victoria.

    PubMed

    Mwanuzi, F; Aalderink, H; Mdamo, L

    2003-04-01

    Lake Victoria has undergone substantial and most negative changes, especially over the last 30 years. One of the driving factors is nutrient enrichment from human activities in the catchment, which is causing eutrophication. This has been associated with, among others, the rapid proliferation of water hyacinth, alga blooms, and with general disruption of the lake ecosystem. Most of pollution to the lake flow via the natural wetlands. In order to understand how wetlands function within the Lake Victoria ecosystem a wetland model has been developed. The main objective of the model is to establish and simulate the buffering processes and capacity of individual wetlands (that is, their ability to absorb sediments, nutrients and pollutants) within the Lake Victoria basin. It was found that in both seasonal and permanent swamp there is a net export of organic matter produced in the wetlands. Most of the inorganic phosphorous were retained in the wetlands (60% to 90% removal) while there was a negative retention of nitrates probably due to the export of organic matter which associate very much with nitrates. PMID:12605942

  2. 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

  3. 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

  4. 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.

  5. Water quality management and sustainability: the experience of Lake Victoria Environmental Management Project (LVEMP)??Tanzania

    NASA Astrophysics Data System (ADS)

    Machiwa, Praxeda K.

    Human health and development are threatened in many parts of the world either from lack of water or poor water quality. Human development has partially contributed to water quality deterioration. In Tanzania, for instance, rapid population growth that caused expansion of agricultural activities, livestock keeping, deforestation, biomass burning and human settlement have exerted pressures within the Lake Victoria Basin. These developments have led to land degradation and increased levels of pollution mainly from non-point sources. The Governments of Kenya, Tanzania and Uganda initiated the program of Lake Victoria Environmental Management Project, (LVEMP), in 1994 to rehabilitate the Lake Ecosystem through restoration and conservation of biodiversity in the lake as well as within the catchment. This paper presents the five years (1997-2002) experience of LVEMP in Tanzania on the issues of water quality; focusing on water pollution, water quality monitoring and LVEMP strategies to accomplish water quality management in the Lake Zone (Kagera, Mara and Mwanza regions). The findings show that non-point source pollution from agricultural practices; as well as unplanned urban settlements contribute more to siltation and eutrophication of the of Lake Victoria than that from point source pollution. Recommendations for water quality management and sustainability are presented.

  6. Mislocalization and Degradation of Human P23H-Rhodopsin-GFP in a Knockin Mouse Model of Retinitis Pigmentosa

    PubMed Central

    Price, Brandee A.; Sandoval, Ivette M.; Chan, Fung; Simons, David L.; Wu, Samuel M.; Wensel, Theodore G.

    2011-01-01

    Purpose. To engineer a knockin mouse model that can be used to monitor the effects of treatments on degradation and mislocalization of proline-to-histidine change at codon 23 (P23H) rhodopsin, a common cause of autosomal dominant retinitis pigmentosa (ADRP). The goal was to introduce a gene that expressed rhodopsin at low levels to avoid rapid retinal degeneration, and with a readily visible tag to make it easy to distinguish from wild type rhodopsin. Methods. One copy of the endogenous mouse rhodopsin gene was replaced with a mutant human rhodopsin gene that encodes P23H-rhodopsin fused to enhanced green fluorescent protein (GFP) at its C terminus. The gene includes a LoxP site in the sequence corresponding to the 5′-untranslated region, which greatly reduces translation efficiency. Characterized are the resulting heterozygous and homozygous P23H-hRho-GFP mouse lines for mRNA and protein expression, P23H-rhodopsin localization in rod cells, effects on visual function, and retinal degeneration. Results. The retinas of heterozygous P23H-hRho-GFP mice are morphologically and functionally very similar to those of wild type mice, and they display little cell death over time. P23H-hRho-GFP mice transcribe the knockin gene as efficiently as the endogenous mouse allele, but they contain much less of the protein product than do knockin mice expressing nonmutant hRho-GFP, indicating that substantial degradation of P23H-rRho-GFP occurs in mouse rod cells. The remaining P23H-hRho-GFP mislocalizes to the inner segment and outer nuclear layer, with only approximately 20% in rod outer segments. Conclusions. P23H-hRho-GFP mice provide a valuable tool for evaluating the efficacy of potential therapies for ADRP that influence the levels or localization of P23H-rhodopsin. PMID:22110080

  7. 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.

  8. 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

  9. 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. PMID:23499733

  10. Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum

    PubMed Central

    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 in vivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCRRH2-RH2-1 or LCRRH2-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCRRH2-RH2-1:GFP)pt112 and Tg(LCRRH2-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. PMID:23499733

  11. 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. PMID:16883477

  12. Isolation, plant colonization potential, and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp. Ph6-gfp

    PubMed Central

    Sun, Kai; Liu, Juan; Gao, Yanzheng; Jin, Li; Gu, Yujun; Wang, Wanqing

    2014-01-01

    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. PMID:24964867

  13. Matrix metalloproteinase 2 fused to GFP, expressed in E. coli, successfully tracked MMP-2 distribution in vivo.

    PubMed

    Azevedo, A; Prado, A F; Issa, J P M; Gerlach, R F

    2016-08-01

    Matrix Metalloproteinases (MMPs) participate in many physiological and pathological processes. One major limitation to a better understanding of the role MMPs play in these processes is the lack of well-characterized chimeric proteins and characterization of their fluorescence. The specialized literature has reported on few constructs bearing MMPs fused to the sequence of the green fluorescent protein (GFP), but none of the described constructs have been intended for expression in bacteria or for purification and use in vivo. This work has tested a recombinant reporter protein containing the MMP-2 catalytic domain fused to GFP in terms of purification efficiency, degradation of substrates in solution and in zymograms, kinetic activity, GFP fluorescence, and GFP fluorescence in whole animals after injection of the purified and lyophilized fluorescent protein. This work has also characterized rhMMP-2 (recombinant human MMP-2) and inactive clones and used them as negative controls in experiments employing catMMP-2/GFP and rhMMP-2. To our knowledge, this is the first study that has fully characterized a chimeric protein with the MMP-2 catalytic domain fused to GFP, that has efficiently purified such protein from bacteria in a single-step, and that has obtained an adequate chimeric protein for injection in animals and tracking of MMP-2 fate and activity in vivo. PMID:27156693

  14. Expression and secretion of a CB4-1 scFv-GFP fusion protein by fission yeast.

    PubMed

    Naumann, Julia Maria; Küttner, Gabriele; Bureik, Matthias

    2011-01-01

    There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion. PMID:20617397

  15. A Fluorogenic Red Fluorescent Protein Heterodimer

    PubMed Central

    Alford, Spencer C.; Abdelfattah, Ahmed S.; Ding, Yidan; Campbell, Robert E.

    2012-01-01

    SUMMARY The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a Kd of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca2+-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis. PMID:22444590

  16. 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

  17. Comparison of Mortality Following Hospitalisation for Isolated Head Injury in England and Wales, and Victoria, Australia

    PubMed Central

    Gabbe, Belinda J.; Lyons, Ronan A.; Lecky, Fiona E.; Bouamra, Omar; Woodford, Maralyn; Coats, Timothy J.; Cameron, Peter A.

    2011-01-01

    Background Traumatic brain injury (TBI) remains a leading cause of death and disability. The National Institute for Health and Clinical Excellence (NICE) guidelines recommend transfer of severe TBI cases to neurosurgical centres, irrespective of the need for neurosurgery. This observational study investigated the risk-adjusted mortality of isolated TBI admissions in England/Wales, and Victoria, Australia, and the impact of neurosurgical centre management on outcomes. Methods Isolated TBI admissions (>15 years, July 2005–June 2006) were extracted from the hospital discharge datasets for both jurisdictions. Severe isolated TBI (AIS severity >3) admissions were provided by the Trauma Audit and Research Network (TARN) and Victorian State Trauma Registry (VSTR) for England/Wales, and Victoria, respectively. Multivariable logistic regression was used to compare risk-adjusted mortality between jurisdictions. Findings Mortality was 12% (749/6256) in England/Wales and 9% (91/1048) in Victoria for isolated TBI admissions. Adjusted odds of death in England/Wales were higher compared to Victoria overall (OR 2.0, 95% CI: 1.6, 2.5), and for cases <65 years (OR 2.36, 95% CI: 1.51, 3.69). For severe TBI, mortality was 23% (133/575) for TARN and 20% (68/346) for VSTR, with 72% of TARN and 86% of VSTR cases managed at a neurosurgical centre. The adjusted mortality odds for severe TBI cases in TARN were higher compared to the VSTR (OR 1.45, 95% CI: 0.96, 2.19), but particularly for cases <65 years (OR 2.04, 95% CI: 1.07, 3.90). Neurosurgical centre management modified the effect overall (OR 1.12, 95% CI: 0.73, 1.74) and for cases <65 years (OR 1.53, 95% CI: 0.77, 3.03). Conclusion The risk-adjusted odds of mortality for all isolated TBI admissions, and severe TBI cases, were higher in England/Wales when compared to Victoria. The lower percentage of cases managed at neurosurgical centres in England and Wales was an explanatory factor, supporting the changes made to the NICE

  18. In vivo protein stabilization based on fragment complementation and a split GFP system

    PubMed Central

    Lindman, Stina; Hernandez-Garcia, Armando; Szczepankiewicz, Olga; Frohm, Birgitta; Linse, Sara

    2010-01-01

    Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state. PMID:21041669

  19. Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.

    PubMed

    Bleckmann, Maren; Schmelz, Stefan; Schinkowski, Christian; Scrima, Andrea; van den Heuvel, Joop

    2016-09-01

    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 baculovirus infected insect cells

  20. Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera.

    PubMed

    García-Mata, R; Bebök, Z; Sorscher, E J; Sztul, E S

    1999-09-20

    Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins. PMID:10491388

  1. Characterization and Dynamics of Aggresome Formation by a Cytosolic Gfp-Chimera✪

    PubMed Central

    García-Mata, Rafael; Bebök, Zsuzsa; Sorscher, Eric J.; Sztul, Elizabeth S.

    1999-01-01

    Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883–1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481–490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end–directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins. PMID:10491388

  2. 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. PMID:25048992

  3. Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization

    PubMed Central

    Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin (Simon); Williams, Melissa; Zaveri, Nurulain T.; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L.

    2015-01-01

    The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [35S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native

  4. 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

  5. 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

  6. Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression.

    PubMed

    Gorfer, Markus; Klaubauf, Sylvia; Bandian, Dragana; Strauss, Joseph

    2007-07-01

    Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome. PMID:17662587

  7. 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

  8. 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

  9. 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

  10. 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.

  11. TRV–GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function

    PubMed Central

    Tian, Ji; Pei, Haixia; Ma, Nan

    2014-01-01

    Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3’ terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV–GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV–GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV–GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75–80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants. PMID:24218330

  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. Short Hydrogen Bonds and Proton Delocalization in Green Fluorescent Protein (GFP)

    PubMed Central

    2015-01-01

    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

  14. Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants

    PubMed Central

    Giraldez, Teresa; Hughes, Thomas E.; Sigworth, Fred J.

    2005-01-01

    The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the α subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The “success rate” of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions. PMID:16260837

  15. On the optical absorption of the anionic GFP chromophore in vacuum, solution, and protein.

    PubMed

    Petrone, Alessio; Caruso, Pasquale; Tenuta, Silvia; Rega, Nadia

    2013-12-21

    In spite of the large number of experimental and theoretical studies, the optical absorption trend of the green fluorescent protein (GFP) chromophore in several environments has not been fully understood. We calculated at the same level of time dependent density functional theory the vertical excitation energy of the anionic GFP chromophore in the protein and in ethanol, dioxane, methanol and water solutions. As result, we reproduced for the first time the experimental trend of the absorption peaks with 0.015 eV as the standard deviation of the accuracy. This systematic error allowed us to analyze with confidence the relative weight of several solvation effects on the vertical excitation energy. Experimental trends not correlated with the solvent polarity were therefore explained with a fine balance of different steric and electronic effects on the photophysics of the chromophore. As an indirect and remarkable result, the present analysis confirms that the optical absorption of the chromophore in the gas phase is close to the value of 2.84 eV extrapolated by Dong et al. (J. Am. Chem. Soc., 2006, 128, 12038), and, as a consequence, that the protein environment induces a red shift of 0.23 eV. PMID:24177429

  16. Proteome-wide screens in Saccharomyces cerevisiae using the yeast GFP collection.

    PubMed

    Chong, Yolanda T; Cox, Michael J; Andrews, Brenda

    2012-01-01

    The budding yeast is a simple and genetically tractable eukaryotic organism. It remains a leading system for functional genomic work and has been the focus of many pioneering efforts, including the systematic construction and analysis of gene deletion mutants. Over the past decade, many large-scale studies have made use of the deletion and other mutant collections to assay genetic interactions, chemical sensitivities, and other phenotypes, contributing enormously to our understanding of gene function. The deletion mutant collection has also been used in cell biological surveys to identify genes that control cell and organelle morphology. One valuable approach for systematic definition of gene function and biological pathways involves global assessment of the localization patterns of the proteins they encode and how these patterns are altered in response to environmental or genetic perturbation. However, proteome-wide, cell biological screens are extremely challenging, from both a technical and computational perspective. The yeast GFP collection, an elegant and unique strain set, is ideal for studying both protein localization and abundance across the proteome ( http://yeastgfp.yeastgenome.org/ ). In this chapter, we outline how the yeast GFP collection has been used to date and discuss approaches for conducting future surveys of the proteome. PMID:22161327

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

    PubMed Central

    Zapata-Hommer, Otilia; Griesbeck, Oliver

    2003-01-01

    Background The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins. Results By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells. Conclusion "T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments. PMID:12769828

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning

    PubMed Central

    Drobizhev, M.; Callis, P. R.; Nifosì, R.; Wicks, G.; Stoltzfus, C.; Barnett, L.; Hughes, T. E.; Sullivan, P.; Rebane, A.

    2015-01-01

    The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement. PMID:26286372

  3. 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

  4. Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability.

    PubMed

    Nischan, Nicole; Herce, Henry D; Natale, Francesco; Bohlke, Nina; Budisa, Nediljko; Cardoso, M Cristina; Hackenberger, Christian P R

    2015-02-01

    The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells. PMID:25521313

  5. 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

  6. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-09-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16023367

  7. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-12-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP)and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immuno-gold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated indifferent axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16456925

  8. 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.

  9. Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

    PubMed

    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

  10. 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

  11. 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.

  12. Metals in Nile perch (Lates niloticus) and suspended particulate matter from Lake Victoria, Tanzania.

    PubMed

    Machiwa, John F

    2005-01-01

    A study was conducted to assess the levels of pollutant metals in suspended particulate matter and Nile perch from Lake Victoria. The metals in particulate matter were determined to ascertain their concentrations at the base of the food chain. Nile perch samples were collected in September 2003 from five major fish processing factories at the shores of Lake Victoria in Mwanza and Musoma. The concentrations of total Hg, Pb, Cd, and Cu were generally low in particulate matter and in most locations were close to or below their limits of detection. The concentrations of Zn were high in suspended particulate matter, the highest being 219.4 +/- 153.0 microg L(-1) found in particulate matter from Nungwe Bay in the southern part of Lake Victoria. Nile perch generally contained low levels of heavy metals; the range for Pb was <0.01-0.08 microg g(-1) ww, Cd was <0.001-0.04 microg g(-1) ww, Cu was 0.01-0.97 microg g(-1) ww, and Zn was <0.01-18.94 microg g(-1) ww. The concentration of total mercury ranged between 31.0 and 684.2 ng g(-1) ww; generally, it was below the Food and Agriculture Organization of the United Nations/World Health Organization (1000 ng total Hg g(-1) ww for piscivorous fish species) maximum allowable level. Indeed, all Nile perch samples that weighed less than 10 kg had less than 200 ng total Hg g(-1) ww and therefore are safe for regular consumption by at-risk groups such as children and pregnant women. Levels of mercury and other heavy metals in Nile perch at present is, therefore, not a severe environmental issue; however, urgent regulatory measures should be taken to minimize metal input into the lake to maintain the current levels in the fish. PMID:16134370

  13. 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

  14. Malaria Vectors in Lake Victoria and Adjacent Habitats in Western Kenya

    PubMed Central

    Minakawa, Noboru; Dida, Gabriel O.; Sonye, George O.; Futami, Kyoko; Njenga, Sammy M.

    2012-01-01

    The prevalence of malaria among the residents of the Lake Victoria basin remains high. The environment associated with the lake may maintain a high number of malaria vectors. Lake habitats including water hyacinths have been suspected to be the source of vectors. This study investigated whether malaria vectors breed in the lake habitats and adjacent backwater pools. Anopheline larvae were collected within the littoral zone of the lake and adjacent pools located along approximately 24.3 km of the lakeshore in western Kenya, and their breeding sites characterized. Three primary vector species, Anopheles arabiensis, Anopheles gambiae s.s. and Anopheles funestus s.s., and three potential vectors, were found in the lake habitats. Unexpectedly, An. arabiensis was the most dominant vector species in the lake sampling sites. Its habitats were uncovered or covered with short grass. A potential secondary malaria vector, Anopheles rivulorum, dominated the water hyacinths in the lake. Most breeding sites in the lake were limited to areas that were surrounded by tall emergent plants, including trees, and those not exposed to waves. Nearly half of adjacent habitats were lagoons that were separated from the lake by sand bars. Lagoons contained a variety of microhabitats. Anopheles arabiensis dominated open habitats, whereas An. funestus s.s. was found mainly in vegetated habitats in lagoons. The current study confirmed that several breeding sites are associated with Lake Victoria. Given that Lake Victoria is the second largest lake in the world, the lake related habitats must be extensive; therefore, making targeted vector control difficult. Further exploration is necessary to estimate the effects of lake associated habitats on malaria transmission so as to inform a rational decision-making process for vector control. PMID:22412913

  15. Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic...

  16. Reconstruction of Eolian Bedforms from Cross-Bedded Strata at Victoria Crater, Meridiani Planum, Mars

    NASA Astrophysics Data System (ADS)

    Hayes, A. G.; Grotzinger, J. P.; Squyres, S. W.; Lewis, K.; Metz, J.; Bell, J. F.; Athena Science Team

    2007-12-01

    Outcrop exposures imaged by the Opportunity rover at Meridiani Planum have depicted cross-bedded strata with geometries and scales similar to eolian and subaqeous deposits on Earth. On Earth preserved cross-strata are rich in geologic information, providing insight into the depositional environment and sediment transport directions. The high-resolution stratigraphy of these cross-bedded strata can be used to reconstruct sedimentary bedforms on Mars to infer formation process and describe the depositional environment. Meter-scale cross bedding at Victoria Crater is similar to terrestrial eolian deposits and is interpreted as a dry dune field. Sets of cross-strata in the Cape St. Vincent and Cape St. Mary sections of Victoria Crater are comparable to Jurassic-age eolian deposits of the western US. The Opportunity Rover has spent ~300 sols traversing 90 degrees of the rim of Victoria crater, obtaining images of rock outcrops exposed by several promontories along the way. The outcrops at the Cape St. Mary and Cape St. Vincent promontories, which are located at opposite ends of the traverse, have proven to be the best examples of meter scale cross-bedding observed on Mars to date. Super-resolution imaging techniques and long baseline stereo observations were utilized during an extended imaging campaign of both outcrop faces. Cape St. Mary is characterized by meter-scale trough-style cross bedding, suggesting sinuous crested dunes with scour pits migrating perpendicular to the outcrop face. Cape St. Vincent, which is striking 110° away from Cape St. Mary, has layering indicative of a single climbing bedform with dune heights of several meters. The findings at Cape St. Mary and Cape St. Vincent are combined with other bedding faces to produce an eolian deposition model for layering exposed at Victoria Crater. Any depositional model used to explain the bedding must conform to an observed N-S paleo-flow direction. In addition to bedded layering, a bright band is observed

  17. A new species of Anabarhynchus (Diptera: Therevidae) from an ocean beach in south east Victoria.

    PubMed

    Ferguson, David J; Yeates, David K

    2014-01-01

    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 Anabarhynchusoceanus sp. n., a species found on ocean beaches in eastern Victoria, Australia. The species shares most characters with the monobasic Anabarhynchuskampmeierae 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

  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. Gambling revenues as a public administration issue: electronic gaming machines in Victoria.

    PubMed

    Pickernell, David; Keast, Robyn; Brown, Kerry; Yousefpour, Nina; Miller, Chris

    2013-12-01

    Gambling activities and the revenues derived have been seen as a way to increase economic development in deprived areas. There are also, however, concerns about the effects of gambling in general and electronic gaming machines (EGMs) in particular, on the resources available to the localities in which they are situated. This paper focuses on the factors that determine the extent and spending of community benefit-related EGM-generated resources within Victoria, Australia, focusing in particular on the relationships between EGM activity and socio-economic and social capital indicators, and how this relates to the community benefit resources generated by gaming. PMID:23065238

  20. 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

  1. Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae.

    PubMed

    Paiva, Sandra; Kruckeberg, Arthur L; Casal, Margarida

    2002-05-01

    Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123 s(-1). It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p-GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Deltadoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease. PMID:11964174

  2. 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.

  3. 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. PMID:24637200

  4. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

    PubMed Central

    Peddie, Christopher J.; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O׳Toole, Peter; Larijani, Banafshe; Collinson, Lucy M.

    2014-01-01

    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. PMID:24637200

  5. 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

  6. Round the bend: a brief history of mental health nursing in Victoria, Australia 1848 to 1950's.

    PubMed

    Sands, Natisha Marina

    2009-06-01

    This paper presents a history of mental health nursing in Victoria, Australia from 1848 to the 1950's, or the asylum years to the era of the mental hospital. The research for this historical overview was conducted as part of a literature review for a mental health nursing doctoral thesis, which included an account of the evolution of the profession from asylum attendant to the present time. The literature reviewed for this project revealed a distinct lack of a coherent, chronological account of the historical development of mental health nursing in Victoria, and this paper seeks to address that knowledge gap. PMID:19499436

  7. Three Licentiates of the Royal College of Surgeons of Edinburgh who were decorated with the Victoria Cross.

    PubMed

    Kaufman, M H

    2011-08-01

    Since the Victoria Cross was introduced in January 1856 by Queen Victoria to reward acts of valour in the face of the enemy, initially during the Crimean War, over 1350 medals have been awarded. Of these, three were awarded to medical officers who had previously gained the Licentiate Diploma of the Royal College of Surgeons of Edinburgh (LRCS Edin) - Valentine Munbee McMaster on 25 September 1857, Henry Thomas Sylvester on 20 November 1857 (although the acts of valour for which he was awarded his VC occurred on two occasions in September 1855) and Campbell Mellis (or Millis) Douglas on 7 May 1867. PMID:21810849

  8. Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation.

    PubMed

    Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping

    2016-09-01

    Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45-/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45-/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45-/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. PMID:27269414

  9. 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

  10. 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

  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. GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria

    PubMed Central

    Paley, Olga; Agnello, Giulia; Cantor, Jason; Yoo, Tae Hyun; Georgiou, George; Stone, Everett

    2014-01-01

    There is significant interest in engineering human amino acid degrading enzymes as non-immunogenic chemotherapeutic agents. We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporter to the catalytic activity of the desired amino acid degrading enzyme in an appropriate E. coli genetic background. The method described here allows facile screening of much larger libraries (106–107) than was previously possible. We demonstrate the application of this technique in the screening of libraries of bacterial and human asparaginases and also for the catalytic optimization of an engineered human methionine gamma lyase. PMID:23423887

  13. A simple method for GFP- and RFP-based dual color single-molecule localization microscopy.

    PubMed

    Platonova, Evgenia; Winterflood, Christian M; Ewers, Helge

    2015-06-19

    The recent development of single-molecule localization-based super-resolution techniques has afforded a resolution in the nanometer range in light microscopy. The ability to resolve biological structures on this scale by multicolor techniques faces significant challenges which have prevented their widespread use. Here, we provide a generic approach for high-quality simultaneous two-color single-molecule localization microscopy imaging of any combination of GFP- and RFP-tagged proteins with the use of nanobodies. Our method addresses a number of common issues related to two-color experiments, including accuracy and density of labeling as well as chromatic aberration and color-crosstalk with only minimal technical requirements. We demonstrate two-color imaging of various nanoscopic structures and show a compound resolution down to the limit routinely achieved only in a single color. PMID:25806422

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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. PMID:21440093

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

    PubMed

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

    2016-08-01

    Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs. PMID:27185381

  1. 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

  2. Pb isotope record over one century in snow from Victoria Land, Antarctica

    NASA Astrophysics Data System (ADS)

    Van de Velde, K.; Vallelonga, P.; Candelone, J.-P.; Rosman, K. J. R.; Gaspari, V.; Cozzi, G.; Barbante, C.; Udisti, R.; Cescon, P.; Boutron, C. F.

    2005-03-01

    Pb and Ba concentrations and Pb isotopic compositions are reported for firn core and snow pit samples from Victoria Land, Antarctica, dating from 1872 AD to 1994 AD. From variations in Pb/Ba ratios and Pb isotopic compositions, two periods of major Pb enhancements were identified, from 1891 to 1908 AD and from 1948 to 1994 AD. The earlier pollution event is attributed to Pb emissions from non-ferrous metal production and coal combustion in the Southern Hemisphere and is in excellent agreement with coincident pollution inputs reported in firn/ice cores from two other regions of Antarctica, at Coats Land and Law Dome. Using Pb isotopic systematics, it was calculated that ˜50% of Pb deposited in Victoria Land in 1897 originated from anthropogenic emission sources. The more recent period of Pb enhancements, from 1948 to 1994 AD, corresponds to the introduction and widespread use of gasoline alkyl Pb additives in automobiles in the Southern Hemisphere, with anthropogenic Pb inputs averaging 60% of total Pb but with large uncertainty. Intra- and inter-annual variations in Pb concentrations and isotopic compositions were evaluated in snow pits samples corresponding to the period 1991-1994. Substantial variations in Pb/Ba and 206Pb/ 207Pb ratios were detected but the absence of a regular seasonal pattern for these parameters suggests that the transport and deposition of aerosols to the Antarctic ice sheet are complex and vary from year to year.

  3. 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.

  4. 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.

  5. 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. PMID:26350290

  6. On the age and origin of the species flock of haplochromine cichlid fishes of Lake Victoria.

    PubMed

    Fryer, G

    2001-06-01

    Recent suggestions concerning the age and origin of the flock of haplochromine cichlid fishes in Lake Victoria (East Africa) are considered. These accept as proven the suggestion that Lake Victoria dried out completely in the Late Pleistocene, was dry for several thousand years, and refilled ca. 12400 years ago. Apart from the fact that other geophysical evidence contradicts this claim, its biological implications, which do likewise, have never been considered by those who have accepted it. Like those of all previous authors who have seized on the presence of the haplochromine flock of perhaps more than 600 species as evidence of extremely rapid evolution since the lake allegedly refilled, the account completely overlooks the fact that any such desiccation must have eliminated not only the haplochromine cichlids but the entire biota of the lake. Nevertheless, its present fauna not only includes the haplochromines but many other endemic organisms that would not be expected, and whose presence and history demand an explanation if the lake did indeed dry out. No such explanation has been offered, nor does such seem possible. The recent interpretation of events is questioned and rejected. PMID:11375102

  7. On the age and origin of the species flock of haplochromine cichlid fishes of Lake Victoria.

    PubMed Central

    Fryer, G.

    2001-01-01

    Recent suggestions concerning the age and origin of the flock of haplochromine cichlid fishes in Lake Victoria (East Africa) are considered. These accept as proven the suggestion that Lake Victoria dried out completely in the Late Pleistocene, was dry for several thousand years, and refilled ca. 12400 years ago. Apart from the fact that other geophysical evidence contradicts this claim, its biological implications, which do likewise, have never been considered by those who have accepted it. Like those of all previous authors who have seized on the presence of the haplochromine flock of perhaps more than 600 species as evidence of extremely rapid evolution since the lake allegedly refilled, the account completely overlooks the fact that any such desiccation must have eliminated not only the haplochromine cichlids but the entire biota of the lake. Nevertheless, its present fauna not only includes the haplochromines but many other endemic organisms that would not be expected, and whose presence and history demand an explanation if the lake did indeed dry out. No such explanation has been offered, nor does such seem possible. The recent interpretation of events is questioned and rejected. PMID:11375102

  8. The Influence of Subsurface Karst Terrain on Hydrology and Hydrogeology in Southwestern Victoria, Australia

    NASA Astrophysics Data System (ADS)

    Perveen, F.; Webb, J.; Dresel, E.; Hekmeijer, P.; Zydor, H.

    2012-12-01

    A detailed study, in collaboration with Department of Primary Industries (DPI), Victoria, has been carried out in three small subcatchments of southwestern Victoria (total area 8.4 km2), which are characterised by varying degrees of influence of a subsurface karst terrain. Lithological logs and downhole geophysics (gamma and bulk conductivity - EM39) on 15 bores within the catchments were supplemented by 2D electrical resistivity vertical profiling, and showed that the middle to late Miocene Port Campbell Limestone is present at shallow depths (~5 m) in two catchments, and somewhat deeper (>70 m) in the third catchment. The limestone is overlain by early Pliocene clay-rich Dorodong Sands. The topography of the third catchment is characterised by shallow closed depressions. Detailed hydrogeological cross-sections using groundwater levels in the bores show closed depressions within the potentiometric surface, that are attributed to the presence of subsurface conduits within the highly permeable limestone, verified by the variable hydraulic conductivity values ( 0.005 - 0.545m/day) obtained from single borehole recovery tests. Stream hydrographs reveal that there is virtually no surface runoff from one subcatchment, due to leakage into a conduit in the underlying limestone. A perched water table is also found in the same area. Thus the study area shows the typical karst features of a highly heterogeneous terrain with massive connectivity between surface water and groundwater regimes, despite the fact that the limestone is overlain by the clay-rich Dorodong Sands.

  9. Was Lates Late? A Null Model for the Nile Perch Boom in Lake Victoria

    PubMed Central

    Downing, Andrea S.; Galic, Nika; Goudswaard, Kees P. C.; van Nes, Egbert H.; Scheffer, Marten; Witte, Frans; Mooij, Wolf M.

    2013-01-01

    Nile perch (Lates niloticus) suddenly invaded Lake Victoria between 1979 and 1987, 25 years after its introduction in the Ugandan side of the lake. Nile perch then replaced the native fish diversity and irreversibly altered the ecosystem and its role to lakeshore societies: it is now a prised export product that supports millions of livelihoods. The delay in the Nile perch boom led to a hunt for triggers of the sudden boom and generated several hypotheses regarding its growth at low abundances – all hypotheses having important implications for the management of Nile perch stocks. We use logistic growth as a parsimonious null model to predict when the Nile perch invasion should have been expected, given its growth rate, initial stock size and introduction year. We find the first exponential growth phase can explain the timing of the perch boom at the scale of Lake Victoria, suggesting that complex mechanisms are not necessary to explain the Nile perch invasion or its timing. However, the boom started in Kenya before Uganda, indicating perhaps that Allee effects act at smaller scales than that of the whole Lake. The Nile perch invasion of other lakes indicates that habitat differences may also have an effect on invasion success. Our results suggest there is probably no single management strategy applicable to the whole lake that would lead to both efficient and sustainable exploitation of its resources. PMID:24204684

  10. The diversity of male nuptial coloration leads to species diversity in Lake Victoria cichlids.

    PubMed

    Miyagi, Ryutaro; Terai, Yohey

    2013-01-01

    The amazing coloration shown by diverse cichlid fish not only fascinates aquarium keepers, but also receives great attention from biologists interested in speciation because of its recently-revealed role in their adaptive radiation in an African lake. We review the important role of coloration in the speciation and adaptive evolution of Lake Victoria cichlids, which have experienced adaptive radiation during a very short evolutionary period. Mature male cichlids display their colors during mate choice. The color of their skin reflects light, and the reflected light forms a color signal that is received by the visual system of females. The adaptive divergence of visual perceptions shapes and diverges colorations, to match the adapted visual perceptions. The divergence of visual perception and coloration indicates that the divergence of color signals causes reproductive isolation between species, and this process leads to speciation. Differences in color signals among coexisting species act to maintain reproductive isolation by preventing hybridization. Thus, the diversity of coloration has caused speciation and has maintained species diversity in Lake Victoria cichlids. PMID:24025243

  11. 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 Central

    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

    2015-01-01

    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. PMID:25972186

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

    PubMed Central

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

    2012-01-01

    Relapse to maladaptive eating habits during dieting is often provoked by stress and there is evidence for a role of ovarian hormones in stress responses and feeding. We studied the role of these hormones in stress-induced reinstatement of food seeking and medial prefrontal cortex (mPFC) neuronal activation in c-fos-GFP transgenic female rats, which express green fluorescent protein (GFP) in strongly activated neurons. Food-restricted ovariectomized or sham-operated c-fos-GFP rats were trained to lever-press for palatable food pellets. Subsequently, lever-pressing was extinguished and reinstatement of food seeking and mPFC neuronal activation was assessed after injections of the pharmacological stressor yohimbine (0.5–2 mg/kg) or pellet priming (1–4 non-contingent pellets). Estrous cycle effects on reinstatement were also assessed in wild-type rats. Yohimbine- and pellet-priming-induced reinstatement was associated with Fos and GFP induction in mPFC; both reinstatement and neuronal activation were minimally affected by ovarian hormones in both c-fos-GFP and wild-type rats. c-fos-GFP transgenic rats were then used to assess glutamatergic synaptic alterations within activated GFP-positive and non-activated GFP-negative mPFC neurons following yohimbine-induced reinstatement of food seeking. This reinstatement was associated with reduced AMPAR/NMDAR current ratios and increased paired-pulse facilitation in activated GFP-positive but not GFP-negative neurons. Together, while ovarian hormones do not appear to play a role in stress-induced relapse of food seeking in our rat model, this reinstatement was associated with unique synaptic alterations in strongly activated mPFC neurons. Our paper introduces the c-fos-GFP transgenic rat as a new tool to study unique synaptic changes in activated neurons during behavior. PMID:22723688

  13. 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, ...

  14. Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFP-containing intermediate filaments.

    PubMed

    Fedoroff, S; White, R; Neal, J; Subrahmanyan, L; Kalnins, V I

    1983-04-01

    When cells from mouse neopallium are grown in colony cultures for 10-12 days, small cells with many processes, resembling normal fibrous astrocytes, form on top of the astrocyte precursor cells independently of the presence of dBcAMP in the culture medium. These cells are distinctly different from the much larger, previously described reactive astrocytes which also form in colony cultures and whose maturation is greatly enhanced by the presence of dBcAMP in the culture medium. Immunofluorescence studies showed that both vimentin-containing and glial filament protein (GFP)-containing intermediate filaments (IF) are present in the small normal fibrous astrocytes as well as in the larger reactive astrocytes. The vimentin-containing IF are assembled first in astrocyte precursor cells, whereas GFP-containing IF are assembled later toward the final stages of astrocyte differentiation both in vivo and in vitro. Thus in respect to the expression of the two types of IF, astrocyte differentiation in vitro closely resembles that in vivo. Parallel studies by electron microscopy showed that the vimentin-positive but GFP-negative astrocyte precursor cells contain single IF or small groups of IF, whereas in the more differentiated normal fibrous astrocytes and reactive astrocytes which are also GFP-positive, additional IF arranged in large bundles are present. PMID:6303521

  15. In vitro cross-species infections using a caprine arthritis encephalitis lentivirus carrying the GFP marker gene.

    PubMed

    Mselli-Lakhal, Laila; Guiguen, François; Greenland, Timothy; Mornex, Jean-François; Chebloune, Yahia

    2007-07-01

    A caprine arthritis encephalitis virus (CAEV), carrying the green fluorescent protein (GFP) into the tat region was recently reported [Mselli-Lakhal, L., Guiguen, F., Greenland, T., Mornex, J.F., Chebloune, Y., 2006. Gene transfer system derived from the caprine arthritis-encephalitis lentivirus. J. Virol. Meth. 136, 177-184]. This construct, called pK2EGFPH replicated to titres up to 10(5)IU/ml on infection of caprine cells, and could be concentrated to 10(6)IU/ml by ultracentrifugation. In the present study, the pK2EGFPH construct was characterized better and used in cross-species infection studies. The pK2EGFPH virus could transduce GFP protein expression both to goat synovial membrane cells and to an immortalized goat milk epithelial cell line. The pK2EGFPH infected cells were demonstrated to express both GFP protein and CAEV viral proteins, as demonstrated by radioimmunoprecipitation and multinucleated cell formation. However GFP expression could not be maintained over passages. This vector was used to investigate cross-species infectious potential of CAEV. The bovine cell lines MDBK and GBK were found to be sensitive to infection while the human cell lines Hela, A431 and THP-1 were not. The pK2EGFPH vector should prove useful in studies of CAEV tropism both in vitro and in vivo. PMID:17386948

  16. Stable expression of a GFP-BSD fusion protein in transfected and blasticidin-selected B. bovis merozoites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Babesia bovis is a tick-borne apicomplexan parasite that causes an acute disease in cattle. This study describes stable expression of an exogenous gfp-bsd gene in B. bovis transformed parasites. Cultured B. bovis infected erythrocytes of the biologically cloned Mo7 strain were transfected by electro...

  17. 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…

  18. 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

  19. 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.

  20. 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.

  1. Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

    PubMed

    Ariotti, Nicholas; Hall, Thomas E; Rae, James; Ferguson, Charles; McMahon, Kerrie-Ann; Martel, Nick; Webb, Robyn E; Webb, Richard I; Teasdale, Rohan D; Parton, Robert G

    2015-11-23

    Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines. PMID:26585296

  2. 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.

  3. Profile of BC College Transfer Students Admitted to the University of Victoria 2003/04 to 2007/08

    ERIC Educational Resources Information Center

    Martell, Jim; Wilson, Alan

    2009-01-01

    This report examines the population of college transfer students who entered the University of Victoria (UVic) from 2003/04 to 2007/08, updating a June 2004 report. Section A provides a general profile of the 6,917 British Columbia (BC) college transfer students who first registered and commenced studies at UVic in any session between summer 2003…

  4. Appendices. Impact and Adequacy; a Report of an Evaluation of Government Post-Primary School Libraries in Victoria.

    ERIC Educational Resources Information Center

    Balson, M.; And Others

    The appendixes to the evaluation of Victoria, Australia's secondary school libraries include lists of the 33 schools visited to gather information and of the interviewees. The names of the 132 high schools with Commonwealth libraries are given with the dates of their openings and maps showing their locations. Copies of survey instruments used to…

  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. 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…

  7. "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…

  8. "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…

  9. 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…

  10. 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.

  11. The Development and Use of Individual Learning Plans for Deaf and Hard of Hearing Students in Victoria, Australia

    ERIC Educational Resources Information Center

    Brown, P. Margaret; Byrnes, Linda J.

    2014-01-01

    This study investigated the Individual Learning Plans of eighty-eight students who were deaf and hard of hearing attending facilities and schools for the deaf in Victoria Australia. The students' assessment and planning portfolios were scrutinised for evidence of formal and informal assessment used to generate goals for the Individual…

  12. "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…

  13. 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…

  14. 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

  15. 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

  16. Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock.

    PubMed

    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

    2003-01-22

    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

  17. 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…

  18. 75 FR 43579 - Exelon Generation Company, LLC; Victoria County Station, Units 1 and 2; Notice of Withdrawal of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-26

    ... receipt and availability of this application was published in the Federal Register (73 FR 56867) on September 30, 2008. On November 6, 2008, a subsequent notice was published in the Federal Register (73 FR... COMMISSION Exelon Generation Company, LLC; Victoria County Station, Units 1 and 2; Notice of Withdrawal...

  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. 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…

  1. 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

  2. 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…

  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. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

    PubMed Central

    Ellison, Thomas J.; Kedes, Dean H.

    2014-01-01

    The availability of reliable recombinant reporter virus systems has been a great boon to the study of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Unexpectedly, we found that expression of the ostensibly constitutive green fluorescent protein (GFP) marker was progressively lost during unselected passage in primary rat mesenchymal precursor cells (MM), despite efficient maintenance of latent viral gene expression and episomal partitioning. This repression of EF1-α promoter-driven GFP expression appeared to be passage-dependent, however, since functionally immortalized MM cells derived from long serial passage retained stable expression of GFP following rKSHV.219 infection. Chromatin analysis of cultures that we had infected in parallel demonstrated an increase in repressive H3K27 tri-methylation across the viral episome with the exception of the LANA control region in MM cells infected at early rather than late passage post-isolation. The silencing of GFP expression in the MM cells was reversible in a dose-dependent fashion by the histone deacetylase inhibitor valproic acid, further implicating cellular silencing on incoming viral genomes, and underscoring potential differences in viral gene regulation between primary and functionally immortalized cells. Furthermore, using multispectral imaging flow cytometry, we also determined that the extent of GFP expression per cell among those that were positive did not correlate with the number of LANA dots per nucleus nor the extent of overall LANA expression per cell. This suggests a more complex mode of local gene regulation, rather than one that simply reflects the relative intracellular viral copy number. In sum, we have demonstrated the significant potential for false-negative data when using a constitutive marker gene as a sole means of evaluating herpesviral infection, especially in primary cells. PMID:25402328

  10. View of portion of Murray River Basin of State of Victoria, Australia

    NASA Technical Reports Server (NTRS)

    1974-01-01

    A near vertical view of a portion of the Murray River Basin area in the northwest corner of the State of Victoria, Australia (35.5S, 142,0E), as seen from the Skylab space station in Earth orbit. Dry Lake Tyrrell appears as pink and white. The Murray River is in the lower right corner of the photograph. A mass of clouds (white) covers a large portion of the left side of the picture. Rectangular fields of varying sizes indicate an area of major agriculture. A difference in soil color (from pink near the river to buff extending outward) is evident. Large uncultivated areas (dark) are either areas of recent burning or areas unsuitable for growing crops.

  11. Recruiting and retaining competent clinical nurses. The Clinical Promotion Project Victoria General Hospital, Halifax, Nova Scotia.

    PubMed

    Grantham, M; Ross, S E; MacKay, R; Banfield, V; Brown, J; Beanlands, H

    1989-06-01

    A career advancement program for nurses has been developed and implemented at the Victoria General Hospital, Halifax, as part of a manpower planning strategy for recruitment and retention of clinical nurses. A competency based performance appraisal system was developed and implemented as part of the program. This system identifies four levels of clinical expertise. Progression through each level indicates that a nurse has achieved a certain level of skill, knowledge and abilities. Demonstrated competence in one level is a prerequisite to promotion to the next level of practice. Implementation of this system provides clinical career opportunities for nurses in direct patient care. This article will provide the nurse administrator with an insight into the development and implementation of a clinical career advancement program. Future articles will described the research evaluation of the Clinical Performance Appraisal System and clinical nurses' reactions to the new system. PMID:2486680

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences.

    PubMed

    Meyer, A; Kocher, T D; Basasibwaki, P; Wilson, A C

    1990-10-11

    Lake Victoria, together with its satellite lakes, harbours roughly 200 endemic forms of cichlid fishes that are classified as 'haplochromines' and yet the lake system is less than a million years old. This 'flock' has attracted attention because of the possibility that it evolved within the lake from one ancestral species and that biologists are thus presented with a case of explosive evolution. Within the past decade, however, morphology has increasingly emphasized the view that the flock may be polyphyletic. We sequenced up to 803 base pairs of mitochondrial DNA from 14 representative Victorian species and 23 additional African species. The flock seems to be monophyletic, and is more akin to that from Lake Malawi than to species from Lake Tanganyika; in addition, it contains less genetic variation than does the human species, and there is virtually no sharing of mitochondrial DNA types among species. These results confirm that the founding event was recent. PMID:2215680

  17. Sir James Mouat, VC KCB FRCS (1815-1899): winner of the first medical Victoria Cross.

    PubMed

    Lawrenson, Ross

    2004-11-01

    Sir James Mouat was the first of 36 doctors to win the Victoria Cross. Born in Kent in 1815, he was educated at University College London before joining the army. After service in India and Ireland he joined the 6th Dragoon Guards as regimental surgeon and served with them throughout the Crimean War. He won his VC at the Battle of Balaclava. After the Crimean War he was appointed principal medical officer to the British troops in the New Zealand Wars. After serving in New Zealand he returned to England and became Inspector General of Hospitals. Surgeon General Mouat retired on 28 April 1876. He was appointed an honorary surgeon to the Queen in 1888, and a Knight Commander of the Order of the Bath in 1894. He died of a stroke on 4 January 1899 at the age of 83. PMID:15486614

  18. 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

  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. 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

  1. 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. PMID:25875201

  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. 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.

  4. 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.

  5. 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

  6. 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

  7. Basin-scale control on the phytoplankton biomass in Lake Victoria, Africa.

    PubMed

    Cózar, Andrés; Bruno, Miguel; Bergamino, Nadia; Ubeda, Bárbara; Bracchini, Luca; Dattilo, Arduino M; Loiselle, Steven A

    2012-01-01

    The relative bio-optical variability within Lake Victoria was analyzed through the spatio-temporal decomposition of a 1997-2004 dataset of remotely-sensed reflectance ratios in the visible spectral range. Results show a regular seasonal pattern with a phase shift (around 2 months) between the south and north parts of the lake. Interannual trends suggested a teleconnection between the lake dynamics and El-Niño phenomena. Both seasonal and interannual patterns were associated to conditions of light limitation for phytoplankton growth and basin-scale hydrodynamics on phytoplankton access to light. Phytoplankton blooms developed during the periods of lake surface warming and water column stability. The temporal shift apparent in the bio-optical seasonal cycles was related to the differential cooling of the lake surface by southeastern monsoon winds. North-south differences in the exposure to trade winds are supported by the orography of the Eastern Great Rift Valley. The result is that surface layer warming begins in the northern part of the lake while the formation of cool and dense water continues in the southern part. The resulting buoyancy field is sufficient to induce a lake-wide convective circulation and the tilting of the isotherms along the north-south axis. Once surface warming spreads over the whole lake, the phytoplankton bloom dynamics are subjected to the internal seiche derived from the relaxation of thermocline tilting. In 1997-98, El-Niño phenomenon weakened the monsoon wind flow which led to an increase in water column stability and a higher phytoplankton optical signal throughout the lake. This suggests that phytoplankton response to expected climate scenarios will be opposite to that proposed for nutrient-limited great lakes. The present analysis of remotely-sensed bio-optical properties in combination with environmental data provides a novel basin-scale framework for research and management strategies in Lake Victoria. PMID:22253837

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. Perceptions of Prescribed Burning in a Local Forest Community in Victoria, Australia

    NASA Astrophysics Data System (ADS)

    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.

  13. 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. PMID:17001507

  14. 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. PMID:26537030

  15. Two new species of zooplanktivorous haplochromine cichlids from Lake Victoria, Tanzania

    PubMed Central

    de Zeeuw, Marnix P.; Westbroek, Irene; van Oijen, Martien J.P.; Witte, Frans

    2013-01-01

    Abstract Two new species of zooplanktivorous haplochromine cichlids from Lake Victoria, Tanzania, are described and illustrated. These species closely resemble each other. Their affinities to other zooplanktivorous haplochromines from Lake Victoria are discussed. Haplochromis argens sp. n., which featured under nicknames (mainly Haplochromis “argens”) in more than 50 papers, was caught both in the Mwanza Gulf and the Emin Pasha Gulf, whereas Haplochromis goldschmidti sp. n. was only found in the Emin Pasha Gulf. Of the latter species only males are available, but it seems unlikely that it represents a case of male colour polymorphism as several presumably unrelated characters differ in sympatry between the two species, suggesting that there is no gene flow. Statistical analysis revealed that the overall difference between the two species is greater than that between the populations from the two locations. Body depth of the two species in sympatry in the Emin Pasha Gulf was more similar than that of Haplochromis goldschmidti sp. n. and the allopatric population of Haplochromis argens sp. n. from the Mwanza Gulf,which mayindicate an overall environmental effect. However, several measurements related to the width of snout and mouth differed more between the populations of the two species in sympatry than between the allopatric populations. In contrast to a group of zooplanktivorous species that recovered successfully after environmental changes in the lake, Haplochromis argens sp. n. is among a group that became extremely rare and probably is in danger of extinction; the conservation status of Haplochromis goldschmidti sp. n. is currently unknown. PMID:23717179

  16. 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.

  17. Elevated East Antarctic outlet glaciers during warmer-than-present climates in southern Victoria Land

    NASA Astrophysics Data System (ADS)

    Swanger, K.; Marchant, D. R.; Schaefer, J. M.; Winckler, G.; Head, J. W.

    2010-12-01

    We document Plio-Pleistocene changes in the level of Taylor Glacier, an outlet glacier in southern Victoria Land that drains Taylor Dome on the periphery of the East Antarctic Ice Sheet (EAIS). Chronologic control comes from 3He cosmogenic-nuclide analyses of 27 boulders sampled from drifts and moraines in Kennar Valley, a small hanging valley that opens onto a peripheral lobe of Taylor Glacier in the Quartermain Mountains. Assuming a constant boulder-erosion rate of 10 cm Myr-1, our preferred age model spans the last 3.1 Myr and calls for stepped ice recession from a local highstand ~200 m above the present Taylor Glacier at the mouth of Kennar Valley. The texture and sedimentology of all mapped moraines and drifts indicate deposition from cold-based ice, analogous with the situation for the modern Taylor Glacier. Correlation of our moraine record with published reports for fluctuations of Taylor Glacier elsewhere in the Quartermain Mountains, and with a dated moraine record for Ferrar Glacier (a second outlet for Taylor Dome) in the nearby Wilkness Mountains, reveals consistent ice-surface changes, highlighting minor, but widespread ice recession in southern Victoria Land since the mid to late Pliocene. The combined records show an atypical relationship with average global temperatures, with higher-than-present ice levels occurring during globally warm periods, including the Pliocene climatic optimum (˜3.1 Ma), Marine Oxygen Isotope Stage (MIS) 31 (~1.1 Ma), and the penultimate interglacial, MIS 5.5 (~120 ka). The combined records also suggest that the rate of ice-surface lowering for both outlet glaciers accelerated after the Mid-Pleistocene transition at ~0.9 Myr.

  18. 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

  19. 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. PMID:25635627

  20. Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms.

    PubMed

    Aviles-Espinosa, Rodrigo; Filippidis, George; Hamilton, Craig; Malcolm, Graeme; Weingarten, Kurt J; Südmeyer, Thomas; Barbarin, Yohan; Keller, Ursula; Santos, Susana I C O; Artigas, David; Loza-Alvarez, Pablo

    2011-01-01

    We present a portable ultrafast Semiconductor Disk Laser (SDL) (or vertical extended cavity surface emitting laser-VECSELs), to be used for nonlinear microscopy. The SDL is modelocked using a quantum-dot semiconductor saturable absorber mirror (SESAM), delivering an average output power of 287 mW, with 1.5 ps pulses at 500 MHz and a central wavelength of 965 nm. Specifically, despite the fact of having long pulses and high repetition rates, we demonstrate the potential of this laser for Two-Photon Excited Fluorescence (TPEF) imaging of in vivo Caenorhabditis elegans (C. elegans) expressing Green Fluorescent Protein (GFP) in a set of neuronal processes and cell bodies. Efficient TPEF imaging is achieved due to the fact that this wavelength matches the peak of the two-photon action cross section of this widely used fluorescent marker. The SDL extended versatility is shown by presenting Second Harmonic Generation images of pharynx, uterus, body wall muscles and its potential to be used to excite other different commercial dyes. Importantly this non-expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices. PMID:21483599

  1. 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. PMID:21654030

  2. Use of an alpha-smooth muscle actin (SMAA) GFP reporter to identify an osteoprogenitor population

    PubMed Central

    Kalajzic, Zana; Li, Haitao; Wang, Li-Ping; Jiang, Xi; Lamothe, Katie; Adams, Douglas J.; Aguila, Hector L.; Rowe, David W.; Kalajzic, Ivo

    2008-01-01

    Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of α-smooth muscle actin (α-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of SMAA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of α-SMA+ cells towards osteoprogenitor lineage. Utilizing α-SMAGFP transgene, α-SMAGFP+ positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the α-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, α-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, α-SMA+/CD45−/Sca1+ progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts. PMID:18571490

  3. 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

  4. Post-Functionalized Polymer Brushes for Bio-Separation: Tuning GFP Adsorption via Functional Group Display

    NASA Astrophysics Data System (ADS)

    Diamanti, Steve; Arifuzzaman, Shafi; Genzer, Jan; Naik, Rajesh; Vaia, Richard

    2007-03-01

    An inexpensive and robust biosensor platform that can be tuned to separate and/or detect complex mixtures of biomolecules while minimizing reagents would be of great use for military, homeland security, and medical diagnostic applications. Gradient surfaces of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes have been previously shown to spatially localize biomolecule binding, while minimizing non-specific adsorption of the same biomolecule on other regions of the gradient specimen. In order to further improve the specificity and to provide latent functionality for detection of the binding events, post-polymerization modification of PHEMA with various functional groups has been investigated. Using standard succinimide-based coupling, hydroxyl pendants of PHEMA brushes were conjugated to oligo-peptides, alkanes and oligo(ethylene glycol) (OEG) through an alpha-terminus primary amine. Ellipsometry, contact angle, XPS and ER-FTIR spectroscopy indicated that coupling occurred with efficiencies ranging from 10-40%. Post-functionalization of PHEMA with OEG and hexadecane allows manipulation of the hydrophilicity of the surface and thus tuning of Green Fluorescent Protein (GFP) binding.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum

    PubMed Central

    Davis, Margaret I.; Puhl, Henry L.

    2011-01-01

    Transgenic mice expressing eGFP under population specific promoters are widely used in neuroscience to identify specific subsets of neurons in situ and as sensors of neuronal activity in vivo. Mice expressing eGFP from a bacterial artificial chromosome under the Nr4a1 promoter have high expression within the basal ganglia, particularly within the striosome compartments and striatal-like regions of the extended amygdala (bed nucleus of the stria terminalis, striatal fundus, central amygdaloid nucleus and intercalated cells). Grossly, eGFP expression is inverse to the matrix marker calbindin 28K and overlaps with mu-opioid receptor immunoreactivity in the striatum. This pattern of expression is similar to Drd1, but not Drd2, dopamine receptor driven eGFP expression in structures targeted by medium spiny neuron afferents. Striosomal expression is strong developmentally where Nr4a1-eGFP expression overlaps with Drd1, TrkB, tyrosine hydroxylase and phospho-ERK, but not phospho-CREB, immunoreactivity in “dopamine islands”. Exposure of adolescent mice to methylphenidate resulted in an increase in eGFP in both compartments in the dorsolateral striatum but eGFP expression remained brighter in the striosomes. To address the role of activity in Nr4a1-eGFP expression, primary striatal cultures were prepared from neonatal mice and treated with forskolin, BDNF, SKF-83822 or high extracellular potassium and eGFP was measured fluorometrically in lysates. eGFP was induced in both neurons and contaminating glia in response to forskolin but SKF-83822, brain derived neurotrophic factor and depolarization increased eGFP in neuronal-like cells selectively. High levels of eGFP were primarily associated with Drd1+ neurons in vitro detected by immunofluorescence; however ∼15% of the brightly expressing cells contained punctate met-enkephalin immunoreactivity. The Nr4a1-GFP mouse strain will be a useful model for examining the connectivity, physiology, activity and development of the

  10. Solvent dependence of two-photon absorption spectra of the enhanced green fluorescent protein (eGFP) chromophore

    NASA Astrophysics Data System (ADS)

    Hosoi, Haruko; Tayama, Ryo; Takeuchi, Satoshi; Tahara, Tahei

    2015-06-01

    Two-photon absorption spectra of 4‧-hydroxybenzylidene-2,3-dimethylimidazolinone, a model chromophore of enhanced green fluorescent protein (eGFP), were measured in various solvents. The two-photon absorption band of its anionic form is markedly blue-shifted from the corresponding one-photon absorption band in all solvents. Moreover, the magnitude of the blue shift varies largely depending on the solvent, which does not accord with the assignment of the two-photon absorption band to the transitions to the vibrationally excited S1 state. Our finding is readily rationalized by considering overlapping contributions of the S1 ← S0 and S2 ← S0 transitions, suggesting the involvement of the S2 state also in two-photon fluorescence of eGFP.

  11. [Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

    PubMed

    Shisha, E N; Korkhovoĭ, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

    2013-01-01

    The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind. PMID:23745358

  12. 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(1) 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(2,3). 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. PMID:26891031

  13. Two-photon targeted recording of GFP-expressing neurons for light responses and live cell imaging in the mouse retina

    PubMed Central

    Wei, Wei; Elstrott, Justin; Feller, Marla B.

    2015-01-01

    Cell type-specific GFP expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches the photoreceptors. To circumvent this problem, we used two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain the light responses as well as the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 minutes, and the retina preparation remains light sensitive and suitable for recording for at least 8 hours. This protocol can also be applied to study retinal neurons labeled with other two-photon-excitable fluorophores. PMID:20595962

  14. Plasmid Stability in Dried Cells of the Desert Cyanobacterium Chroococcidiopsis and its Potential for GFP Imaging of Survivors on Earth and in Space

    NASA Astrophysics Data System (ADS)

    Billi, Daniela

    2012-06-01

    Two GFP-based plasmids, namely pTTQ18-GFP-pDU1mini and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1mini-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecASyn distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecASyn structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

  15. In Vivo Imaging and Differential Localization of Lipid-Modified GFP-Variant Fusions in Embryonic Stem Cells and Mice

    PubMed Central

    Rhee, Jerry M.; Pirity, Melinda K.; Lackan, Chantal S.; Long, Jonathan Z.; Kondoh, Gen; Takeda, Junji; Hadjantonakis, Anna-Katerina

    2010-01-01

    Summary The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts. PMID:16604528

  16. Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology

    PubMed Central

    Al-Homsi, Lamis; Al-Okla, Souad; Abbady, Abdul Q.

    2015-01-01

    Mutacin I, a bacteriocin produced by streptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. This work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology. The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein (sfGFP), resulting in a high yield expression of soluble sfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition site and separated from the carrier sfGFP by nickel affinity and gel filtration chromatography. An additional advantage of this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin. Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide. PMID:26668664

  17. Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein.

    PubMed

    Bleve, Gianluca; Zacheo, Giuseppe; Cappello, Maria Stella; Dellaglio, Franco; Grieco, Francesco

    2005-08-15

    GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1-GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1-GFP or GFP-Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters. PMID:15813700

  18. Diversity of Retinal Ganglion Cells Identified by Transient GFP Transfection in Organotypic Tissue Culture of Adult Marmoset Monkey Retina

    PubMed Central

    Moritoh, Satoru; Komatsu, Yusuke; Yamamori, Tetsuo; Koizumi, Amane

    2013-01-01

    The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs) besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC) have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus) monkey as a “super-model” of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells), and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]). By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina. PMID:23336011

  19. Effects of in situ cobalt ion addition on the activity of a gfp-oph fusion protein: the fermentation kinetics.

    PubMed

    Wu, C F; Valdes, J J; Bentley, W E

    2001-01-01

    The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor. PMID:11485418

  20. Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli.

    PubMed

    Jaspers, M C; Meier, C; Zehnder, A J; Harms, H; van der Meer, J R

    2001-08-01

    Diffusion of octane from oily droplets in different microscale settings was measured using Escherichia coli expressing the stable green fluorescent protein (GFP) from the alkB promoter of Pseudomonas oleovorans. GFP fluorescence intensities were determined quantitatively at the single-cell level after 1.0 or 2.5 h incubation and compared with different calibration series using known concentrations of octane. By immobilizing the E. coli sensor cells on the bottom glass plate of a microscope flow chamber, it was possible to monitor the diffusion process for octane in aqueous solution as a function of time and distance from non-aqueous phase droplets of octane alone or oily octane mixtures. When a gas phase was included in the flow chambers, octane transport could be demonstrated from the oily mixtures to the cells through both gas and liquid phase. Assays of non-immobilized sensor cells in microdroplets in the presence or absence of soil particles incubated with octane through the vapour phase revealed a slight reduction in the total amount of induced E. coli cells in the presence of soil. Our results indicate the power of using GFP-marked single-cell biosensors in determining microscale bioavailability of organic pollutants. PMID:11578312

  1. A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

    PubMed Central

    Sheridan, Douglas L; Hughes, Thomas E

    2004-01-01

    Background There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation. Results We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R). Conclusions These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation. PMID:15317651

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

    PubMed

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

    2016-01-01

    Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression. PMID:27547572

  3. Characterization of directly transformed weedy Brassica rapa and introgressed B. rapa with Bt cry1Ac and gfp genes.

    PubMed

    Moon, Hong S; Halfhill, Matthew D; Good, Laura L; Raymer, Paul L; Neal Stewart, C

    2007-07-01

    Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa x B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds. PMID:17333014

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

    PubMed Central

    Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady

    2016-01-01

    Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression. PMID:27547572

  5. ROSSMAP; Regional Seismic Stratigraphic Correlations in the Victoria Land Basin and the Timing of Rifting Episodes

    NASA Astrophysics Data System (ADS)

    Davy, B. W.; Henrys, S. A.; Wilson, T. J.; Fielding, C. R.; Levy, R. H.; Andrill Mis-Science Team

    2010-12-01

    ROSSMAP aims to produce a new series of digital seismic stratigraphic and structural maps for the Ross Sea region, and develop reconstructions of past sedimentary volumes and paleo-bathymetry that will be used in numerical models to help identify tectonic and climate feedbacks. In particular, ROSSMAP will define targets for future drilling initiatives in the Ross Sea region, and provide a legacy of web-based electronic databases that will be available to other researcher. Interpretation in the Victoria Land Basin (VLB) has focused on establishing links to new and existing drill holes. Several regional seismic reflectors were mapped throughout the southern portion of the Victoria Land Basin (VLB) and are tied to the ANDRILL McMurdo Sound drill cores. The age of one prominent reflector (Ri - red, correlative with RSU-2) is 4.7-4.3 Ma based on a tie to AND-1B and extensively mapped in the VLB. Mapping, together with quantitative biostratigraphic correlation techniques, indicates that the red reflector is associated with relative sea-level fall resulting from ice-sheet growth and/or local tectonic uplift. Correlative sediments preserved in AND-1B indicate regional climatic warmth, which suggests that the red reflector is more likely related to a tectonic event. Furthermore, the onset of sediment accumulation at CIROS-2 post-dates 4.5 Ma, suggesting that local subsidence and creation of accommodation space began at this time. Two other stratigraphic horizons (Rh and Rg) have been extensively mapped where seismic data image above the seafloor multiple. Reflector Rh (dark green and correlative with RSU-4a marks a basin-wide unconformity. Near the western margin of the Victoria Land Basin, this horizon is a marked angular discordance with angularity increasing westward. In AND-1B Rh is correlated with the base of a ~180-m-thick interval of late Miocene-early Pliocene, pyrite-cemented, high-velocity volcanic sandstone and mudstone. Volcanic rocks in a tongue extending from

  6. Rock Glaciers in the Upper Basin of Bermejo River, Santa Victoria, Argentina

    NASA Astrophysics Data System (ADS)

    Ahumada, A. L.; Íbáñez Palacios, G. P.; Toledo, M. A.; Páez, S. V.

    2013-05-01

    The Bermejo River is an international basin in the Tropical Andes, Santa Victoria range. The Santa Victoria range extend in N-S sense (22°08' and 23°00' South latitude between 65° and 65°30' West longitude). It maxim height is about 5.055 m s.n.m. and it average height is 3000 m s.n.m. There have been localize intact rock glaciers (active and inactive) and relict. The rock glaciers are frozen water reservoir and regulate the water regime in the high mountain over the world. The global warming effects have been express in this region with average annual air temperatures in increase: The series of temperatures of La Quiaca weather station, years 1911-2010, shows an increase about 1° C (from 9,14° C to 10,08° C) since 70' decade to actuality. This study aims to contribute to the survey of intact rock glaciers and the suite of accompanying landforms. The geographic identification and location of rock glaciers have made through the interpretation of aerial photographs and LandSat 7 and Aster Images in comparison with Google Earth using GIS and visual interpretation technics. This information has controlled with fieldwork in several campaigns. The rock glaciers are classified (in base of it activity) in actives, inactives and relict. The first and the second class were grouped in a category called intact rock glaciers for simplify it identification. The unified categories are water-storing. In the fieldwork have determinated the following aspects: 1- Active processes determination (needle ice, lenticular ice formation, etc.) and its sedimentary products: sucrose structure, inverted gradation, extrusion, open work gravels. 2- Location and geomorphologic measurement of rock glaciers and periglacial forms. There are identified 318 intact rock glaciers in high basin of Bermejo river, Sub-basins Condado river, Los Toldos-Lipeo river and Iruya river, Santa Victoria range. The actives rock glaciers, indicators of discontinuous permafrost, are installed from 4300 m s

  7. 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

  8. Biologically relevant physical measurements in the ice-free valleys of southern Victoria Land: soil temperature profiles and ultraviolet radiation

    NASA Technical Reports Server (NTRS)

    Nienow, J. A.; Meyer, M. A.; Friedmann, E. I. (Principal Investigator)

    1986-01-01

    As part of the ongoing comprehensive study of the cryptoendolithic microbial community in the ice-free valleys of southern Victoria Land, thermal properties of the soil and the ultraviolet radiation regime were measured. Although soil temperature profiles have been measured in the ice-free valleys (e.g., Cameron et al. 1970; Cameron 1972), these are the first such data from higher elevations. This is apparently the first time the ultraviolet radiation regime has been measured in the Antarctic.

  9. Association of a New Wolbachia Strain with, and Its Effects on, Leptopilina victoriae, a Virulent Wasp Parasitic to Drosophila spp.

    PubMed Central

    Gueguen, Gwenaelle; Onemola, Bodunde

    2012-01-01

    Wolbachia bacteria are ubiquitous intracellular bacteria of arthropods. Often considered reproductive parasites, they can benefit certain host species. We describe a new Wolbachia strain from Leptopilina victoriae, a Drosophila wasp. The strain is closely related to Wolbachia from Culex sp. Located to the posterior poles of oocytes, it manipulates its host's reproduction by inducing a male development type of cytoplasmic incompatibility. We also report its diverse effects on the wasp's life history traits. PMID:22685158

  10. 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. PMID:25395445

  11. Legacy and currently used pesticides in the atmospheric environment of Lake Victoria, East Africa.

    PubMed

    Arinaitwe, Kenneth; Kiremire, Bernard T; Muir, Derek C G; Fellin, Phil; Li, Henrik; Teixeira, Camilla; Mubiru, Drake N

    2016-02-01

    The Lake Victoria watershed has extensive agricultural activity with a long history of pesticide use but there is limited information on historical use or on environmental levels. To address this data gap, high volume air samples were collected from two sites close to the northern shore of Lake Victoria; Kakira (KAK) and Entebbe (EBB). The samples, to be analyzed for pesticides, were collected over various periods between 1999 and 2004 inclusive (KAK 1999-2000, KAK 2003-2004, EBB 2003 and EBB 2004 sample sets) and from 2008 to 2010 inclusive (EBB 2008, EBB 2009 and EBB 2010 sample sets). The latter sample sets (which also included precipitation samples) were also analyzed for currently used pesticides (CUPs) including chlorpyrifos, chlorthalonil, metribuzin, trifluralin, malathion and dacthal. Chlorpyrifos was the predominant CUP in air samples with average concentrations of 93.5, 26.1 and 3.54 ng m(-3) for the EBB 2008, 2009, 2010 sample sets, respectively. Average concentrations of total endosulfan (ΣEndo), total DDT related compounds (ΣDDTs) and hexachlorocyclohexanes (ΣHCHs) ranged from 12.3-282, 22.8-130 and 3.72-81.8 pg m(-3), respectively, for all the sample sets. Atmospheric prevalence of residues of persistent organic pollutants (POPs) increased with fresh emissions of endosulfan, DDT and lindane. Hexachlorobenzene (HCB), pentachlorobenzene (PeCB) and dieldrin were also detected in air samples. Transformation products, pentachloroanisole, 3,4,5-trichloroveratrole and 3,4,5,6-tetrachloroveratrole, were also detected. The five most prevalent compounds in the precipitation samples were in the order chlorpyrifos>chlorothalonil>ΣEndo>ΣDDTs>ΣHCHs with average fluxes of 1123, 396, 130, 41.7 and 41.3 ng m(-2)sample(-1), respectively. PeCB exceeded HCB in precipitation samples. The reverse was true for air samples. Backward air trajectories suggested transboundary and local emission sources of the analytes. The results underscore the need for a concerted

  12. New constraints on fluid sources in orogenic gold deposits, Victoria, Australia

    NASA Astrophysics Data System (ADS)

    Fu, Bin; Kendrick, Mark A.; Fairmaid, Alison M.; Phillips, David; Wilson, Christopher J. L.; Mernagh, Terrence P.

    2012-03-01

    Fluid inclusion microthermometry, Raman spectroscopy and noble gas plus halogen geochemistry, complemented by published stable isotope data, have been used to assess the origin of gold-rich fluids in the Lachlan Fold Belt of central Victoria, south-eastern Australia. Victorian gold deposits vary from large turbidite-hosted `orogenic' lode and disseminated-stockwork gold-only deposits, formed close to the metamorphic peak, to smaller polymetallic gold deposits, temporally associated with later post-orogenic granite intrusions. Despite the differences in relative timing, metal association and the size of these deposits, fluid inclusion microthermometry indicates that all deposits are genetically associated with similar low-salinity aqueous, CO2-bearing fluids. The majority of these fluid inclusions also have similar 40Ar/36Ar values of less than 1500 and 36Ar concentrations of 2.6-58 ppb (by mass) that are equal to or much greater than air-saturation levels (1.3-2.7 ppb). Limited amounts of nitrogen-rich fluids are present at a local scale and have the highest measured 40Ar/36Ar values of up to 5,700, suggesting an external or distinct source compared to the aqueous fluids. The predominance of low-salinity aqueous-carbonic fluids with low 40Ar/36Ar values, in both `orogenic' and `intrusion-related' gold deposits, is attributed to fluid production from common basement volcano-sedimentary sequences and fluid interaction with sedimentary cover rocks (turbidites). Aqueous fluid inclusions in the Stawell-Magdala deposit of western Victoria (including those associated with N2) preserve mantle-like Br/Cl and I/Cl values. In contrast, fluid inclusions in deposits in the eastern structural zones, which contain more abundant shales, have elevated molar I/Cl ratios with maximum values of 5,170 × 10-6 in the Melbourne Zone. Br/I ratios in this zone range from 0.5 to 3.0 that are characteristic of fluid interaction with organic-rich sediments. The maximum I/Cl and characteristic

  13. Elevated East Antarctic outlet glaciers during warmer-than-present climates in southern Victoria Land

    NASA Astrophysics Data System (ADS)

    Swanger, Kate M.; Marchant, David R.; Schaefer, Joerg M.; Winckler, Gisela; Head, James W.

    2011-10-01

    We document Plio-Pleistocene changes in the level of Taylor Glacier, an outlet glacier in southern Victoria Land that drains Taylor Dome on the periphery of the East Antarctic Ice Sheet (EAIS). Chronologic control comes from 3He cosmogenic-nuclide analyses of 27 boulders sampled from drifts and moraines in Kennar Valley, a small hanging valley that opens onto a peripheral lobe of Taylor Glacier in the Quartermain Mountains. Assuming a constant boulder-erosion rate of 10 cm Myr -1, our preferred age model spans the last 3.1 Myr and calls for stepped ice recession from a local highstand ~ 200 m above the present base of Taylor Glacier at the mouth of Kennar Valley. The texture and sedimentology of all mapped moraines and drifts indicate deposition from cold-based ice, analogous with the modern Taylor Glacier at the mouth of Kennar Valley. The Kennar Valley glacial record shows an uncharacteristic relationship with average global temperatures, exhibiting higher-than-present ice levels during globally warm periods, including the Pliocene climatic optimum (~ 3.1 Ma) and Marine Oxygen Isotope Stage (MIS) 31 (~ 1.07 Ma). The Kennar Valley record also suggests that the rate of ice-surface lowering accelerated after the mid-Pleistocene transition at ~ 0.9 Ma. Correlation of our moraine record with published reports for fluctuations of Taylor Glacier elsewhere in the Quartermain Mountains, and with a dated moraine record from Ferrar Glacier (a second outlet for Taylor Dome), reveals similar ice-surface changes, highlighting minor, but widespread ice recession in southern Victoria Land since the mid- to late-Pliocene. Our record for minimal variability in the East Antarctic Ice Sheet contrasts with recent data from nearby marine cores that call for dynamic fluctuations in the volume of grounded ice in the Ross Embayment, and significant reduction of the West Antarctic Ice Sheet (WAIS) during warmer-than-present intervals. Taken together, these records from the Ross Embayment

  14. Impact of immigrant pastoral herds to fringing wetlands of lake Victoria in Magu district Mwanza region, Tanzania

    NASA Astrophysics Data System (ADS)

    Hongo, H.; Masikini, M.

    The assessment of impacts of pastoral herds to the fringing wetlands of Lake Victoria in Magu district in Mwanza region was carried out in 1999/2000. Lamadi village located along Speke Gulf of Lake Victoria was chosen. The main farming systems in the area are agriculture, agro-pastoralism, and pastoralism. The wetlands are heavily used for livestock grazing during the dry season. Since 1990s the area has been experiencing a high influx of immigrant pastoral herds from drought prone districts. The increasing livestock numbers have led into serious degradation of wetlands. The type of damages includes: soil erosion, loss of vegetation cover and deforestation. This lead to pollution of Lake Victoria along the Speke gulf in particular as the wetlands was buffering a lot of pollutants from the catchments. The range condition at Lamadi was rated fair. The carrying capacity of rangelands was estimated at 3.57-6.75 ha/LU and the wetlands were seriously degraded causing heavy soil erosion and environmental pollution during rainy season. It was recommended to raise people’s awareness on conservation of environment and mobilise communities to take responsibility on management of the environmental resources.

  15. Improved expression of recombinant GFP using a replicating vector based on Beet curly top virus in leaf-disks and infiltrated Nicotiana benthamiana leaves.

    PubMed

    Kim, Kyung Il; Sunter, Garry; Bisaro, David M; Chung, In Sik

    2007-05-01

    Recombinant green fluorescent protein (GFP) with a molecular mass of 29 kDa was transiently expressed in Agrobacterium-inoculated leaf-disks prepared from Nicotiana benthamiana plants. Expression of GFP from the Cauliflower mosaic virus (CaMV) 35 S promoter within a replicating vector based on the geminivirus Beet curly top virus (BCTV) was more than 3 times higher than from a control, non-replicating vector. Use of the Cassava vein mosaic virus (CsVMV) promoter in the BCTV replicating vector increased the expression of recombinant GFP 320% at the transcript level, compared to use of the control CaMV 35 S promoter. Expression of recombinant GFP from Agrobacterium-inoculated leaf-disks of N. benthamiana was further enhanced up to 240% in the presence of post-transcriptional gene silencing suppressor p19. PMID:17294255

  16. Weathered stony meteorites from Victoria Land, Antarctica, as possible guides to rock weathering on Mars

    NASA Technical Reports Server (NTRS)

    Gooding, J. L.

    1984-01-01

    Parallel studies of Martian geomorphic features and their analogs on Earth continue to be fruitful in deciphering the geologic history of Mars. In the context of rock weathering, the Earth-analog approach is admirably served by the study of meteorites recovered from ice sheets in Antarctica. The weathering environment of Victoria Land possesses several Mars-like attributes. Four of the five Antarctic meteorites being studied contain rust and EETA79005 further possesses a conspicuous, dark, weathering rind on one side. Secondary minerals (rust and salts) occur both on the surfaces and interiors of some of the samples and textural evidence indicates that such secondary mineralization contributed to physical weathering (by salt riving) of the rocks. Several different rust morphologies occur and emphasis is being placed on identifying the phase compositions of the various rust occurrances. A thorough understanding of terrestrial weathering features of the meteorites is a prerequisite for identifying possible Martian weathering features (if such features exist) that might be postulated to occur in some meteorites.

  17. Fatal injury in tree felling and related activities, Victoria, Australia 1992-2007.

    PubMed

    Brodie, Lisa Ruth; Ibrahim, Joseph Elias

    2010-02-01

    This study aims to examine fatalities resulting from tree felling and related activities in Victoria, Australia, involving work and do-it-yourself (DIY) activities, 1992-2007. Case identification was undertaken using coronial databases. A manual review of coroners' findings of closed cases was performed. Data collected and examined comprised demographics, occupation, incident location, activity, equipment used, injury mechanism and cause of death. Sixty-two cases were identified during the 16-year period; over 50% comprised DIY deaths (n=33). All but one victim was male. The median age for paid workers was less than for DIY (43 years vs 59 years). One-third of work activities were performed by persons outside professional tree-felling industries. While commercial forestry and logging industries experience a high fatality rate in Australia, non-professionals are also vulnerable to tree-felling injury. Study findings identified in excess of 70% of fatal incidents involved persons not employed within a relevant industry. Prevention efforts must focus on safety beyond workplaces and certain industries alone to reduce these deaths. PMID:20179037

  18. Corporatization as a means of improving water quality: the experience in Victoria, Australia.

    PubMed

    Martin, Narelle

    Factors including fragmentation, a lack of direction, poor accountability, poor water quality, and a sizable state government subsidy contributed to the rural water industry in Victoria, Australia, in 1993. In 1993 the state government set out parameters for reform to change the size, structure, performance, and culture of the water industry. The path taken was not privatization, but corporatization. Tools used included amalgamation of organizations; separating water provisions from local government; changing the composition and reporting mechanisms of the boards; establishing clear benchmarks and performance criteria; making information publicly available; and providing a commercial orientation. The outcomes of the reforms were to be a focus on water quality and effluent management. In 2001, 15 water authorities were in place. There were significant improvements in accountability, finances, and performance. The authorities provided information on performance to both the state and the public. Reductions of operating costs have been in the range of 20-35%, with savings put back into new infrastructure. Water quality has significantly improved in a number of parameters and effluent management has also improved. This paper describes the challenges faced before the reform process, the reforms initiated, and the outcomes. It argues that privatization is not the only path to improvement: Developing a corporate structure and accountability can also deliver substantial improvements. PMID:15371224

  19. Single- versus multi-vehicle bicycle road crashes in Victoria, Australia.

    PubMed

    Boufous, Soufiane; de Rome, Liz; Senserrick, Teresa; Ivers, Rebecca Q

    2013-10-01

    The aim of the study is to compare trends, circumstances and outcomes of single- versus multi-vehicle bicycle on-road crashes in Victoria, Australia, through the analysis of police records and hospital admissions between January 2004 and December 2008. The results show that over 80% of on-road single-vehicle bicycle crashes occurred as a result of the cyclist losing control of the bicycle with the remainder involving collisions with objects. Compared with multi-vehicle crashes, single-vehicle crashes were more likely to occur in the dark, in wet conditions and in rural areas. Over half of the cyclists hospitalised as result of on-road crashes were injured in single-vehicle crashes and this proportion seems to be increasing over time. Single-vehicle crashes were associated with hospitalised injuries as severe as those resulting from multivehicle crashes. The findings highlight the significant burden of serious injury associated with single-vehicle bicycle road crashes. Further research is needed to investigate in greater detail the risk factors of these crashes and the effectiveness of countermeasures to reduce their burden. PMID:23435306

  20. Opportunity Takes a Last Look at Rock Exposure Before Heading to 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars Exploration Rover Opportunity recently stopped to analyze an exposure of rock near 'Beagle Crater,' on a target nicknamed 'Baltra.' Nearly 100 sols, or Martian days, had passed since Opportunity had last analyzed one of the now-familiar rock exposures seen on the Plains of Meridiani. The rover ground a 3-millimeter-deep (0.12-inch-deep) hole in the rock using the rock abrasion tool on sol 893 (July 29, 2006) while stationed about 25 meters (82 feet) from the southwest rim of Beagle Crater.

    Scientists wanted to analyze the outcrop one more time before driving the rover onto the ring of smooth material surrounding 'Victoria Crater.' Opportunity's analysis showed the rock to be very similar in its elemental composition to other exposures encountered during the rover's southward trek across Meridiani Planum.

    Opportunity acquired the image data shown here shortly after noon on Mars on sol 896 (Aug. 1, 2006) with the panoramic camera (Pancam), after backing up 1 meter (3.3 feet) from Baltra to assure that the target was in sunlight. This is an approximately true-color Pancam image, generated from mathematical combinations of calibrated left-eye images using filters ranging from 432-nanometer to 753-nanometer wavelengths.

  1. Opportunity Takes a Last Look at Rock Exposure Before Heading to 'Victoria Crater'(Microscopic

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars Exploration Rover Opportunity recently stopped to analyze an exposure of rock near 'Beagle Crater,' on a target nicknamed 'Baltra.' Nearly 100 sols, or Martian days, had passed since Opportunity had last analyzed one of the now-familiar rock exposures seen on the Plains of Meridiani. The rover ground a 3-millimeter-deep (0.12-inch-deep) hole in the rock using the rock abrasion tool on sol 893 (July 29, 2006) while stationed about 25 meters (82 feet) from the southwest rim of Beagle Crater.

    Scientists wanted to analyze the outcrop one more time before driving the rover onto the ring of smooth material surrounding 'Victoria Crater.' Opportunity's analysis showed the rock to be very similar in its elemental composition to other exposures encountered during the rover's southward trek across Meridiani Planum.

    Opportunity's microscopic imager acquired this view on sol 894 (July 30, 2006) while the target was fully shadowed. The view shows an area about 6 centimeters (2.4 inches) across, just spanning the diameter of the hole ground into Baltra. The image resolution of 30 microns per pixel makes it possible to see features as small as 0.1 millimeter (0.004 inch).

  2. Opportunity Takes a Last Look at Rock Exposure Before Heading to 'Victoria Crater' (False Color)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars Exploration Rover Opportunity recently stopped to analyze an exposure of rock near 'Beagle Crater,' on a target nicknamed 'Baltra.' Nearly 100 sols, or Martian days, had passed since Opportunity had last analyzed one of the now-familiar rock exposures seen on the Plains of Meridiani. The rover ground a 3-millimeter-deep (0.12-inch-deep) hole in the rock using the rock abrasion tool on sol 893 (July 29, 2006) while stationed about 25 meters (82 feet) from the southwest rim of Beagle Crater.

    Scientists wanted to analyze the outcrop one more time before driving the rover onto the ring of smooth material surrounding 'Victoria Crater.' Opportunity's analysis showed the rock to be very similar in its elemental composition to other exposures encountered during the rover's southward trek across Meridiani Planum.

    Opportunity acquired the image data shown here shortly after noon on Mars on sol 896 (Aug. 1, 2006) with the panoramic camera (Pancam), after backing up 1 meter (3.3 feet) from Baltra to assure that the target was in sunlight. This is a false-color image, made using the Pancam's 753-nanometer, 535-nanometer, and 432-nanometer filters. The false color emphasizes differences in rock and soil materials.

  3. Review of the materials-chemistry models in the VICTORIA code

    NASA Astrophysics Data System (ADS)

    Olander, D. R.; Mubayi, Vinod

    Recently a Peer Review Committee established by the US Nuclear Regulatory Commission issued its report on VICTORIA, a detailed mechanistic computer code that addresses the release of fission products from degraded reactor fuel and the ex-core behavior of the released material. The present paper summarizes the findings of the report relevant to the materials-chemistry aspects of the code that apply chiefly to the high-temperature, steam-hydrogen environment of the degrading fuel and cladding. In addition to reviewing the theoretical foundation of the mechanisms used in the code, the assessment utilizes the `test problem' technique. In this method, the code (or its methods) are applied to very simple fuel and cladding systems with variations of input conditions, code flags, and material properties. The output is judged against expected behavior for each change. The topics treated include the thermochemical database, the code's method of treating interphase equilibria, the model of fission product release, and the treatment of cladding oxidation and hydrogen production.

  4. What can legacy datasets tell us about soil quality trends? Soil acidity in Victoria

    NASA Astrophysics Data System (ADS)

    Marchant, B. P.; Crawford, D. M.; Robinson, N. J.

    2015-07-01

    Purpose-built soil monitoring networks have been established in many countries to identify where soil functionality is threatened and to target remediation initiatives. An alternative to purpose-built soil monitoring networks is to use legacy soils information. Such information yields almost instant assessments of soil change but the results should be interpreted with caution since the information was not collected with monitoring in mind. We assess the threat of soil acidification in Victoria using two legacy datasets: (i) the Victorian Soils Information System (VSIS) which is a repository of the results of soil analyses conducted for scientific purposes since the 1950s and (ii) a database of 75 000 routine soil test results requested by farmers between 1973 and 1993. We find that the VSIS measurements are clustered in space and time and are therefore suitable for local rather than broad-scale assessments of soil change. The farmers’ results have better spatial and temporal coverage and space-time models can be used to quantify the spatial and temporal trends in the pH measurements. However, careful validation of these findings is required since we do not completely understand how the measured paddocks were selected and we cannot be certain that sampling or laboratory protocols have not changed with time.

  5. Characterisation of bacterioplankton communities in the meltwater ponds of Bratina Island, Victoria Land, Antarctica.

    PubMed

    Archer, Stephen D J; McDonald, Ian R; Herbold, Craig W; Cary, Stephen C

    2014-08-01

    A unique collection of Antarctic aquatic environments (meltwater ponds) lies in close proximity on the rock and sediment-covered undulating surface of the McMurdo Ice Shelf, near Bratina Island (Victoria Land, Antarctica). During the 2009-10 mid-austral summer, sets of discrete water samples were collected across the vertical geochemical gradients of five meltwater ponds (Egg, P70E, Legin, Salt and Orange) for geochemical and microbial community structure analysis. Bacterial DNA fingerprints (using Automated Ribosomal Intergenic Spacer Analysis) statistically clustered communities within ponds based on anosim (R = 0.766, P = 0.001); however, one highly stratified pond (Egg) had two distinct depth-related bacterial communities (R = 0.975, P = 0.008). 454 pyrosequencing at three depths within Egg also identified phylum level shifts and increased diversity with depth, Bacteroidetes being the dominant phyla in the surface sample and Proteobacteria being dominant in the bottom two depths. best analysis, which attempts to link community structure and the geochemistry of a pond, identified conductivity and pH individually, and to a lesser extent Ag(109) , NO2 and V(51) as dominant influences to the microbial community structure in these ponds. Increasing abundances of major halo-tolerant OTUs across the strong conductivity gradient reinforce it as the primary driver of community structure in this study. PMID:24862286

  6. Highly Heterogeneous Soil Bacterial Communities around Terra Nova Bay of Northern Victoria Land, Antarctica

    PubMed Central

    Lim, Hyoun Soo; Hong, Soon Gyu; Kim, Ji Hee; Lee, Joohan; Choi, Taejin; Ahn, Tae Seok; Kim, Ok-Sun

    2015-01-01

    Given the diminished role of biotic interactions in soils of continental Antarctica, abiotic factors are believed to play a dominant role in structuring of microbial communities. However, many ice-free regions remain unexplored, and it is unclear which environmental gradients are primarily responsible for the variations among bacterial communities. In this study, we investigated the soil bacterial community around Terra Nova Bay of Victoria Land by pyrosequencing and determined which environmental variables govern the bacterial community structure at the local scale. Six bacterial phyla, Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, Cyanobacteria, and Bacteroidetes, were dominant, but their relative abundance varied greatly across locations. Bacterial community structures were affected little by spatial distance, but structured more strongly by site, which was in accordance with the soil physicochemical compositions. At both the phylum and species levels, bacterial community structure was explained primarily by pH and water content, while certain earth elements and trace metals also played important roles in shaping community variation. The higher heterogeneity of the bacterial community structure found at this site indicates how soil bacterial communities have adapted to different compositions of edaphic variables under extreme environmental conditions. Taken together, these findings greatly advance our understanding of the adaption of soil bacterial populations to this harsh environment. PMID:25799273

  7. Short-term droughts forecast using Markov chain model in Victoria, Australia

    NASA Astrophysics Data System (ADS)

    Rahmat, Siti Nazahiyah; Jayasuriya, Niranjali; Bhuiyan, Muhammed A.

    2016-04-01

    A comprehensive risk management strategy for dealing with drought should include both short-term and long-term planning. The objective of this paper is to present an early warning method to forecast drought using the Standardised Precipitation Index (SPI) and a non-homogeneous Markov chain model. A model such as this is useful for short-term planning. The developed method has been used to forecast droughts at a number of meteorological monitoring stations that have been regionalised into six (6) homogenous clusters with similar drought characteristics based on SPI. The non-homogeneous Markov chain model was used to estimate drought probabilities and drought predictions up to 3 months ahead. The drought severity classes defined using the SPI were computed at a 12-month time scale. The drought probabilities and the predictions were computed for six clusters that depict similar drought characteristics in Victoria, Australia. Overall, the drought severity class predicted was quite similar for all the clusters, with the non-drought class probabilities ranging from 49 to 57 %. For all clusters, the near normal class had a probability of occurrence varying from 27 to 38 %. For the more moderate and severe classes, the probabilities ranged from 2 to 13 % and 3 to 1 %, respectively. The developed model predicted drought situations 1 month ahead reasonably well. However, 2 and 3 months ahead predictions should be used with caution until the models are developed further.

  8. Chlorine-36 tracing of salinity sources in the Dry Valleys of Victoria Land, Antarctica

    NASA Astrophysics Data System (ADS)

    Carlson, Catherine A.; Phillips, Fred M.; Elmore, David; Bentley, Harold W.

    1990-02-01

    Chlorine-36 was used to trace the origins of salts in six saline lakes in the Dry Valleys of Southern Victoria Land, Antarctica. Characteristic 36Cl signatures were estimated for the various potential chloride sources, which include atmospheric deposition, rock weathering, seawater, and deep ground water. 36Cl /Cl ratios were measured in natural waters and salts from the Dry Valleys. Dilute lake waters (Cl - < 100 mg/l) were found to have 36Cl /Cl ratios in the range 100 × 10 -15 to 1,700 × 10 -15, whereas saline waters (Cl - > 1000 mg/l) had ratios in the range 9 × 10 -15 to 40 × 10 -15. Simple mixing models were employed to quantify the relative contributions of the various chloride sources to Lake Vanda and Don Juan Pond. These results show that Lake Vanda has received its chloride from both deep ground water and the Onyx River. Don Juan Pond has received nearly all its chloride from deep ground water, probably ultimately from rock-water interaction. Deep ground water is the principal source of chloride to the lakes of Wright Valley. However, preliminary data suggest that marine-derived salts or relict sea water may be a significant source of chloride to the lakes of Taylor Valley, implying a possible recent marine invasion that did not affect Wright Valley.

  9. Chlorine-36 tracing of salinity sources in the dry valleys of Victoria land, Antarctica

    SciTech Connect

    Carlson, C.A.; Phillips, F.M. ); Elmore, D. ); Bentley, H.W. )

    1990-02-01

    Chlorine-36 was used to trace the origins of salts in six saline lakes in the Dry Valleys of Southern Victoria Land, Antarctica. Characteristic {sup 36}Cl signatures were estimated for the various potential chloride sources, which include atmospheric deposition, rock weathering, seawater, and deep ground water. {sup 36}Cl/Cl ratios were measured in natural waters and salts from the Dry Valleys. Dilute lake waters (Cl{sup {minus}} < 100 mg/l) were found to have {sup 36}Cl/Cl ratios in the range 100 {times} 10{sup {minus}15} to 1,700 {times} 10{sup {minus}15}, whereas saline waters (Cl{sup {minus}} > 1000 mg/l) had ratios in the range 9 {times} 10{sup {minus}15} to 40 {times} 10{sup {minus}15}. Simple mixing models were employed to quantify the relative contributions of the various chloride sources to Lake Vanda and Don Juan Pond. These results show that Lake Vanda has received its chloride from both deep ground water and the Onyx River. Don Juan Pond has received nearly all its chloride from deep ground water, probably ultimately from rock-water interaction. Deep ground water is the principal sources of chloride to the lakes of Wright Valley. However, preliminary data suggest that marine-derived salts or relict sea water may be a significant sources of chloride to the lakes of Taylor Valley, implying a possible recent marine invasion that did not affect Wright Valley.

  10. An overview of Australia's Phytophthora species assemblage in natural ecosystems recovered from a survey in Victoria.

    PubMed

    Dunstan, William A; Howard, Kay; StJ Hardy, Giles E; Burgess, Treena I

    2016-06-01

    Although Phytophthora species cause serious diseases worldwide, until recently the main focus on disease in natural ecosystems in southern Australia has been on the distribution and impact of P. cinnamomi. However, new Phytophthora pathogens have emerged from natural ecosystems, and there is a need to better understand the diversity and distribution of these species in our natural forests, woodlands and heathlands. From a survey along a 70 km pipeline easement in Victoria, Phytophthora species were isolated from 249 rhizosphere samples and 25 bait bags deployed in 21 stream, river, or wetland locations. Of the 186 Phytophthora isolates recovered, 130 were identified to species based on ITS sequence data. Ninety-five isolates corresponded to 13 described Phytophthora species while additionally 35 isolates were identified as Clade 6 hybrids. Phytophthora cinnamomi was the most common species isolated (31 %), followed by P. elongata (6 %), both species were only recovered from soil. Samples from sites with the highest soil moisture at the time of sampling had the highest yield of isolates. Consistent with other studies throughout the world, Clade 6 species and their hybrids dominated water samples, although many of these species were also recovered less frequently from soil samples. Many of the species recovered in this study have not previously been reported from eastern Australia, reinforcing that Phytophthora species are widespread, abundant and diverse in natural ecosystems. We have probably been underestimating Phytophthora diversity in Australia. PMID:27433440

  11. Recent trends in benzodiazepine use by injecting drug users in Victoria and Tasmania.

    PubMed

    Fry, Craig L; Bruno, Raimondo B

    2002-12-01

    To address the lack of data on patterns of benzodiazepine use among injecting drug users (IDU) in Victoria and Tasmania, convenience samples of 152 Melbourne and 100 Hobart IDU were recruited from needle and syringe programme outlets and administered a structured survey on patterns of benzodiazepine use, injection-related health problems and drug use history. Most respondents had used benzodiazepines during the preceding 6 months, and more than one-third (Melbourne 36%, 95% CI, 28-44; Hobart 37%, 95% CI, 27-47) had injected benzodiazepines during this period. Diazepam was the preferred benzodiazepine for those using orally, while intravenous benzodiazepine users preferred to inject temazepam. Benzodiazepine injection for Melbourne IDU was related to greater levels of injection-related health problems. Patterns of benzodiazepine use amongst Melbourne and Hobart IDU are different to that in other Australian jurisdictions, with available data suggesting that prevalence of injection may be increasing. Ongoing monitoring of benzodiazepine injection, together with in-depth studies of supply characteristics and health impacts in jurisdictions where significant trends are detected is needed. Consideration of regulatory, supply, education and training options for the prevention of benzodiazepine injection is also indicated. PMID:12537706

  12. Forensic odontology, part 3. The Australian bushfires - Victoria state, February 2009.

    PubMed

    Hinchliffe, J

    2011-04-01

    This paper aims to demonstrate the stages in the disaster victim identification of those who lost their lives in the Australian bushfires that raged across the state of Victoria in February 2009. Communities were damaged or destroyed leaving families distressed and homeless, and as the number of deaths increased the Disaster Victim Identification (DVI) teams were activated, with plans evolving to deal with this emergency. The identification process was challenging due to many factors, such as the dangers and difficulties involved in body recovery and the charring and commingling of remains. It would take several months of careful work to identify the dead using a multidisciplinary approach. The impact of this incident will have long-lasting consequences for the families and communities involved. At the time of writing all but one of the 173 victims had been identified, mostly by dental methods: quite remarkable when only small fragments of the dental structures remained in many cases. This article is based on the author's personal experience working to assist the organised and experienced Australian Dental DVI Team. PMID:21475281

  13. Diversity of Conotoxin Gene Superfamilies in the Venomous Snail, Conus victoriae

    PubMed Central

    Robinson, Samuel D.; Safavi-Hemami, Helena; McIntosh, Lachlan D.; Purcell, Anthony W.; Norton, Raymond S.; Papenfuss, Anthony T.

    2014-01-01

    Animal venoms represent a vast library of bioactive peptides and proteins with proven potential, not only as research tools but also as drug leads and therapeutics. This is illustrated clearly by marine cone snails (genus Conus), whose venoms consist of mixtures of hundreds of peptides (conotoxins) with a diverse array of molecular targets, including voltage- and ligand-gated ion channels, G-protein coupled receptors and neurotransmitter transporters. Several conotoxins have found applications as research tools, with some being used or developed as therapeutics. The primary objective of this study was the large-scale discovery of conotoxin sequences from the venom gland of an Australian cone snail species, Conus victoriae. Using cDNA library normalization, high-throughput 454 sequencing, de novo transcriptome assembly and annotation with BLASTX and profile hidden Markov models, we discovered over 100 unique conotoxin sequences from 20 gene superfamilies, the highest diversity of conotoxins so far reported in a single study. Many of the sequences identified are new members of known conotoxin superfamilies, some help to redefine these superfamilies and others represent altogether new classes of conotoxins. In addition, we have demonstrated an efficient combination of methods to mine an animal venom gland and generate a library of sequences encoding bioactive peptides. PMID:24505301

  14. Modelling catchment management impact on in-stream phosphorus loads in northern Victoria.

    PubMed

    Vigiak, O; Rattray, D; McInnes, J; Newham, L T H; Roberts, A M

    2012-11-15

    Phosphorus pollution severely impairs the water quality of rivers in Australia and worldwide. Conceptual models have proved useful to assess management impact on phosphorus loads, particularly in data-sparse environments. This paper develops and evaluates the coupling of a point-scale model (HowLeaky2008) to a catchment scale model (CatchMODS) to enhance modelling of farm management impacts on in-stream phosphorus loads. The model was tested in two adjacent catchments in northern Victoria (Avon-Richardson and Avoca), Australia. After calibration of the in-stream attenuation parameter against measurements at gauging stations, the model simulated specific annual phosphorus loads across the catchments well (Nash-Sutcliffe model efficiency of 0.52 in the Avon-Richardson and 0.83 for the Avoca catchment). Phosphorus loads at both catchment outlets under current conditions were estimated at 7 t y(-1) and were dominated by field exports. Changes to farm management practices, i.e. the use of perennial pastures in grazing systems and zero-tillage in cropping systems were estimated to reduce phosphorus load by 31% in the Avon-Richardson catchment and 19% in the Avoca catchment, relative to current practices (annual pasture and minimum tillage). The model afforded a major improvement in conceptual modelling by explicit simulation of the impacts of soil and climatic conditions on field-scale exports and by placing them in the context of landscape processes. PMID:22796756

  15. Nuclear forensic analysis of uranium oxide powders interdicted in Victoria, Australia

    DOE PAGESBeta

    Kristo, Michael Joseph; Keegan, Elizabeth; Colella, Michael; Williams, Ross; Lindvall, Rachel; Eppich, Gary; Roberts, Sarah; Borg, Lars; Gaffney, Amy; Plaue, Jonathan; et al

    2015-04-13

    Nuclear forensic analysis was conducted on two uranium samples confiscated during a police investigation in Victoria, Australia. The first sample, designated NSR-F-270409-1, was a depleted uranium powder of moderate purity (~1000 μg/g total elemental impurities). The chemical form of the uranium was a compound similar to K2(UO2)3O4·4H2O. While aliquoting NSR-F-270409-1 for analysis, the body and head of a Tineid moth was discovered in the sample. The second sample, designated NSR-F-270409-2, was also a depleted uranium powder. It was of reasonably high purity (~380 μg/g total elemental impurities). The chemical form of the uranium was primarily UO3·2H2O, with minor phases ofmore » U3O8 and UO2. While aliquoting NSR-F-270409-2 for analysis, a metal staple of unknown origin was discovered in the sample. The presence of 236U and 232U in both samples indicates that the uranium feed stocks for these samples experienced a neutron flux at some point in their history. The reactor burn-up calculated from the isotopic composition of the uranium is consistent with that of spent fuel from natural uranium (NU) fueled Pu production. These nuclear forensic conclusions allow us to categorically exclude Australia as the origin of the material and greatly reduce the number of candidate sources.« less

  16. A 16 year study of injuries to professional kickboxers in the state of Victoria, Australia

    PubMed Central

    Zazryn, T; Finch, C; McCrory, P

    2003-01-01

    Objectives: To determine the rate and type of injuries occurring to registered professional kickboxers in Victoria, Australia over a 16 year period. Methods: Data describing all fight outcomes and injuries sustained during competition for the period August 1985 to August 2001 were obtained from the Victorian Professional Boxing and Combat Sports Board. Results: A total of 382 injuries were recorded from 3481 fight participations, at an injury rate of 109.7 injuries per 1000 fight participations. The most common body region injured was the head/neck/face (52.5%), followed by the lower extremities (39.8%). Specifically, injuries to the lower leg (23.3%), the face (19.4%), and intracranial injury (17.2%) were the most common. Over 64% of the injuries were superficial bruising or lacerations. Conclusion: The nature of kickboxing, whereby kicking the opponent is the prime movement and the head a prime target, is reflected in the distributions of body regions most commonly injured by participants. Further research into injury patterns in different styles of kickboxing and the mechanism of injury occurrence is required. Exposure adjusted prospective studies are needed to monitor injury rates over time. PMID:14514540

  17. Education does not slow cognitive decline with aging: 12-year evidence from the victoria longitudinal study.

    PubMed

    Zahodne, Laura B; Glymour, M Maria; Sparks, Catharine; Bontempo, Daniel; Dixon, Roger A; MacDonald, Stuart W S; Manly, Jennifer J

    2011-11-01

    Although the relationship between education and cognitive status is well-known, evidence regarding whether education moderates the trajectory of cognitive change in late life is conflicting. Early studies suggested that higher levels of education attenuate cognitive decline. More recent studies using improved longitudinal methods have not found that education moderates decline. Fewer studies have explored whether education exerts different effects on longitudinal changes within different cognitive domains. In the present study, we analyzed data from 1014 participants in the Victoria Longitudinal Study to examine the effects of education on composite scores reflecting verbal processing speed, working memory, verbal fluency, and verbal episodic memory. Using linear growth models adjusted for age at enrollment (range, 54-95 years) and gender, we found that years of education (range, 6-20 years) was strongly related to cognitive level in all domains, particularly verbal fluency. However, education was not related to rates of change over time for any cognitive domain. Results were similar in individuals older or younger than 70 at baseline, and when education was dichotomized to reflect high or low attainment. In this large longitudinal cohort, education was related to cognitive performance but unrelated to cognitive decline, supporting the hypothesis of passive cognitive reserve with aging. PMID:21923980

  18. Education Does Not Slow Cognitive Decline with Aging: 12-Year Evidence from the Victoria Longitudinal Study

    PubMed Central

    Zahodne, L.B.; Glymour, M.M.; Sparks, C.; Bontempo, D.; Dixon, R.A.; MacDonald, S.W.S.; Manly, J.J.

    2012-01-01

    Although the relationship between education and cognitive status is well-known, evidence regarding whether education moderates the trajectory of cognitive change in late life is conflicting. Early studies suggested that higher levels of education attenuate cognitive decline. More recent studies using improved longitudinal methods have not found that education moderates decline. Few studies have explored whether education exerts different effects on longitudinal changes within different cognitive domains. In the present study, we analyzed data from 1,023 participants in the Victoria Longitudinal Study to examine the effects of education on composite scores reflecting verbal processing speed, working memory, verbal fluency, and verbal episodic memory. Using linear growth models adjusted for age at enrollment (range: 55–94) and gender, we found that years of education (range: 6–20) was strongly related to cognitive level in all domains, particularly verbal fluency. However, education was not related to rates of change over time for any cognitive domain. Results were similar in individuals older or younger than 70 at baseline, and when education was dichotomized to reflect high or low attainment. In this large longitudinal cohort, education was related to cognitive performance but unrelated to cognitive decline, supporting the hypothesis of passive cognitive reserve with aging. PMID:21923980

  19. Integrating environmental gap analysis with spatial conservation prioritization: a case study from Victoria, Australia.

    PubMed

    Sharafi, Seyedeh Mahdieh; Moilanen, Atte; White, Matt; Burgman, Mark

    2012-12-15

    Gap analysis is used to analyse reserve networks and their coverage of biodiversity, thus identifying gaps in biodiversity representation that may be filled by additional conservation measures. Gap analysis has been used to identify priorities for species and habitat types. When it is applied to identify gaps in the coverage of environmental variables, it embodies the assumption that combinations of environmental variables are effective surrogates for biodiversity attributes. The question remains of how to fill gaps in conservation systems efficiently. Conservation prioritization software can identify those areas outside existing conservation areas that contribute to the efficient covering of gaps in biodiversity features. We show how environmental gap analysis can be implemented using high-resolution information about environmental variables and ecosystem condition with the publicly available conservation prioritization software, Zonation. Our method is based on the conversion of combinations of environmental variables into biodiversity features. We also replicated the analysis by using Species Distribution Models (SDMs) as biodiversity features to evaluate the robustness and utility of our environment-based analysis. We apply the technique to a planning case study of the state of Victoria, Australia. PMID:22935646

  20. Reprint of: Revised magnetostratigraphic chronologies for New Harbour drill cores, southern Victoria Land, Antarctica

    NASA Astrophysics Data System (ADS)

    Ohneiser, Christian; Wilson, Gary

    2012-10-01

    The Taylor Valley (DVDP-10, -11) and Ferrar Fiord (CIROS-2) drill cores offer a window into the evolution of southern Victoria Land glaciers and the Antarctic climate system during the late-Neogene. Here we present new magnetostratigraphic chronologies, which we use to correlate the drill core successions with onshore dry-valleys geomorphic records and offshore deep-ocean records. Magnetostratigraphies were constructed using stepwise AF and/or thermal demagnetisation of discrete specimens from the drill cores. Correlation of magnetostratigraphies with the magnetic polarity timescale was guided by biostratigraphic and radiometric constraints. We recognise five styles of sedimentation in the Taylor/Ferrar fiords, which we correlate with discrete climate phases. During the latest Miocene-early Pliocene, wet based glaciers filled the Taylor and Ferrar fiords with active sedimentation in the Taylor Fiord and erosion of basement rocks in the Ferrar Fiord. Glaciers retreated during the Pliocene warm period leaving open marine conditions and deep fiords (> 300 m) at a time when the Ross Sea was free of ice and sea surface temperatures around Antarctica were at least 5 °C warmer than today. We recognise the first significant cooling in DVDP-11 post 2.6 Ma by a shift to current winnowed sediments sourced from the Ross Sea rather than from East Antarctic glaciers. Post 1.7 Ma, lacustrine sediments were deposited behind ice-dammed lakes, which formed when West Antarctic ice expanded and grounded across the Ross Embayment and abutted the Transantarctic Mountains.