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Sample records for bioluminescence reporter gene

  1. Bioluminescent bacteria: lux genes as environmental biosensors

    OpenAIRE

    Nunes-Halldorson,Vânia da Silva; Duran,Norma Letícia

    2003-01-01

    Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in env...

  2. Uptake kinetics and biodistribution of C-14-D-luciferin-a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction : impact on bioluminescence based reporter gene imaging

    NARCIS (Netherlands)

    Berger, Frank; Paulmurugan, Ramasamy; Bhaumik, Srabani; Gambhir, Sanjiv Sam

    2008-01-01

    Purpose Firefly luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter

  3. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  4. Bioluminescent bacteria: lux genes as environmental biosensors

    Directory of Open Access Journals (Sweden)

    Nunes-Halldorson Vânia da Silva

    2003-01-01

    Full Text Available Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in environmental studies, with special emphasis on the Microtox toxicity bioassay. Also, the general ecological significance of bioluminescence will be addressed.

  5. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging

    International Nuclear Information System (INIS)

    Ponomarev, Vladimir; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Tourkova, Vilia; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Ivanova, Anna; Blasberg, Ronald; Balatoni, Julius; Bornmann, William; Gelovani Tjuvajev, Juri

    2004-01-01

    Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (Δ45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Δ45HSV1-tk/GFP/luciferase (Δ45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Δ45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Δ45-TGL cells compared to nontransduced control cells. The Ki of 14 C-FIAU was 0.49±0.02, 0.51±0.03, and 0.003±0.001 ml/min/g in U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [ 131 I]FIAU (7.4 MBq/animal) or [ 124 I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity

  6. Dual monitoring using 124I-FIAU and bioluminescence for HSV1-tk suicide gene therapy

    International Nuclear Information System (INIS)

    Lee, T. S.; Kim, J. H.; Kwon, H. C.

    2007-01-01

    Herpes simplex virus type I thymidine kinase (HSV-tk) is the most common reporter gene and is used in cancer gene therapy with a prodrug nucleoside analog, ganciclovir (GCV). The aim of this study is to evaluate therapeutic efficacy of suicide gene therapy with 2'-fluoro-2'-deoxy-1-D-arabinofuranosyl-5-[ 124 I] iodouracil ( 124 I - FIAU) and bioluminescence in retrovirally HSV -tk and firefly luciferase transduced hepatoma model. The HSV -tk and firefly luciferase (Luc) was retrovirally transduced and expressed in MCA rat Morris hepatoma cells. Nude mice with subcutaneous tumors, MCA and MCA-TK-Luc, were subjected to GCV treatment (50mg/Kg/d intraperitoneally) for 5 day. PET imaging and biodistribution with ( 124 I-FIAU) were performed at before and after initiation of therapy with GCV. Bioluminescent signal was also measured during GCV treatment. Before GCV treatment, no significant difference in tumor volume was found in tumors between MCA and MCA-TK-Luc. After GCV treatment, tumor volume of MCA-TK-Luc markedly reduced compared to that of MCA. In biodistribution study, 124 I-FIAU uptake after GCV therapy significantly decreased compared with pretreatment levels (34.8 13.67 %ID/g vs 7.6 2.59 %ID/g) and bioluminescent signal was also significantly decreased compared with pretreatment levels. In small animal PET imaging, 124 I-FIAU selectively localized in HSV -tk expressing tumor and the therapeutic efficacy of GCV treatment was evaluated by 124 I-FIAU PET imaging. 124 I-FIAU PET and bioluminescence imaging in HSV-tk suicide gene therapy were effective to evaluate the therapeutic response. 124 I-FIAU may serve as an efficient and selective agent for monitoring of transduced HSV1-tk gene expression in vivo in clinical trials

  7. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    Directory of Open Access Journals (Sweden)

    Daniell Henry

    2011-06-01

    Full Text Available Abstract Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun" delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI methods. Results Plasmid DNA carrying the firefly luciferase (LUC reporter gene under the control of the human Cytomegalovirus (CMV promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter using different DNA Loading Ratios (DLRs, and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50 at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results

  8. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  9. Dual monitoring using {sup 124}I-FIAU and bioluminescence for HSV1-tk suicide gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, T. S.; Kim, J. H.; Kwon, H. C. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)] (and others)

    2007-07-01

    Herpes simplex virus type I thymidine kinase (HSV-tk) is the most common reporter gene and is used in cancer gene therapy with a prodrug nucleoside analog, ganciclovir (GCV). The aim of this study is to evaluate therapeutic efficacy of suicide gene therapy with 2'-fluoro-2'-deoxy-1-D-arabinofuranosyl-5-[{sup 124}I] iodouracil ({sup 124}I - FIAU) and bioluminescence in retrovirally HSV -tk and firefly luciferase transduced hepatoma model. The HSV -tk and firefly luciferase (Luc) was retrovirally transduced and expressed in MCA rat Morris hepatoma cells. Nude mice with subcutaneous tumors, MCA and MCA-TK-Luc, were subjected to GCV treatment (50mg/Kg/d intraperitoneally) for 5 day. PET imaging and biodistribution with ({sup 124}I-FIAU) were performed at before and after initiation of therapy with GCV. Bioluminescent signal was also measured during GCV treatment. Before GCV treatment, no significant difference in tumor volume was found in tumors between MCA and MCA-TK-Luc. After GCV treatment, tumor volume of MCA-TK-Luc markedly reduced compared to that of MCA. In biodistribution study, {sup 124}I-FIAU uptake after GCV therapy significantly decreased compared with pretreatment levels (34.8 13.67 %ID/g vs 7.6 2.59 %ID/g) and bioluminescent signal was also significantly decreased compared with pretreatment levels. In small animal PET imaging, {sup 124}I-FIAU selectively localized in HSV -tk expressing tumor and the therapeutic efficacy of GCV treatment was evaluated by {sup 124}I-FIAU PET imaging. {sup 124}I-FIAU PET and bioluminescence imaging in HSV-tk suicide gene therapy were effective to evaluate the therapeutic response. {sup 124}I-FIAU may serve as an efficient and selective agent for monitoring of transduced HSV1-tk gene expression in vivo in clinical trials.

  10. Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters.

    Science.gov (United States)

    Rogers, Kelly L; Stinnakre, Jacques; Agulhon, Cendra; Jublot, Delphine; Shorte, Spencer L; Kremer, Eric J; Brûlet, Philippe

    2005-02-01

    Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca(2+)-sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260-7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable 'real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell-cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies.

  11. Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR).

    Science.gov (United States)

    Crosby, Priya; Hoyle, Nathaniel P; O'Neill, John S

    2017-12-13

    Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

  12. Bioluminescence imaging of β cells and intrahepatic insulin gene activity under normal and pathological conditions.

    Directory of Open Access Journals (Sweden)

    Tokio Katsumata

    Full Text Available In diabetes research, bioluminescence imaging (BLI has been applied in studies of β-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of β cells, we generated a bacterial artificial chromosome (BAC transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse. BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-lucVUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP, the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the β-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the β-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of β-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet β cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions.

  13. A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

    Science.gov (United States)

    Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

  14. Attenuated Bioluminescent Brucella melitensis Mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) Confer Protection in Mice

    OpenAIRE

    Rajashekara, Gireesh; Glover, David A.; Banai, Menachem; O'Callaghan, David; Splitter, Gary A.

    2006-01-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B....

  15. Sensitive in situ monitoring of a recombinant bioluminescent Yersinia enterocolitica reporter mutant in real time on Camembert cheese.

    Science.gov (United States)

    Maoz, Ariel; Mayr, Ralf; Bresolin, Geraldine; Neuhaus, Klaus; Francis, Kevin P; Scherer, Siegfried

    2002-11-01

    Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10 degrees C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm(2). Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a "real-product" status, and at a low temperature.

  16. Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence.

    Science.gov (United States)

    Nackerdien, Zeena E; Keynan, Alexander; Bassler, Bonnie L; Lederberg, Joshua; Thaler, David S

    2008-02-27

    The light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy. The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh) strains altered in quorum sensing on the one hand, and bioluminescence on the other. By hypothesis, growth rate is energy limited: mutants deficient in quorum sensing grow faster because wild type quorum sensing unleashes bioluminescence and bioluminescence diverts energy. Findings reported here confirm a role for bioluminescence in limiting Vh growth rate, at least under the conditions tested. However, the results argue that the bioluminescence is insufficient to explain the relationship of growth rate and quorum sensing in Vh. A Vh mutant null for all genes encoding the bioluminescence pathway grew faster than wild type but not as fast as null mutants in quorum sensing. Vh quorum sensing mutants showed altered growth rates that do not always rank with their relative increase or decrease in bioluminescence. In addition, the cell-free culture fluids of a rapidly growing Vibrio parahaemolyticus (Vp) strain increased the growth rate of wild type Vh without significantly altering Vh's bioluminescence. The same cell-free culture fluid increased the bioluminescence of Vh quorum mutants. The effect of quorum sensing on Vh growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate.

  17. Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence.

    Directory of Open Access Journals (Sweden)

    Zeena E Nackerdien

    2008-02-01

    Full Text Available The light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy.The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh strains altered in quorum sensing on the one hand, and bioluminescence on the other. By hypothesis, growth rate is energy limited: mutants deficient in quorum sensing grow faster because wild type quorum sensing unleashes bioluminescence and bioluminescence diverts energy. Findings reported here confirm a role for bioluminescence in limiting Vh growth rate, at least under the conditions tested. However, the results argue that the bioluminescence is insufficient to explain the relationship of growth rate and quorum sensing in Vh. A Vh mutant null for all genes encoding the bioluminescence pathway grew faster than wild type but not as fast as null mutants in quorum sensing. Vh quorum sensing mutants showed altered growth rates that do not always rank with their relative increase or decrease in bioluminescence. In addition, the cell-free culture fluids of a rapidly growing Vibrio parahaemolyticus (Vp strain increased the growth rate of wild type Vh without significantly altering Vh's bioluminescence. The same cell-free culture fluid increased the bioluminescence of Vh quorum mutants.The effect of quorum sensing on Vh growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate.

  18. Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay.

    Science.gov (United States)

    Sajayan, Arya; Seghal Kiran, G; Priyadharshini, S; Poulose, Navya; Selvin, Joseph

    2017-09-01

    A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0-10.0) and temperatures up to 100 °C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3 mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B. cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

    Science.gov (United States)

    Lin, J W; Lu, H C; Chen, H Y; Weng, S F

    1997-10-09

    Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

  20. Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

    International Nuclear Information System (INIS)

    Golberg, Karina; Elbaz, Amit; McNeil, Ronald; Kushmaro, Ariel; Geddes, Chris D.; Marks, Robert S.

    2014-01-01

    We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a specific promoter incorporated with a bioluminescence reporter gene. The silver deposition process was first optimized to provide optimal nanoparticle size in the reaction time dependence with fluorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched non-silver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-field distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic field distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a re-excitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially useful for cases in which reporter luminescence signals are very weak

  1. Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Golberg, Karina, E-mail: karingo@bgu.ac.il; Elbaz, Amit [Ben-Gurion University of the Negev, Avram and Stella Goldstein-Goren Department of Biotechnology Engineering (Israel); McNeil, Ronald [The Institute of Fluorescence, University of Maryland Baltimore County (United States); Kushmaro, Ariel [Ben-Gurion University of the Negev, Avram and Stella Goldstein-Goren Department of Biotechnology Engineering (Israel); Geddes, Chris D. [The Institute of Fluorescence, University of Maryland Baltimore County (United States); Marks, Robert S., E-mail: rsmarks@bgu.ac.il [Ben-Gurion University of the Negev, Avram and Stella Goldstein-Goren Department of Biotechnology Engineering (Israel)

    2014-12-15

    We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a specific promoter incorporated with a bioluminescence reporter gene. The silver deposition process was first optimized to provide optimal nanoparticle size in the reaction time dependence with fluorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched non-silver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-field distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic field distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a re-excitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially useful for cases in which reporter luminescence signals are very weak.

  2. Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-Vibrio symbiosis.

    Science.gov (United States)

    Heath-Heckman, Elizabeth A C; Peyer, Suzanne M; Whistler, Cheryl A; Apicella, Michael A; Goldman, William E; McFall-Ngai, Margaret J

    2013-04-02

    The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut.

  3. Circular polarization observed in bioluminescence

    NARCIS (Netherlands)

    Wijnberg, Hans; Meijer, E.W.; Hummelen, J.C.; Dekkers, H.P.J.M.; Schippers, P.H.; Carlson, A.D.

    1980-01-01

    While investigating circular polarization in luminescence, and having found it in chemiluminescence, we have studied bioluminescence because it is such a widespread and dramatic natural phenomenon. We report here that left and right lanterns of live larvae of the fireflies, Photuris lucicrescens and

  4. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    Science.gov (United States)

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2013-05-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

  5. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium

    Directory of Open Access Journals (Sweden)

    Hannah M. Read

    2016-06-01

    Full Text Available Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC and enterohaemorrhagic E. coli (EHEC infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169 in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.

  6. Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

    Directory of Open Access Journals (Sweden)

    Casey A Maguire

    2013-01-01

    Full Text Available Bioluminescence imaging (BLI has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc and Renilla or Gaussia (Gluc luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL delivered using an adeno-associated viral vector (AAV on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

  7. Detection of organic compounds with whole-cell bioluminescent bioassays.

    Science.gov (United States)

    Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven; Sayler, Gary

    2014-01-01

    Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices.

  8. Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon

    Directory of Open Access Journals (Sweden)

    Lawrence Mark L

    2010-07-01

    Full Text Available Abstract Background Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. Results We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. Conclusions This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.

  9. Radionuclide reporter gene imaging

    International Nuclear Information System (INIS)

    Min, Jung Joon

    2004-01-01

    Recent progress in the development of non-invasive imaging technologies continues to strengthen the role of molecular imaging biological research. These tools have been validated recently in variety of research models, and have been shown to provide continuous quantitative monitoring of the location(s), magnitude, and time-variation of gene expression. This article reviews the principles, characteristics, categories and the use of radionuclide reporter gene imaging technologies as they have been used in imaging cell trafficking, imaging gene therapy, imaging endogenous gene expression and imaging molecular interactions. The studies published to date demonstrate that reporter gene imaging technologies will help to accelerate model validation as well as allow for clinical monitoring of human diseases

  10. Radionuclide reporter gene imaging

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon [School of Medicine, Chonnam National Univ., Gwangju (Korea, Republic of)

    2004-04-01

    Recent progress in the development of non-invasive imaging technologies continues to strengthen the role of molecular imaging biological research. These tools have been validated recently in variety of research models, and have been shown to provide continuous quantitative monitoring of the location(s), magnitude, and time-variation of gene expression. This article reviews the principles, characteristics, categories and the use of radionuclide reporter gene imaging technologies as they have been used in imaging cell trafficking, imaging gene therapy, imaging endogenous gene expression and imaging molecular interactions. The studies published to date demonstrate that reporter gene imaging technologies will help to accelerate model validation as well as allow for clinical monitoring of human diseases.

  11. Bioluminescent bioreporter sensing of foodborne toxins

    Science.gov (United States)

    Fraley, Amanda C.; Ripp, Steven; Sayler, Gary S.

    2004-06-01

    Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.

  12. Imaging reporter gene for monitoring gene therapy

    International Nuclear Information System (INIS)

    Beco, V. de; Baillet, G.; Tamgac, F.; Tofighi, M.; Weinmann, P.; Vergote, J.; Moretti, J.L.; Tamgac, G.

    2002-01-01

    Scintigraphic images can be obtained to document gene function at cellular level. This approach is presented here and the use of a reporter gene to monitor gene therapy is described. Two main ways are presented: either the use of a reporter gene coding for an enzyme the action of which will be monitored by radiolabeled pro-drug, or a cellular receptor gene, the action of which is documented by a radio labeled cognate receptor ligand. (author)

  13. Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion.

    Science.gov (United States)

    Wollenberg, M S; Preheim, S P; Polz, M F; Ruby, E G

    2012-03-01

    This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains of Vibrio fischeri. These 'dark' V. fischeri strains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light production in dim strains of luminous bacteria. Surprisingly, the entire lux locus (eight genes) was absent in over 97% of these dark V. fischeri strains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within the V. fischeri clade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the model V. fischeri host, the Hawaiian bobtail squid Euprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  14. Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

    Science.gov (United States)

    Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

    2017-10-01

    Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

  15. GMO detection using a bioluminescent real time reporter (BART of loop mediated isothermal amplification (LAMP suitable for field use

    Directory of Open Access Journals (Sweden)

    Kiddle Guy

    2012-04-01

    Full Text Available Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART for determination of genetically modified (GM maize target DNA at low levels of contamination (0.1-5.0% GM using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading

  16. Bioluminescence imaging of Chlamydia muridarum ascending infection in mice.

    Directory of Open Access Journals (Sweden)

    Jessica Campbell

    Full Text Available Chlamydial pathogenicity in the upper genital tract relies on chlamydial ascending from the lower genital tract. To monitor chlamydial ascension, we engineered a luciferase-expressing C. muridarum. In cells infected with the luciferase-expressing C. muridarum, luciferase gene expression and enzymatic activity (measured as bioluminescence intensity correlated well along the infection course, suggesting that bioluminescence can be used for monitoring chlamydial replication. Following an intravaginal inoculation with the luciferase-expressing C. muridarum, 8 of 10 mice displayed bioluminescence signal in the lower with 4 also in the upper genital tracts on day 3 after infection. By day 7, all 10 mice developed bioluminescence signal in the upper genital tracts. The bioluminescence signal was maintained in the upper genital tract in 6 and 2 mice by days 14 and 21, respectively. The bioluminescence signal was no longer detectable in any of the mice by day 28. The whole body imaging approach also revealed an unexpected airway infection following the intravaginal inoculation. Although the concomitant airway infection was transient and did not significantly alter the genital tract infection time courses, caution should be taken during data interpretation. The above observations have demonstrated that C. muridarum can not only achieve rapid ascending infection in the genital tract but also cause airway infection following a genital tract inoculation. These findings have laid a foundation for further optimizing the C. muridarum intravaginal infection murine model for understanding chlamydial pathogenic mechanisms.

  17. BLProt: Prediction of bioluminescent proteins based on support vector machine and relieff feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar

    2011-08-17

    Background: Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence.Results: In this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated.Conclusion: BLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. 2011 Kandaswamy et al; licensee BioMed Central Ltd.

  18. BLProt: Prediction of bioluminescent proteins based on support vector machine and relieff feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar; Pugalenthi, Ganesan; Hazrati, Mehrnaz Khodam; Kalies, Kai-Uwe; Martinetz, Thomas

    2011-01-01

    Background: Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence.Results: In this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated.Conclusion: BLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. 2011 Kandaswamy et al; licensee BioMed Central Ltd.

  19. Bioluminescent organs of two deep-sea arrow worms, Eukrohnia fowleri and Caecosagitta macrocephala, with further observations on Bioluminescence in chaetognaths.

    Science.gov (United States)

    Thuesen, Erik V; Goetz, Freya E; Haddock, Steven H D

    2010-10-01

    Bioluminescence in the deep-sea chaetognath Eukrohnia fowleri is reported for the first time, and behavioral, morphological, and chemical characteristics of bioluminescence in chaetognaths are examined. Until this study, the only known species of bioluminescent chaetognath was Caecosagitta macrocephala. The luminescent organ of that species is located on the ventral edge of each anterior lateral fin, whereas that of E. fowleri runs across the center of the tail fin on both dorsal and ventral sides. Scanning electron microscopy showed that the bioluminescent organs of both species consist of hexagonal chambers containing elongate ovoid particles-the organelles holding bioluminescent materials. No other luminous organism is known to use hexagonal packing to hold bioluminescent materials. Transmission electron microscopy of particles from C. macrocephala revealed a densely packed paracrystalline matrix punctuated by globular inclusions, which likely correspond to luciferin and luciferase, respectively. Both species use unique luciferases in conjunction with coelenterazine for light emission. Luciferase of C. macrocephala becomes inactive after 30 min, but luciferase of E. fowleri is highly stable. Although C. macrocephala has about 90 times fewer particles than E. fowleri, it has a similar bioluminescent capacity (total particle volume) due to its larger particle size. In situ observations of C. macrocephala from a remotely operated vehicle revealed that the luminous particles are released to form a cloud. The discovery of bioluminescence in a second chaetognath phylogenetically distant from the first highlights the importance of bioluminescence among deep-sea organisms.

  20. Bioluminescence imaging: a shining future for cardiac regeneration

    Science.gov (United States)

    Roura, Santiago; Gálvez-Montón, Carolina; Bayes-Genis, Antoni

    2013-01-01

    Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright. PMID:23402217

  1. Assessment of chitosan-affected metabolic response by peroxisome proliferator-activated receptor bioluminescent imaging-guided transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Chia-Hung Kao

    Full Text Available Chitosan has been widely used in food industry as a weight-loss aid and a cholesterol-lowering agent. Previous studies have shown that chitosan affects metabolic responses and contributes to anti-diabetic, hypocholesteremic, and blood glucose-lowering effects; however, the in vivo targeting sites and mechanisms of chitosan remain to be clarified. In this study, we constructed transgenic mice, which carried the luciferase genes driven by peroxisome proliferator-activated receptor (PPAR, a key regulator of fatty acid and glucose metabolism. Bioluminescent imaging of PPAR transgenic mice was applied to report the organs that chitosan acted on, and gene expression profiles of chitosan-targeted organs were further analyzed to elucidate the mechanisms of chitosan. Bioluminescent imaging showed that constitutive PPAR activities were detected in brain and gastrointestinal tract. Administration of chitosan significantly activated the PPAR activities in brain and stomach. Microarray analysis of brain and stomach showed that several pathways involved in lipid and glucose metabolism were regulated by chitosan. Moreover, the expression levels of metabolism-associated genes like apolipoprotein B (apoB and ghrelin genes were down-regulated by chitosan. In conclusion, these findings suggested the feasibility of PPAR bioluminescent imaging-guided transcriptomic analysis on the evaluation of chitosan-affected metabolic responses in vivo. Moreover, we newly identified that downregulated expression of apoB and ghrelin genes were novel mechanisms for chitosan-affected metabolic responses in vivo.

  2. Hv 1 Proton Channels in Dinoflagellates: Not Just for Bioluminescence?

    Science.gov (United States)

    Kigundu, Gabriel; Cooper, Jennifer L; Smith, Susan M E

    2018-04-26

    Bioluminescence in dinoflagellates is controlled by H V 1 proton channels. Database searches of dinoflagellate transcriptomes and genomes yielded hits with sequence features diagnostic of all confirmed H V 1, and show that H V 1 is widely distributed in the dinoflagellate phylogeny including the basal species Oxyrrhis marina. Multiple sequence alignments followed by phylogenetic analysis revealed three major subfamilies of H V 1 that do not correlate with presence of theca, autotrophy, geographic location, or bioluminescence. These data suggest that most dinoflagellates express a H V 1 which has a function separate from bioluminescence. Sequence evidence also suggests that dinoflagellates can contain more than one H V 1 gene. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

    Science.gov (United States)

    Rajashekara, Gireesh; Glover, David A; Banai, Menachem; O'Callaghan, David; Splitter, Gary A

    2006-05-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

  4. Bioluminescence in vivo imaging of autoimmune encephalomyelitis predicts disease

    Directory of Open Access Journals (Sweden)

    Steinman Lawrence

    2008-02-01

    Full Text Available Abstract Background Experimental autoimmune encephalomyelitis is a widely used animal model to understand not only multiple sclerosis but also basic principles of immunity. The disease is scored typically by observing signs of paralysis, which do not always correspond with pathological changes. Methods Experimental autoimmune encephalomyelitis was induced in transgenic mice expressing an injury responsive luciferase reporter in astrocytes (GFAP-luc. Bioluminescence in the brain and spinal cord was measured non-invasively in living mice. Mice were sacrificed at different time points to evaluate clinical and pathological changes. The correlation between bioluminescence and clinical and pathological EAE was statistically analyzed by Pearson correlation analysis. Results Bioluminescence from the brain and spinal cord correlates strongly with severity of clinical disease and a number of pathological changes in the brain in EAE. Bioluminescence at early time points also predicts severity of disease. Conclusion These results highlight the potential use of bioluminescence imaging to monitor neuroinflammation for rapid drug screening and immunological studies in EAE and suggest that similar approaches could be applied to other animal models of autoimmune and inflammatory disorders.

  5. Bioorthogonal chemistry in bioluminescence imaging.

    Science.gov (United States)

    Godinat, Aurélien; Bazhin, Arkadiy A; Goun, Elena A

    2018-05-18

    Bioorthogonal chemistry has developed significant over the past few decades, to the particular benefit of molecular imaging. Bioluminescence imaging (BLI) along with other imaging modalities have significantly benefitted from this chemistry. Here, we review bioorthogonal reactions that have been used to signific antly broaden the application range of BLI. Copyright © 2018. Published by Elsevier Ltd.

  6. Smartphone-based low light detection for bioluminescence application

    Science.gov (United States)

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation ...

  7. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  8. Bioluminescent system for dynamic imaging of cell and animal behavior

    Energy Technology Data Exchange (ETDEWEB)

    Hara-Miyauchi, Chikako [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198 (Japan); Department of Biophysics and Biochemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Tsuji, Osahiko [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582 (Japan); Hanyu, Aki [Division of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo 135-8550 (Japan); Okada, Seiji [Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Yasuda, Akimasa [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582 (Japan); Fukano, Takashi [Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198 (Japan); Akazawa, Chihiro [Department of Biophysics and Biochemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakamura, Masaya [Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582 (Japan); Imamura, Takeshi [Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295 (Japan); Core Research for Evolutional Science and Technology, The Japan Science and Technology Corporation, Tokyo 135-8550 (Japan); Matsuzaki, Yumi [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Okano, Hirotaka James, E-mail: hjokano@jikei.ac.jp [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Division of Regenerative Medicine Jikei University School of Medicine, Tokyo 150-8461 (Japan); and others

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. Black-Right-Pointing-Pointer ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. Black-Right-Pointing-Pointer ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. Black-Right-Pointing-Pointer ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.

  9. Bioluminescent system for dynamic imaging of cell and animal behavior

    International Nuclear Information System (INIS)

    Hara-Miyauchi, Chikako; Tsuji, Osahiko; Hanyu, Aki; Okada, Seiji; Yasuda, Akimasa; Fukano, Takashi; Akazawa, Chihiro; Nakamura, Masaya; Imamura, Takeshi; Matsuzaki, Yumi; Okano, Hirotaka James

    2012-01-01

    Highlights: ► We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. ► ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. ► ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. ► ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.

  10. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research

    Directory of Open Access Journals (Sweden)

    Giulia eSiciliano

    2015-05-01

    Full Text Available The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria.

  11. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research.

    Science.gov (United States)

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria.

  12. Radionuclide reporter gene imaging for cardiac gene therapy

    International Nuclear Information System (INIS)

    Inubushi, Masayuki; Tamaki, Nagara

    2007-01-01

    In the field of cardiac gene therapy, angiogenic gene therapy has been most extensively investigated. The first clinical trial of cardiac angiogenic gene therapy was reported in 1998, and at the peak, more than 20 clinical trial protocols were under evaluation. However, most trials have ceased owing to the lack of decisive proof of therapeutic effects and the potential risks of viral vectors. In order to further advance cardiac angiogenic gene therapy, remaining open issues need to be resolved: there needs to be improvement of gene transfer methods, regulation of gene expression, development of much safer vectors and optimisation of therapeutic genes. For these purposes, imaging of gene expression in living organisms is of great importance. In radionuclide reporter gene imaging, ''reporter genes'' transferred into cell nuclei encode for a protein that retains a complementary ''reporter probe'' of a positron or single-photon emitter; thus expression of the reporter genes can be imaged with positron emission tomography or single-photon emission computed tomography. Accordingly, in the setting of gene therapy, the location, magnitude and duration of the therapeutic gene co-expression with the reporter genes can be monitored non-invasively. In the near future, gene therapy may evolve into combination therapy with stem/progenitor cell transplantation, so-called cell-based gene therapy or gene-modified cell therapy. Radionuclide reporter gene imaging is now expected to contribute in providing evidence on the usefulness of this novel therapeutic approach, as well as in investigating the molecular mechanisms underlying neovascularisation and safety issues relevant to further progress in conventional gene therapy. (orig.)

  13. BarTeL, a Genetically Versatile, Bioluminescent and Granule Neuron Precursor-Targeted Mouse Model for Medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Gregory M Shackleford

    Full Text Available Medulloblastomas are the most common malignant pediatric brain tumor and have been divided into four major molecular subgroups. Animal models that mimic the principal molecular aberrations of these subgroups will be important tools for preclinical studies and allow greater understanding of medulloblastoma biology. We report a new transgenic model of medulloblastoma that possesses a unique combination of desirable characteristics including, among others, the ability to incorporate multiple and variable genes of choice and to produce bioluminescent tumors from a limited number of somatic cells within a normal cellular environment. This model, termed BarTeL, utilizes a Barhl1 homeobox gene promoter to target expression of a bicistronic transgene encoding both the avian retroviral receptor TVA and an eGFP-Luciferase fusion protein to neonatal cerebellar granule neuron precursor (cGNP cells, which are cells of origin for the sonic hedgehog (SHH subgroup of human medulloblastomas. The Barhl1 promoter-driven transgene is expressed strongly in mammalian cGNPs and weakly or not at all in mature granule neurons. We efficiently induced bioluminescent medulloblastomas expressing eGFP-luciferase in BarTeL mice by infection of a limited number of somatic cGNPs with avian retroviral vectors encoding the active N-terminal fragment of SHH and a stabilized MYCN mutant. Detection and quantification of the increasing bioluminescence of growing tumors in young BarTeL mice was facilitated by the declining bioluminescence of their uninfected maturing cGNPs. Inclusion of eGFP in the transgene allowed enriched sorting of cGNPs from neonatal cerebella. Use of a single bicistronic avian vector simultaneously expressing both Shh and Mycn oncogenes increased the medulloblastoma incidence and aggressiveness compared to mixed virus infections. Bioluminescent tumors could also be produced by ex vivo transduction of neonatal BarTeL cerebellar cells by avian retroviruses and

  14. Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

    International Nuclear Information System (INIS)

    Yokawa, Satoru; Suzuki, Takahiro; Inouye, Satoshi; Inoh, Yoshikazu; Suzuki, Ryo; Kanamori, Takao; Furuno, Tadahide; Hirashima, Naohide

    2017-01-01

    We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.

  15. Effects of Epigenetic Modulation on Reporter Gene Expression: Implications for Stem Cell Imaging

    Science.gov (United States)

    Krishnan, Manickam; Park, Jinha M.; Cao, Feng; Wang, Dongxu; Paulmurugan, Ramasay; Tseng, Jeffrey R.; Gonzalgo, Mark L.; Gambhir, Sanjiv S.; Wu, Joseph C.

    2013-01-01

    Tracking stem cell localization, survival, differentiation, and proliferation following transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3–8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage-1 (843±28) to passage-20 (250±10) to passage-40 (44±3) to passage-60 (3±1 RLU/µg) (P<0.05 vs. passage-1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared to trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator) (P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 weeks compared to 1 week for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation. PMID:16246867

  16. Bactérias bioluminescentes: os genes lux como biosensores ambientais

    OpenAIRE

    Nunes-Halldorson, Vânia da Silva; Duran, Norma Letícia

    2003-01-01

    Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in env...

  17. Monitoring and quantitative assessment of tumor burden using in vivo bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.-C. [Cancer Research Division, National Health Research Institute, Miaoli 350, Taiwan (China); Hwang, Jeng-Jong [Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China)]. E-mail: jjhwang@ym.edu.tw; Ting, G. [Cancer Research Division, National Health Research Institute, Miaoli 350, Taiwan (China); Tseng, Y.-L. [Taiwan Liposome Company, Taipei 115, Taiwan (China); Wang, S.-J. [Department of Nuclear Medicine, Veterans General Hospital, Taipei 112, Taiwan (China); Whang-Peng, J. [Cancer Research Division, National Health Research Institute, Miaoli 350, Taiwan (China)

    2007-02-01

    In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating tumor growth. In this study, the kinetic of tumor growth has been assessed in C26 colon carcinoma bearing BALB/c mouse model. The ability of BLI to noninvasively quantitate the growth of subcutaneous tumors transplanted with C26 cells genetically engineered to stably express firefly luciferase and herpes simplex virus type-1 thymidine kinase (C26/tk-luc). A good correlation (R {sup 2}=0.998) of photon emission to the cell number was found in vitro. Tumor burden and tumor volume were monitored in vivo over time by quantitation of photon emission using Xenogen IVIS 50 and standard external caliper measurement, respectively. At various time intervals, tumor-bearing mice were imaged to determine the correlation of in vivo BLI to tumor volume. However, a correlation of BLI to tumor volume was observed when tumor volume was smaller than 1000 mm{sup 3} (R {sup 2}=0.907). {gamma} Scintigraphy combined with [{sup 131}I]FIAU was another imaging modality used for verifying the previous results. In conclusion, this study showed that bioluminescence imaging is a powerful and quantitative tool for the direct assay to monitor tumor growth in vivo. The dual reporter genes transfected tumor-bearing animal model can be applied in the evaluation of the efficacy of new developed anti-cancer drugs.

  18. Monitoring and quantitative assessment of tumor burden using in vivo bioluminescence imaging

    International Nuclear Information System (INIS)

    Chen, C.-C.; Hwang, Jeng-Jong; Ting, G.; Tseng, Y.-L.; Wang, S.-J.; Whang-Peng, J.

    2007-01-01

    In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating tumor growth. In this study, the kinetic of tumor growth has been assessed in C26 colon carcinoma bearing BALB/c mouse model. The ability of BLI to noninvasively quantitate the growth of subcutaneous tumors transplanted with C26 cells genetically engineered to stably express firefly luciferase and herpes simplex virus type-1 thymidine kinase (C26/tk-luc). A good correlation (R 2 =0.998) of photon emission to the cell number was found in vitro. Tumor burden and tumor volume were monitored in vivo over time by quantitation of photon emission using Xenogen IVIS 50 and standard external caliper measurement, respectively. At various time intervals, tumor-bearing mice were imaged to determine the correlation of in vivo BLI to tumor volume. However, a correlation of BLI to tumor volume was observed when tumor volume was smaller than 1000 mm 3 (R 2 =0.907). γ Scintigraphy combined with [ 131 I]FIAU was another imaging modality used for verifying the previous results. In conclusion, this study showed that bioluminescence imaging is a powerful and quantitative tool for the direct assay to monitor tumor growth in vivo. The dual reporter genes transfected tumor-bearing animal model can be applied in the evaluation of the efficacy of new developed anti-cancer drugs

  19. Smartphone-based low light detection for bioluminescence application

    OpenAIRE

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an an...

  20. High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.

    Directory of Open Access Journals (Sweden)

    Michelle Cronin

    Full Text Available The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v. administered to mice bearing subcutaneous (s.c FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

  1. Investigation progress of PET reporter gene imaging

    International Nuclear Information System (INIS)

    Chen Yumei; Huang Gang

    2006-01-01

    Molecular imaging for gene therapy and gene expression has been more and more attractive, while the use of gene therapy has been widely investigated and intense research have allowed it to the clinical setting in the last two-decade years. In vivo imaging with positron emission tomography (PET) by combination of appropriate PET reporter gene and PET reporter probe could provide qualitative and quantitative information for gene therapy. PET imaging could also obtain some valuable parameters not available by other techniques. This technology is useful to understand the process and development of gene therapy and how to apply it into clinical practice in the future. (authors)

  2. MRI Reporter Genes for Noninvasive Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Caixia Yang

    2016-05-01

    Full Text Available Magnetic resonance imaging (MRI is one of the most important imaging technologies used in clinical diagnosis. Reporter genes for MRI can be applied to accurately track the delivery of cell in cell therapy, evaluate the therapy effect of gene delivery, and monitor tissue/cell-specific microenvironments. Commonly used reporter genes for MRI usually include genes encoding the enzyme (e.g., tyrosinase and β-galactosidase, the receptor on the cells (e.g., transferrin receptor, and endogenous reporter genes (e.g., ferritin reporter gene. However, low sensitivity limits the application of MRI and reporter gene-based multimodal imaging strategies are common including optical imaging and radionuclide imaging. These can significantly improve diagnostic efficiency and accelerate the development of new therapies.

  3. Repeated and Widespread Evolution of Bioluminescence in Marine Fishes.

    Directory of Open Access Journals (Sweden)

    Matthew P Davis

    Full Text Available Bioluminescence is primarily a marine phenomenon with 80% of metazoan bioluminescent genera occurring in the world's oceans. Here we show that bioluminescence has evolved repeatedly and is phylogenetically widespread across ray-finned fishes. We recover 27 independent evolutionary events of bioluminescence, all among marine fish lineages. This finding indicates that bioluminescence has evolved many more times than previously hypothesized across fishes and the tree of life. Our exploration of the macroevolutionary patterns of bioluminescent lineages indicates that the present day diversity of some inshore and deep-sea bioluminescent fish lineages that use bioluminescence for communication, feeding, and reproduction exhibit exceptional species richness given clade age. We show that exceptional species richness occurs particularly in deep-sea fishes with intrinsic bioluminescent systems and both shallow water and deep-sea lineages with luminescent systems used for communication.

  4. Downstream reporter gene imaging for signal transduction pathway of dopamine type 2 receptor

    International Nuclear Information System (INIS)

    Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung

    2004-01-01

    The Dopamine 2 receptor (D2R) signal pathway regulates gene expression by phosphorylation of proteins including cAMP reponse element-binding protein (CREB), a transcription factor. In this study, we developed a reporter strategy using the GAL4 fusion CREB to assess the phosphorylation of CREB, one of the targets of the D2R signal transduction pathway. We used three plasmids: GAL4 fusion transactivator (pCMV-CREB), firefly luciferase reporter with GAL4 binding sites (pG5-FLUC), and D2R plasmid (pCMV-D2R). Group 1 293T cells were transiently transfected with pCMV-CREB and pG5-FLUC, and group 2 cells were transfected with all three plasmids. Transfected cells were stimulated with different concentrations of dopamine (0-200 M). For animal studies, group 1 and 2 cells (1x10 6 ) were subcutaneously injected on the left and right thigh of six nude mice, respectively. Dopamine stimiulation was performed with intraperitoneal injection of L-DOPA incombination with carbidopa, a peripheral DOPA decarboxylase inhibitor. Bioluminescence optical imaging studies were performed before and after L-DOPA injection. In cell culture studies, group 1 cells showed strong luciferase activity which implies direct activation of the signaling pathway due to growth factors contained in culture medium. Group 2 cells showed strong luciferase activity and a further increase after administration of dopamine. In animal studies, group 1 and 2 cells showed bioluminescence signal before L-DOPA injection, but signal from group 2 cells significantly increased 12 h after L-DOPA injection. The signal from group 1 cells disappeared thereafter, but group 2 cells continued to show signal until 36 h of L-DOPA injection. This study demonstrates imaging of the D2R signal transduction pathway and should be useful for noninvasive imaging of downstream effects of G-coupled protein pathways

  5. Rapid detection of E. Coli O157:H7 by IFAST and ATP bioluminescence assay for water analysis

    CSIR Research Space (South Africa)

    Ngamsom, B

    2016-10-01

    Full Text Available The present investigation reports isolation and detection of E. coli O157:H7 employing a simple and portable microfluidic device based on immiscible filtration assisted by surface tension (IFAST) and adenosine triphosphate (ATP) bioluminescence...

  6. A Transgenic Tri-Modality Reporter Mouse

    OpenAIRE

    Yan, Xinrui; Ray, Pritha; Paulmurugan, Ramasamy; Tong, Ricky; Gong, Yongquan; Sathirachinda, Ataya; Wu, Joseph C.; Gambhir, Sanjiv S.

    2013-01-01

    Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent...

  7. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    Science.gov (United States)

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

  8. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

    Science.gov (United States)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work

  9. In vivo cell tracking with bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong Cheol [Dept. of Nuclear Medicine, Kyungpook National University School of Medicine and Hospital, Daegu (Korea, Republic of)

    2015-03-15

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  10. Chemistry and biology of insect bioluminescence

    International Nuclear Information System (INIS)

    Colepicolo Neto, P.; Bechara, E.J.H.

    1984-01-01

    Basic aspects on the Chemistry and Biology of bioluminescence are reviewed, with emphasis on insects. Data from the investigation of Lampyridae (fireflies) are collected from literature. With regard to Elateridae (click beetles) and Phengodidae (rail road worms), the least explored families of luminescent insects, new data are presented on the following aspects: (i) 'in vivo' emission spectra, (ii) chemical nature of the luciferin, (iii) conection between bioluminescence and 'oxygen toxicity' as a result of molecular oxygen storage and (iv) the role of light emission by larvae and pupae. (Author) [pt

  11. In vivo bioluminescence imaging of cell differentiation in biomaterials: a platform for scaffold development.

    Science.gov (United States)

    Bagó, Juli R; Aguilar, Elisabeth; Alieva, Maria; Soler-Botija, Carolina; Vila, Olaia F; Claros, Silvia; Andrades, José A; Becerra, José; Rubio, Nuria; Blanco, Jerónimo

    2013-03-01

    In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.

  12. Bacteria as part of bioluminescence emission at the deep ANTARES station (North-Western Mediterranean Sea) during a one-year survey

    Science.gov (United States)

    Martini, S.; Michotey, V.; Casalot, L.; Bonin, P.; Guasco, S.; Garel, M.; Tamburini, C.

    2016-10-01

    Bioluminescent bacteria have been studied during a one-year survey in 2011 at the deep ANTARES site (Northwestern Mediterranean Sea, 2000 m depth). The neutrino underwater telescope ANTARES, located at this station, has been used to record the bioluminescence at the same depth. Together with these data, environmental variables (potential temperature, salinity, nutrients, dissolved organic carbon and oxygen) have been characterized in water samples. The year 2011 was characterized by relatively stable conditions, as revealed by minor variability in the monitored oceanographic variables, by low bioluminescence and low current speed. This suggests weak eukaryote participation and mainly non-stimulated light emission. Hence, no processes of dense water have affected the ANTARES station during this survey. Abundance of bioluminescent bacteria belonging to Photobacterium genus, measured by qPCR of the luxF gene, ranged from 1.4×102 to 7.2×102 genes mL-1. Their effective activity was confirmed through mRNA luxF quantification. Our results reveal that bioluminescent bacteria appeared more active than the total counterpart of bacteria, suggesting an ecological benefit of this feature such as favoring interaction with macro-organisms. Moreover, these results show that part of the bioluminescence, recorded at 2000 m depth over one year, could be due to bioluminescent bacteria in stable hydrological conditions.

  13. Advances in study of reporter gene imaging for monitoring gene therapy

    International Nuclear Information System (INIS)

    Mu Chuanjie; Zhou Jiwen

    2003-01-01

    To evaluate the efficiency of gene therapy, it is requisite to monitor localization and expression of the therapeutic gene in vivo. Monitoring expression of reporter gene using radionuclide reporter gene technique is the best method. Adenoviral vectors expressing reporter gene are constructed using gene fusion, bicistronic, double promoter or bidirectional transcriptional recombination techniques, and transferred into target cells and tissues, then injected radiolabeled reporter probes which couple to the reporter genes. The reporter genes can be imaged invasively, repeatedly, quantitatively with γ-camera, PET and SPECT. Recently, several reporter gene and reporter probe systems have been used in studies of gene therapy. The part of them has been used for clinic trials

  14. Comparison of Acute Toxicity of Algal Metabolites Using Bioluminescence Inhibition Assay

    Directory of Open Access Journals (Sweden)

    Hansa Jeswani

    2015-01-01

    Full Text Available Microalgae are reported to degrade hazardous compounds. However, algae, especially cyanobacteria are known to produce secondary metabolites which may be toxic to flora, fauna and human beings. The aim of this study was selection of an appropriate algal culture for biological treatment of biomass gasification wastewater based on acute toxicity considerations. The three algae that were selected were Spirulina sp., Scenedesmus abundans and a fresh water algal consortium. Acute toxicity of the metabolites produced by these algal cultures was tested at the end of log phase using the standard bioluminescence inhibition assay based on Vibrio fischeri NRRLB 11174. Scenedesmus abundans and a fresh water algal consortium dominated by cyanobacteria such as Phormidium, Chroococcus and Oscillatoria did not release much toxic metabolites at the end of log phase and caused only about 20% inhibition in bioluminescence. In comparison, Spirulina sp. released toxic metabolites and caused 50% bioluminescence inhibition at 3/5 times dilution of the culture supernatant (EC50.

  15. Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris

    Science.gov (United States)

    Francis, Warren R; Powers, Meghan L; Haddock, Steven H D

    2014-01-01

    Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow–orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C15H10O5, which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence. PMID:24760626

  16. Bioluminescence lights the way to food safety

    Science.gov (United States)

    Brovko, Lubov Y.; Griffiths, Mansel W.

    2003-07-01

    The food industry is increasingly adopting food safety and quality management systems that are more proactive and preventive than those used in the past which have tended to rely on end product testing and visual inspection. The regulatory agencies in many countries are promoting one such management tool, Hazard Analysis Critical Control Point (HACCP), as a way to achieve a safer food supply and as a basis for harmonization of trading standards. Verification that the process is safe must involve microbiological testing but the results need not be generated in real-time. Of all the rapid microbiological tests currently available, the only ones that come close to offering real-time results are bioluminescence-based methods. Recent developments in application of bioluminescence for food safety issues are presented in the paper. These include the use of genetically engineered microorganisms with bioluminescent and fluorescent phenotypes as a real time indicator of physiological state and survival of food-borne pathogens in food and food processing environments as well as novel bioluminescent-based methods for rapid detection of pathogens in food and environmental samples. Advantages and pitfalls of the methods are discussed.

  17. Bioluminescent hydrocarbonclastic bacteria of the Niger Delta ...

    African Journals Online (AJOL)

    Utilization of three petroleum hydrocarbons (Mobil SAE 40 Engine Oil, Diesel and Bonny light Crude Oil) by four bioluminescent bacteria (Vibrio harveyi, V. fisheri, Photobacterium leiognathi and P. Phosphoreum isolated from the Bonny estuary in the Niger Delta, Nigeria was investigated. Microbial utilization was monitored ...

  18. Heterogeneity in quorum sensing-regulated bioluminescence of Vibrio harveyi.

    Science.gov (United States)

    Anetzberger, Claudia; Pirch, Torsten; Jung, Kirsten

    2009-07-01

    Quorum sensing (QS) refers to the ability of bacterial populations to read out the local environment for cell density and to collectively activate gene expression. Vibrio harveyi, one of the best characterized model organisms in QS, was used to address the question how single cells behave within a QS-activated community in a homogeneous environment. Analysis of the QS-regulated bioluminescence of a wild type strain revealed that even at high cell densities only 69% of the cells of the population produced bioluminescence, 25% remained dark and 6% were dead. Moreover, light intensities greatly varied from cell to cell at high population density. Addition of autoinducer to a bright liquid culture of V. harveyi increased the percentage of luminescent cells up to 98%, suggesting that V. harveyi produces and/or keeps the autoinducers at non-saturating concentrations. In contrast, all living cells of a constitutive QS-active mutant (DeltaluxO) produced light. We also found that QS affects biofilm formation in V. harveyi. Our data provide first evidence that a heterogeneous population produces more biofilm than a homogeneous one. It is suggested that even a QS-committed population of V. harveyi takes advantage of heterogeneity, which extends the current view of QS-regulated uniformity.

  19. Reporter gene imaging: potential impact on therapy

    International Nuclear Information System (INIS)

    Serganova, Inna; Blasberg, Ronald

    2005-01-01

    Positron emission tomography (PET)-based molecular-genetic imaging in living organisms has enjoyed exceptional growth over the past 5 years; this is particularly striking since it has been identified as a new discipline only within the past decade. Positron emission tomography is one of three imaging technologies (nuclear, magnetic resonance and optical) that has begun to incorporate methods that are established in molecular and cell biology research. The convergence of these disciplines and the wider application of multi-modality imaging are at the heart of this success story. Most current molecular-genetic imaging strategies are 'indirect,' coupling a 'reporter gene' with a complimentary 'reporter probe.' Reporter gene constructs can be driven by constitutive promoter elements and used to monitor gene therapy vectors and the efficacy of trans gene targeting and transduction, as well as to monitor adoptive cell-based therapies. Inducible promoters can be used as 'sensors' to regulate the magnitude of reporter gene expression and can be used to provide information about endogenous cell processes. Reporter systems can also be constructed to monitor mRNA stabilization and specific protein-protein interactions. Promoters can be cell specific and restrict transgene expression to certain tissue and organs. The translation of reporter gene imaging to specific clinical applications is discussed. Several examples that have potential for patient imaging studies in the near future include monitoring adenoviral-based gene therapy, oncolytic herpes virus therapy, adoptive cell-based therapies and Salmonella-based tumor-targeted cancer therapy and imaging. The primary translational applications of noninvasive in vivo reporter gene imaging are likely to be (a) quantitative monitoring of the gene therapy vector and the efficacy of transduction in clinical protocols, by imaging the location, extent and duration of transgene expression; (b) monitoring cell trafficking, targeting

  20. Smartphone-based low light detection for bioluminescence application

    Science.gov (United States)

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time.

  1. Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.

    Science.gov (United States)

    Amaral, D T; Arnoldi, F G C; Rosa, S P; Viviani, V R

    2014-08-01

    Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity.

    Science.gov (United States)

    Xu, Tingting; Close, Dan M; Webb, James D; Ripp, Steven A; Sayler, Gary S

    2013-10-28

    Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

  3. Controlled field release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and control

    Energy Technology Data Exchange (ETDEWEB)

    Ripp, S.; Nivens, D.E.; Ahn, Y.; Werner, C.; Jarrell, J. IV; Easter, J.P.; Cox, C.D.; Burlage, R.S.; Sayler, G.S.

    2000-03-01

    Pseudomonas fluorescens HK44 represents the first genetically engineered microorganism approved for field testing in the United States for bioremediation purposes. Strain HK44 harbors an introduced lux gene fused within a naphthalene degradative pathway, thereby allowing this recombinant microbe to bioluminescent as it degrades specific polyaromatic hydrocarbons such as naphthalene. The bioremediation process can therefore be monitored by the detection of light. P. fluorescens HK44 was inoculated into the vadose zone of intermediate-scale, semicontained soil lysimeters contaminated with naphthalene, anthracene, and phenanthrene, and the population dynamics were followed over an approximate 2-year period in order to assess the long-term efficacy of using strain HK44 for monitoring and controlling bioremediation processes. Results showed that P. fluorescens HK44 was capable of surviving initial inoculation into both hydrocarbon contaminated and uncontaminated soils and was recoverable from these soils 660 days post inoculation. It was also demonstrated that strain HK44 was capable of generating bioluminescence in response to soil hydrocarbon bioavailability. Bioluminescence approaching 166,000 counts/s was detected in fiber optic-based biosensor devices responding to volatile polyaromatic hydrocarbons, while a portable photomultiplier module detected bioluminescence at an average of 4300 counts/s directly from soil-borne HK44 cells within localized treatment areas. The utilization of lux-based bioreporter microorganisms therefore promises to be a viable option for in situ determination of environmental contaminant bioavailability and biodegradation process monitoring and control.

  4. Promising Biological Indicator of Heavy Metal Pollution: Bioluminescent Bacterial Strains Isolated and Characterized from Marine Niches of Goa, India.

    Science.gov (United States)

    Thakre, Neha A; Shanware, Arti S

    2015-09-01

    In present study, several marine water samples collected from the North Goa Beaches, India for isolation of luminescent bacterial species. Isolates obtained labelled as DP1-5 and AB1-6. Molecular characterization including identification of a microbial culture using 16S rRNA gene based molecular technique and phylogenetic analysis confirmed that DP3 & AB1 isolates were Vibrio harveyi. All of the isolates demonstrated multiple metal resistances in terms of growth, with altered luminescence with variable metal concentration. Present investigations were an attempt towards exploring and reporting an updated diversity of bioluminescent bacterial species from various sites around the Goa, India which would be explored in future for constructing luminescence based biosensor for efficiently monitoring the level of hazardous metals in the environment.

  5. Advances in study of molecular imaging reporte gene systems

    International Nuclear Information System (INIS)

    Wu Tao; An Rui

    2010-01-01

    The use of molecular imaging reporter gene systems has allowed gene therapy to move from the laboratory to the clinical application, which provides methodology to monitor the expression of therapeutic gene noninvasively and achieve quantitative outcome in vivo. Recently, the radionuclide reporter gene still is the focus many studies, but MRI and optical reporter gene have gradually played a important part in reporter gene systems. On the basis of combination of multi-subject, for example applied chemistry and molecular biology, more and more new modified reporter genes and molecular probes have spread out. This paper mainly introduces the advantages and disadvantages of reporter gene system and development trends. (authors)

  6. Effect of electromagnetic fields on the bacteria bioluminescent activity

    International Nuclear Information System (INIS)

    Berzhanskaya, L.Yu.; Berzhanskij, V.N.; Beloplotova, O.Yu.

    1995-01-01

    The effect of electromagnetic field with frequency from 36.2 to 55.9 GHz on bioluminescence activity of bacterium were investigated. Electromagnetic field results in decrease of bioluminescence, which depends from frequency. The electromagnetic field adaptation time is higher of intrinsic time parameters of bioluminescence system. The effect has nonthermal nature. It is suggested that electromagnetic field influence connects with structure rearrangements near cell emitter. 8 refs.; 3 figs

  7. a positive control plasmid for reporter gene assay

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-04

    Jul 4, 2008 ... qualification as a positive control for luciferase reporter gene assays. Key words: Reporter gene plasmid, luciferase assay, cytomegalovirus promoter/enhancer, human melanoma cell line. INTRODUCTION. Reporter genes, often called reporters, have become a precious tool in studies of gene expression ...

  8. Stimulated bioluminescence by fluid shear stress associated with pipe flow

    Energy Technology Data Exchange (ETDEWEB)

    Cao Jing; Wang Jiangan; Wu Ronghua, E-mail: caojing981@126.com [Col. of Electronic Eng., Naval University of Engineering, Wuhan 430033 (China)

    2011-01-01

    Dinoflagellate can be stimulated bioluminescence by hydrodynamic agitation. Two typical dinoflagellate (Lingulodinium polyedrum and Pyrocystis noctiluca) was choosed to research stimulated bioluminescence. The bioluminescence intensity and shear stress intensity were measured using fully developed pipe flow. There is shear stress threshold to agitate organism bioluminescence. From these experiment, the response thresholds of the stimulated bioluminscence always occurred in laminar flows at a shear stress level of 0.6-3 dyn/cm{sup 2}. At the same time, the spectral characteristc of dinoflagellate was recorded, the wavelength of them is about 470nm, and the full width at half maximum is approximate 30nm.

  9. In Vivo Imaging of Retinoic Acid Receptor Activity using a Sodium/Iodide Symporter and Luciferase Dual Imaging Reporter Gene

    Directory of Open Access Journals (Sweden)

    Min Kyung So

    2004-07-01

    Full Text Available Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/NL. After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.

  10. The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.

    Directory of Open Access Journals (Sweden)

    Katie J Herbst

    2009-05-01

    Full Text Available Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA.We present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc variants in a population of cells and also in single cells. Using 10 microM of luciferase substrate and 10 microM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (+/-15% and 54% (+/-14% of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8.The identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell

  11. Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells.

    Directory of Open Access Journals (Sweden)

    Tingting Xu

    Full Text Available Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity

  12. A Trp53fl/flPtenfl/fl mouse model of undifferentiated pleomorphic sarcoma mediated by adeno-Cre injection and in vivo bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Marisa R Buchakjian

    Full Text Available Genetic mouse models of soft tissue sarcoma provide critical insights into disease pathophysiology, which are oftentimes unable to be extracted from human tumor samples or xenograft models. In this study we describe a mouse model of soft tissue sarcoma mediated by adenoviral-Cre recombinase injection into Trp53fl/fl/Ptenfl/fl lox-stop-lox luciferase mice. Injection of adenovirus expressing Cre recombinase, either subcutaneously or intramuscularly in two experimental groups, results in viral infection and gene recombination with 100% penetrance within the first 24 hours following injection. Luciferase expression measured by real-time bioluminescence imaging increases over time, with an initial robust increase following viral injection, followed by a steady rise over the next several weeks as primary tumors develop and grow. Intramuscular injections were more commonly associated with evidence of systemic viral distribution than subcutaneous injections. All mice developed soft tissue sarcomas at the primary injection site, with histological examination identifying 93% of tumors as invasive pleomorphic sarcomas based on microscopic morphology and immunohistochemical expression of sarcoma markers. A lymphocytic infiltrate was present in 64% of the sarcomas in this immunocompetent model and 71% of tumors expressed PD-L1. This is the first report of a viral-Cre mediated Trp53/Pten mouse model of undifferentiated pleomorphic sarcoma. The bioluminescence imaging feature, along with high penetrance of the model and its immunological characteristics, makes it suited for pre-clinical studies of soft tissue sarcoma.

  13. Reporter gene bioassays in environmental analysis.

    Science.gov (United States)

    Köhler, S; Belkin, S; Schmid, R D

    2000-01-01

    In parallel to the continuous development of increasingly more sophisticated physical and chemical analytical technologies for the detection of environmental pollutants, there is a progressively more urgent need also for bioassays which report not only on the presence of a chemical but also on its bioavailability and its biological effects. As a partial fulfillment of that need, there has been a rapid development of biosensors based on genetically engineered bacteria. Such microorganisms typically combine a promoter-operator, which acts as the sensing element, with reporter gene(s) coding for easily detectable proteins. These sensors have the ability to detect global parameters such as stress conditions, toxicity or DNA-damaging agents as well as specific organic and inorganic compounds. The systems described in this review, designed to detect different groups of target chemicals, vary greatly in their detection limits, specificity, response times and more. These variations reflect on their potential applicability which, for most of the constructs described, is presently rather limited. Nevertheless, present trends promise that additional improvements will make microbial biosensors an important tool for future environmental analysis.

  14. Advancing Molecular Therapies through In Vivo Bioluminescent Imaging

    Directory of Open Access Journals (Sweden)

    Anton McCaffrey

    2003-04-01

    Full Text Available Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLI is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

  15. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research

    OpenAIRE

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have bee...

  16. Development of Optical Molecular Imaging System for the Acquisition of Bioluminescence Signals from Small Animals

    International Nuclear Information System (INIS)

    Lee, Byeong Il; Kim, Hyeon Sik; Jeong, Hye Jin; Lee, Hyung Jae; Moon, Seung Min; Kwon, Seung Young; Jeong, Shin Young; Bom, Hee Seung; Min, Jung Joon; Choi, Eun Seo

    2009-01-01

    Optical imaging is providing great advance and improvement in genetic and molecular imaging of animals and humans. Optical imaging system consists of optical imaging devices, which carry out major function for monitoring, tracing, and imaging in most of molecular in-vivo researches. In bio-luminescent imaging, small animals containing luciferase gene locally irradiate light, and emitted photons transmitted through skin of the small animals are imaged by using a high sensitive charged coupled device (CCD) camera. In this paper, we introduced optical imaging system for the image acquisition of bio-luminescent signals emitted from small animals. In the system, Nikon lens and four LED light sources were mounted at the inside of a dark box. A cooled CCD camera equipped with a control module was used. We tested the performance of the optical imaging system using effendorf tube and light emitting bacteria which injected intravenously into CT26 tumor bearing nude mouse. The performance of implemented optical imaging system for bio-luminescence imaging was demonstrated and the feasibility of the system in small animal imaging application was proved. We anticipate this system could be a useful tool for the molecular imaging of small animals adaptable for various experimental conditions in future

  17. Bioluminescent imaging: a critical tool in pre-clinical oncology research.

    LENUS (Irish Health Repository)

    O'Neill, Karen

    2010-02-01

    Bioluminescent imaging (BLI) is a non-invasive imaging modality widely used in the field of pre-clinical oncology research. Imaging of small animal tumour models using BLI involves the generation of light by luciferase-expressing cells in the animal following administration of substrate. This light may be imaged using an external detector. The technique allows a variety of tumour-associated properties to be visualized dynamically in living models. The increasing use of BLI as a small-animal imaging modality has led to advances in the development of xenogeneic, orthotopic, and genetically engineered animal models expressing luciferase genes. This review aims to provide insight into the principles of BLI and its applications in cancer research. Many studies to assess tumour growth and development, as well as efficacy of candidate therapeutics, have been performed using BLI. More recently, advances have also been made using bioluminescent imaging in studies of protein-protein interactions, genetic screening, cell-cycle regulators, and spontaneous cancer development. Such novel studies highlight the versatility and potential of bioluminescent imaging in future oncological research.

  18. Bacillus subtilis spore survival and expression of germination-induced bioluminescence after prolonged incubation under simulated Mars atmospheric pressure and composition: implications for planetary protection and lithopanspermia

    Science.gov (United States)

    Nicholson, Wayne L.; Schuerger, Andrew C.

    2005-01-01

    Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7-18 mbar) and composition (100% CO(2)) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes.

  19. Assessing the bioavailability of organic contaminants using a novel bioluminescent biosensor

    International Nuclear Information System (INIS)

    Keane, A.; Phoenix, P.; Lau, P.C.K.; Ghoshal, S.

    2002-01-01

    The limited rate and extent of biodegradation in contaminated soils is often attributed to a lack of bioavailability of hydrophobic organic compounds. To date, the majority of studies aimed at assessing bioavailability and modes of bacterial uptake have relied upon quantification of microbial degradation rates in comparison to rates of dissolution or desorption in corresponding abiotic systems. Several studies have indicated the possibility of a direct uptake mechanism for sorbed or separate phase compounds. However, there is a lack of direct evidence to support these claims. To address the need for a direct measurement technique for microbial bioavailability, we have constructed a whole-cell bioluminescent biosensor, Pseudomonas putida F1G4 (PpF1G4), by fusing lux genes that encode for bioluminescence to the solvent efflux pump (sep) promoter element in PpF1G4, which is induced by the presence of target organic compounds. When the biosensor microorganism is exposed to an inducing compound, the bioluminescence system is activated and the cell produces an intensity of visible light (λ = 495 nm) that is directly related to the level of exposure to the contaminant. Batch experiments were carried out to assess whether the biosensor is able to sense the presence of toluene, a representative target compound, contained in a NAPL. Preliminary results show that while PpF1G4 responds to toluene in the aqueous phase, the biosensor does not appear to emit a significant bioluminescence signal in response to the toluene present in the NAPL. Ongoing research is focusing on optimizing the experimental procedure to fully explore this issue. (author)

  20. In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

    Science.gov (United States)

    Fang, Mingzhu; Kang, Hwan-Goo; Park, Youngil; Estrella, Brian; Zarbl, Helmut

    2017-09-28

    The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both

  1. Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Xiujun Fan

    Full Text Available BACKGROUND: Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts. METHODOLOGY/PRINCIPAL FINDINGS: Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3. Animals were examined for Fluc expression by live bioluminescence imaging (BLI at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages. CONCLUSIONS/SIGNIFICANCE: These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early

  2. BIOLUMINESCENCE: TEACHING BIOCHEMISTRY BEYOND THE UNIVERSITY WALLS

    Directory of Open Access Journals (Sweden)

    Ana Paula Jesus de Almeida

    2016-11-01

    Full Text Available INTRODUCTION: The use of video in teaching and learning processes provides a challenging environment, able to stimulate the intellect and facilitate understanding in life science studies. Videos can be of extraordinary importance in education and dissemination of knowledge, contributing to greater learning, but is rarely used and exploited properly, especially for teaching biochemistry. Biochemistry is considered complex because it involves many molecular structures and processes, especially considering the number of events and molecules involved in the metabolism. OBJECTIVES: This study aimed to introduce biochemistry for the students of basic education using the theme "Light, Science and Life" in a playful and fun way. MATERIALS AND METHODS: A video about bioluminescence was designed and prepared aiming to use it as a support for learning biochemistry by students of basic education of public schools located in Salvador, Bahia. In order to prepare the video, undergraduate students initially revised the literature in order to acquire proper knowledge, and along with their teacher advisor worked the elaboration of texts, textbook and questionnaire and applied at school. DISCUSSION AND RESULTS: Analysis the qualitative results of the experiment on the preparation and use of the video about "Bioluminescence" focused mainly on the content of biochemistry linked to theme Light, Science and Life, and demonstrated the importance of such work in the teaching-learning process. The dynamics used allowed greater interaction between students and teacher, and the teaching of biochemistry in a fun way beyond the university walls. CONCLUSION: The teaching through recreational resources, e.g. videos and other educational strategies that foster learning should be encouraged from basic education, always bearing in order to transmit through these teaching methods the main concepts covered in biochemistry.

  3. Bioluminescent bacteria have potential as a marker of drowning in seawater: two immersed cadavers retrieved near estuaries.

    Science.gov (United States)

    Kakizaki, Eiji; Kozawa, Shuji; Sakai, Masahiro; Yukawa, Nobuhiro

    2009-03-01

    We detected numerous bioluminescent bacteria in blood samples from two cadavers that had been immersed in estuarine environments. Autopsy, diatomaceous and toxicological findings indicated death by drowning, which agreed with environmental aspects and the findings of police investigations. Bioluminescent bacteria appeared in blood samples cultured on selective agar containing 2%, 3% and 4% NaCl after about 18h. Blood from the left side of the heart, the right side of the heart and the femoral vein generated 7.0 x 10(2), 2.0 x 10(4) and 8.0 x 10(2) cfu/ml of blood (case 1), and 1.8 x 10(4), 1.1 x 10(3) and 2.5 x 10(1) cfu/ml (case 2) of bioluminescent colonies, respectively, in agar containing 4% NaCl. Homologous analysis based on the 16S rRNA gene also identified the bioluminescent colonies as Vibrio fischeri and V. harveyi, which normally inhabit seawater. This simple assay might serve as an additional indicator to support a conclusion of death by drowning together with the diatom test.

  4. Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae

    Science.gov (United States)

    Knafo, Steven; Prendergast, Andrew; Thouvenin, Olivier; Figueiredo, Sophie Nunes; Wyart, Claire

    2017-01-01

    The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry. PMID:29130058

  5. Developmentally regulated expression of reporter gene in adult ...

    Indian Academy of Sciences (India)

    pression of reporter gene in adult brain specific GAL4 enhancer traps of. Drosophila ... genes based on their expression pattern, thus enabling us to overcome the ... order association and storage centres of olfactory learning and memory, and ...

  6. Action of γ-radiation on bioluminescence of Noctiluca miliaris

    International Nuclear Information System (INIS)

    Tokarev, Yu.N.

    1976-01-01

    Results of the study in the action of various doses of irradiation on the bioluminescence of Noctiluca miliaris are presented. The doses are found that stimulate the bioluminescence and the dose - effect curves are obtained. It has been shown that stimulation of Noctiluca luminescence by γ-radiation is not of a constant character and extinguishes after a period of time determined by a dose rate

  7. Symplectin evolved from multiple duplications in bioluminescent squid

    DEFF Research Database (Denmark)

    Francis, Warren R.; Christianson, Lynne M.; Haddock, Steven H.D.

    2017-01-01

    The squid Sthenoteuthis oualaniensis, formerly Symplectoteuthis oualaniensis, generates light using the luciferin coelenterazine and a unique enzyme, symplectin. Genetic information is limited for bioluminescent cephalopod species, so many proteins, including symplectin, occur in public databases...... functioning is conserved across essentially all members of the protein family, even those unlikely to be used for bioluminescence. Conversely, active site residues involved in pantetheinase catalysis are also conserved across essentially all of these proteins, suggesting that symplectin may have multiple...

  8. Far red bioluminescence from two deep-sea fishes.

    Science.gov (United States)

    Widder, E A; Latz, M I; Herring, P J; Case, J F

    1984-08-03

    Spectral measurements of red bioluminescence were obtained from the deep-sea stomiatoid fishes Aristostomias scintillans (Gilbert) and Malacosteus niger (Ayres). Red luminescence from suborbital light organs extends to the near infrared, with peak emission at approximately 705 nanometers in the far red. These fishes also have postorbital light organs that emit blue luminescence with maxima between 470 and 480 nanometers. The red bioluminescence may be due to an energy transfer system and wavelength-selective filtering.

  9. Biological water quality monitoring using chemiluminescent and bioluminescent techniques

    Science.gov (United States)

    Thomas, R. R.

    1978-01-01

    Automated chemiluminescence and bioluminescence sensors were developed for the continuous monitoring of microbial levels in water supplies. The optimal chemical procedures were determined for the chemiluminescence system to achieve maximum sensitivity. By using hydrogen peroxide, reaction rate differentiation, ethylene diamine tetraacetic acid (EDTA), and carbon monoxide pretreatments, factors which cause interference were eliminated and specificity of the reaction for living and dead bacteria was greatly increased. By employing existing technology with some modifications, a sensitive and specific bioluminescent system was developed.

  10. Human reporter genes: potential use in clinical studies

    Energy Technology Data Exchange (ETDEWEB)

    Serganova, Inna [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Ponomarev, Vladimir [Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Blasberg, Ronald [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States)], E-mail: blasberg@neuro1.mskcc.org

    2007-10-15

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  11. Human reporter genes: potential use in clinical studies

    International Nuclear Information System (INIS)

    Serganova, Inna; Ponomarev, Vladimir; Blasberg, Ronald

    2007-01-01

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  12. Bathyphotometer bioluminescence potential measurements: A framework for characterizing flow agitators and predicting flow-stimulated bioluminescence intensity

    Science.gov (United States)

    Latz, Michael I.; Rohr, Jim

    2013-07-01

    Bathyphotometer measurements of bioluminescence are used as a proxy for the abundance of luminescent organisms for studying population dynamics; the interaction of luminescent organisms with physical, chemical, and biological oceanographic processes; and spatial complexity especially in coastal areas. However, the usefulness of bioluminescence measurements has been limited by the inability to compare results from different bathyphotometer designs, or even the same bathyphotometer operating at different volume flow rates. The primary objective of this study was to compare measurements of stimulated bioluminescence of four species of cultured dinoflagellates, the most common source of bioluminescence in coastal waters, using two different bathyphotometer flow agitators as a function of bathyphotometer volume flow rate and dinoflagellate concentration. For both the NOSC and BIOLITE flow agitators and each species of dinoflagellate tested, there was a critical volume flow rate, above which average bioluminescence intensity, designated as bathyphotometer bioluminescence potential (BBP), remained relatively constant and scaled directly with dinoflagellate cell concentration. At supra-critical volume flow rates, the ratio of BIOLITE to NOSC BBP was nearly constant for the same species studied, but varied between species. The spatial pattern and residence time of flash trajectories within the NOSC flow agitator indicated the presence of dominant secondary recirculating flows, where most of the bioluminescence was detected. A secondary objective (appearing in the Appendix) was to study the feasibility of using NOSC BBP to scale flow-stimulated bioluminescence intensity across similar flow fields, where the contributing composition of luminescent species remained the same. Fully developed turbulent pipe flow was chosen because it is hydrodynamically well characterized. Average bioluminescence intensity in a 2.54-cm i.d. pipe was highly correlated with wall shear stress and

  13. Melatonin Entrains PER2::LUC Bioluminescence Circadian Rhythm in the Mouse Cornea

    Science.gov (United States)

    Baba, Kenkichi; Davidson, Alec J.; Tosini, Gianluca

    2015-01-01

    Purpose Previous studies have reported the presence of a circadian rhythm in PERIOD2::LUCIFERASE (PER2::LUC) bioluminescence in mouse photoreceptors, retina, RPE, and cornea. Melatonin (MLT) modulates many physiological functions in the eye and it is believed to be one of the key circadian signals within the eye. The aim of the present study was to investigate the regulation of the PER2::LUC circadian rhythm in mouse cornea and to determine the role played by MLT. Methods Corneas were obtained from PER2::LUC mice and cultured to measure bioluminescence rhythmicity in isolated tissue using a Lumicycle or CCD camera. To determine the time-dependent resetting of the corneal circadian clocks in response to MLT or IIK7 (a melatonin type 2 receptor, MT2, agonist) was added to the cultured corneas at different times of the day. We also defined the location of the MT2 receptor within different corneal layers using immunohistochemistry. Results A long-lasting bioluminescence rhythm was recorded from cultured PER2::LUC cornea and PER2::LUC signal was localized to the corneal epithelium and endothelium. MLT administration in the early night delayed the cornea rhythm, whereas administration of MLT at late night to early morning advanced the cornea rhythm. Treatment with IIK7 mimicked the MLT phase-shifting effect. Consistent with these results, MT2 immunoreactivity was localized to the corneal epithelium and endothelium. Conclusions Our work demonstrates that MLT entrains the PER2::LUC bioluminescence rhythm in the cornea. Our data indicate that the cornea may represent a model to study the molecular mechanisms by which MLT affects the circadian clock. PMID:26207312

  14. Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado

    Directory of Open Access Journals (Sweden)

    ETELVINO J.H. BECHARA

    Full Text Available ABSTRACT Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera belonging to the Lampyridae (fireflies, Elateridae (click-beetles, and Phengodidae (railroad-worms families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the “luminous termite mounds” in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

  15. Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado.

    Science.gov (United States)

    Bechara, Etelvino J H; Stevani, Cassius V

    2018-01-01

    Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

  16. In Vivo Imaging with Bioluminescent Enterovirus 71 Allows for Real-Time Visualization of Tissue Tropism and Viral Spread.

    Science.gov (United States)

    Caine, Elizabeth A; Osorio, Jorge E

    2017-03-01

    Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis. IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to

  17. Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy.

    Science.gov (United States)

    Suzuki, Kazushi; Onishi, Takahito; Nakada, Chieko; Takei, Shunsuke; Daniels, Matthew J; Nakano, Masahiro; Matsuda, Tomoki; Nagai, Takeharu

    2018-05-18

    Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca 2+ indicator GmNL(Ca 2+ ), and its application in a customized microscope for high-throughput drug screening. GmNL(Ca 2+ ) gives a 140% signal change with Ca 2+ , and can image drug-induced changes of Ca 2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca 2+ ) with this adaptation, we could image spontaneous Ca 2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

  18. Assays for noninvasive imaging of reporter gene expression

    International Nuclear Information System (INIS)

    Gambhir, S.S.; Barrio, J.R.; Herschman, H.R.; Phelps, M.E.

    1999-01-01

    Repeated, noninvasive imaging of reporter gene expression is emerging as a valuable tool for monitoring the expression of genes in animals and humans. Monitoring of organ/cell transplantation in living animals and humans, and the assessment of environmental, behavioral, and pharmacologic modulation of gene expression in transgenic animals should soon be possible. The earliest clinical application is likely to be monitoring human gene therapy in tumors transduced with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene. Several candidate assays for imaging reporter gene expression have been studied, utilizing cytosine deaminase (CD), HSV1-tk, and dopamine 2 receptor (D2R) as reporter genes. For the HSV1-tk reporter gene, both uracil nucleoside derivatives (e.g., 5-iodo-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil [FIAU] labeled with 124 I, 131 I ) and acycloguanosine derivatives {e.g., 8-[ 18 F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (8-[ 18 F]-fluoroganciclovir) ([ 18 F]FGCV), 9-[(3-[ 18 F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG)} have been investigated as reporter probes. For the D2R reporter gene, a derivative of spiperone {3-(2'-[ 18 F]-Fluoroethyl)spiperone ([ 18 F]FESP)} has been used with positron emission tomography (PET) imaging. In this review, the principles and specific assays for imaging reporter gene expression are presented and discussed. Specific examples utilizing adenoviral-mediated delivery of a reporter gene as well as tumors expressing reporter genes are discussed

  19. Oculogryphus chenghoiyanae sp. n. (Coleoptera, Lampyridae: a new ototretine firefly from Hong Kong with descriptions of its bioluminescent behavior and ultraviolet-induced fluorescence in females

    Directory of Open Access Journals (Sweden)

    Vor Yiu

    2018-02-01

    Full Text Available The first Oculogryphus species with associated males and female was found in Hong Kong and is described as new: O. chenghoiyanae sp. n. Adults of both sexes were collected live in the field and their bioluminescent behavior is reported for the first time in the genus. The captive males emit weak and continuous light from a pair of light spots on abdominal ventrite 6 or do so when disturbed. The larviform (highly paedomorphic females can glow brightly from a pair of light-emitting organs on the abdomen. The females of Oculogryphus and Stenocladius are to date the only documented representatives of paedomorphism in ototretine fireflies. The finding is consistent with the evidence from male morphology and bioluminescent behavior, supporting the close relationship between the two genera. A key to the Oculogryphus species is provided. The Oculogryphus females can fluoresce with a blue-green light through the whole body under ultraviolet illumination, a phenomenon reported in the Lampyridae for the first time. The co-occurrence of bioluminescence and fluorescence is rare in terrestrial ecosystems, previously known only in some millipedes (Diplopoda. The fluorescence and bioluminescence abilities of Oculogryphus females are functionally independent: abdominal light-emitting organs producing bright yellowish green light while the body wall fluoresces with blue-green light. In contrast, fluorescence and bioluminescence in millipedes are biochemically linked, like in some jellyfish (Cnidaria: Medusozoa.

  20. A portable bioluminescence engineered cell-based biosensor for on-site applications.

    Science.gov (United States)

    Roda, Aldo; Cevenini, Luca; Michelini, Elisa; Branchini, Bruce R

    2011-04-15

    We have developed a portable biosensing device based on genetically engineered bioluminescent (BL) cells. Cells were immobilized on a 4 × 3 multiwell cartridge using a new biocompatible matrix that preserved their vitality. Using a fiber optic taper, the cartridge was placed in direct contact with a cooled CCD sensor to image and quantify the BL signals. Yeast and bacterial cells were engineered to express recognition elements, whose interaction with the analyte led to luciferase expression, via reporter gene technology. Three different biosensors were developed. The first detects androgenic compounds using yeast cells carrying a green-emitting P. pyralis luciferase regulated by the human androgen receptor and a red mutant of the same species as internal vitality control. The second biosensor detects two classes of compounds (androgens and estrogens) using yeast strains engineered to express green-or red-emitting mutant firefly luciferases in response to androgens or estrogens, respectively. The third biosensor detects lactose analogue isopropyl β-d-1-thiogalactopyranoside using two E. coli strains. One strain exploits the lac operon as recognition element for the expression of P. pyralis luciferase. The other strain serves as a vitality control expressing Gaussia princeps luciferase, which requires a different luciferin substrate. The immobilized cells were stable for up to 1 month. The analytes could be detected at nanomolar levels with good precision and accuracy when the specific signal was corrected using the internal vitality control. This portable device can be used for on-site multiplexed bioassays for different compound classes. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

    Science.gov (United States)

    Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

    2016-10-01

    The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

  2. Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus

    NARCIS (Netherlands)

    Daghighi, Seyedmojtaba; Sjollema, Jelmer; Harapanahalli, Akshay; Dijkstra, Rene J. B.; van der Mei, Henny C.; Busscher, Henk J.

    2015-01-01

    Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in

  3. What Orthopaedic Operating Room Surfaces Are Contaminated With Bioburden? A Study Using the ATP Bioluminescence Assay.

    Science.gov (United States)

    Richard, Raveesh Daniel; Bowen, Thomas R

    2017-07-01

    surfaces had bioburden. The ATP RLUs (mean ± SD) are reported for each surface in ascending order: the OR preparation table (postdraping; 8.3 ± 3.4), inside the sterilized pan (9.2 ± 5.5), the inside of the Bair Hugger™ hose (212.5 ± 155.7), supply closet countertops (281.7 ± 236.7), OR light handles (647.8 ± 903.7), the OR preparation table (predraping; 1054 ± 387.5), the Clark-socket attachment (1135.7 ± 705.3), patient positioners used for total hip and spine positioning (1201.7 ± 1144.9), Bovie machine buttons (1264.5 ± 638.8), Bair Hugger™ buttons (1340.8 ± 1064.1), tourniquet machine buttons (1666.5 ± 2144.9), computer keyboard (1810.8 ± 929.6), and the right side of the OR table headboard (2539 ± 5635.8). ATP bioluminescence is a novel method to measure cleanliness within the orthopaedic OR and can help identify environmental trouble spots that can potentially lead to increased infection rates. Future studies correlating ATP bioluminescence findings with microbiology cultures could add to the clinical utility of this technology. Surfaces such as the undersurface of the OR table headboard, Bair Hugger™ buttons, and tourniquet machine buttons should be routinely cleansed as part of an institutional protocol. Although correlation between ATP bioluminescence and clinical infection was not evaluated in this study, it is the subject of future research. Specifically, evaluating microbiology samples taken from these environmental surfaces and correlating them with increased bioburden found with ATP bioluminescence technology can help promote improved surgical cleaning practices.

  4. In vivo bioluminescence imaging using orthotopic xenografts towards patient's derived-xenograft Medulloblastoma models.

    Science.gov (United States)

    Asadzadeh, Fatemeh; Ferrucci, Veronica; DE Antonellis, Pasqualino; Zollo, Massimo

    2017-03-01

    Medulloblastoma is a cerebellar neoplasia of the central nervous system. Four molecular subgrups have been identified (MBWNT, MBSHH, MBgroup3 and MBgroup4) with distinct genetics and clinical outcome. Among these, MBgroup3-4 are highly metastatic with the worst prognosis. The current standard therapy includes surgery, radiation and chemotherapy. Thus, specific treatments adapted to cure those different molecular subgroups are needed. The use of orthotopic xenograft models, together with the non-invasive in vivo biolumiscence imaging (BLI) technology, is emerging during preclinical studies to test novel therapeutics for medulloblastoma treatment. Orthotopic MB xenografts were performed by injection of Daoy-luc cells, that had been previously infected with lentiviral particles to stably express luciferase gene, into the fourth right ventricle of the cerebellum of ten nude mice. For the implantation, specific stereotactic coordinates were used. Seven days after the implantation the mice were imaged by acquisitions of bioluminescence imaging (BLI) using IVIS 3D Illumina Imaging System (Xenogen). Tumor growth was evaluated by quantifying the bioluminescence signals using the integrated fluxes of photons within each area of interest using the Living Images Software Package 3.2 (Xenogen-Perkin Elmer). Finally, histological analysis using hematoxylin-eosin staining was performed to confirm the presence of tumorigenic cells into the cerebellum of the mice. We describe a method to use the in vivo bioluminescent imaging (BLI) showing the potential to be used to investigate the potential antitumorigenic effects of a drug for in vivo medulloblastoma treatment. We also discuss other studies in which this technology has been applied to obtain a more comprehensive knowledge of medulloblastoma using orthotopic xenograft mouse models. There is a need to develop patient's derived-xenograft (PDX) model systems to test novel drugs for medulloblastoma treatment within each molecular sub

  5. In Vivo Bioluminescence Imaging for Longitudinal Monitoring of Inflammation in Animal Models of Uveitis.

    Science.gov (United States)

    Gutowski, Michal B; Wilson, Leslie; Van Gelder, Russell N; Pepple, Kathryn L

    2017-03-01

    We develop a quantitative bioluminescence assay for in vivo longitudinal monitoring of inflammation in animal models of uveitis. Three models of experimental uveitis were induced in C57BL/6 albino mice: primed mycobacterial uveitis (PMU), endotoxin-induced uveitis (EIU), and experimental autoimmune uveitis (EAU). Intraperitoneal injection of luminol sodium salt, which emits light when oxidized, provided the bioluminescence substrate. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software. Bioluminescence on day zero was compared to bioluminescence on the day of peak inflammation for each model. Longitudinal bioluminescence imaging was performed in EIU and EAU. In the presence of luminol, intraocular inflammation generates detectable bioluminescence in three mouse models of uveitis. Peak bioluminescence in inflamed PMU eyes (1.46 × 105 photons/second [p/s]) was significantly increased over baseline (1.47 × 104 p/s, P = 0.01). Peak bioluminescence in inflamed EIU eyes (3.18 × 104 p/s) also was significantly increased over baseline (1.09 × 104 p/s, P = 0.04), and returned to near baseline levels by 48 hours. In EAU, there was a nonsignificant increase in bioluminescence at peak inflammation. In vivo bioluminescence may be used as a noninvasive, quantitative measure of intraocular inflammation in animal models of uveitis. Primed mycobacterial uveitis and EIU are both acute models with robust anterior inflammation and demonstrated significant changes in bioluminescence corresponding with peak inflammation. Experimental autoimmune uveitis is a more indolent posterior uveitis and generated a more modest bioluminescent signal. In vivo imaging system bioluminescence is a nonlethal, quantifiable assay that can be used for monitoring inflammation in animal models of uveitis.

  6. Advances of reporter gene imaging monitoring stem cell therapy

    International Nuclear Information System (INIS)

    Pei Zhijun; Zhang Yongxue

    2010-01-01

    Stem cell transplantation in the treatment of various tissue damage or degenerative diseases are research hotspots both at home and abroad. However, ignorance of the homing, differentiation and functional expression of the stem cell in vivo influence the further development of stem cell therapy. As an important component of molecular imaging technology, reporter gene imaging dynamically monitors the change of stem cell in vivo via monitoring the expression of transfected reporter gene. This paper briefly describes the latest research progress and the future development trend of the monitoring of reporter gene imaging in stem cell therapy in vivo. (authors)

  7. Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

    International Nuclear Information System (INIS)

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Wu, Anna M.; Braun, Jonathan

    2009-01-01

    Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124 I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 ± 0.2, 15.2 ± 1.3, and 4.6 ± 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 ± 0.8%ID/g. The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. (orig.)

  8. Novel peptide chemistry in terrestrial animals: natural luciferin analogues from the bioluminescent earthworm Fridericia heliota.

    Science.gov (United States)

    Dubinnyi, Maxim A; Tsarkova, Aleksandra S; Petushkov, Valentin N; Kaskova, Zinaida M; Rodionova, Natalja S; Kovalchuk, Sergey I; Ziganshin, Rustam H; Baranov, Mikhail S; Mineev, Konstantin S; Yampolsky, Ilia V

    2015-03-02

    We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. [Activation of the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones in the presence of nitrofurans and NO generators].

    Science.gov (United States)

    Zaĭtseva, Iu V; Granik, V G; Belik, A S; Koksharova, O A; Khmel', I A

    2010-01-01

    Nitrofurans (nitrofurazone, nitrofurantoin, furazidin, nifuroxazide), and nitric oxide generators (sodium nitroprusside and isosorbide mononitrate) in subinhibitory concentrations were shown to significantly increase the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones, signaling molecules of Quorum Sensing (QS) regulatory systems. The highest activation of bioluminescence (up to 250-400 fold) was observed in the presence of nitrofurazone on E. coli DH5alpha biosensors containing lux-reporter plasmids pSB401 or pSB536. However, this activation was not specifically associated with the functioning of QS systems. We suggest that the effect observed results from a direct action of nitrofurans and NO donors on the process of bioluminescence. The data indicate the necessity of using the biosensors that make it possible to detect specific effects of substances tested on QS regulation.

  10. Horse species symposium: a novel approach to monitoring pathogen progression during uterine and placental infection in the mare using bioluminescence imaging technology and lux-modified bacteria.

    Science.gov (United States)

    Ryan, P L; Christiansen, D L; Hopper, R M; Walters, F K; Moulton, K; Curbelo, J; Greene, J M; Willard, S T

    2011-05-01

    Uterine and placental infections are the leading cause of abortion, stillbirth, and preterm delivery in the mare. Whereas uterine and placental infections in women have been studied extensively, a comprehensive examination of the pathogenic processes leading to this unsatisfactory pregnancy outcome in the mare has yet to be completed. Most information in the literature relating to late-term pregnancy loss in mares is based on retrospective studies of clinical cases submitted for necropsy. Here we report the development and application of a novel approach, whereby transgenically modified bacteria transformed with lux genes of Xenorhabdus luminescens or Photorhabdus luminescens origin and biophotonic imaging are utilized to better understand pathogen-induced preterm birth in late-term pregnant mares. This technology uses highly sensitive bioluminescence imaging camera systems to localize and monitor pathogen progression during tissue invasion by measuring the bioluminescent signatures emitted by the lux-modified pathogens. This method has an important advantage in that it allows for the potential tracking of pathogens in vivo in real time and over time, which was hitherto impossible. Although the application of this technology in domestic animals is in its infancy, investigators were successful in identifying the fetal lungs, sinuses, nares, urinary, and gastrointestinal systems as primary tissues for pathogen invasion after experimental infection of pregnant mares with lux-modified Escherichia coli. It is important that pathogens were not detected in other vital organs, such as the liver, brain, and cardiac system. Such precision in localizing sites of pathogen invasion provides potential application for this novel approach in the development of more targeted therapeutic interventions for pathogen-related diseases in the equine and other domestic species.

  11. Cloning-free regulated monitoring of reporter and gene expression

    Directory of Open Access Journals (Sweden)

    Demirkaya Omer

    2009-03-01

    Full Text Available Abstract Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

  12. GAL4 enhancer trap strains with reporter gene expression during ...

    Indian Academy of Sciences (India)

    the development of adult brain in Drosophila melanogaster. C. R. VENKATESH ... vous system (CNS), at different time points during the pupal stage—a critical .... in frontal view, with further reduced reporter gene expression. Orthodenticle and ...

  13. Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.; Eremeeva, Elena V.; Malikova, Natalia P.; Cheng, Chongyun; Liu, Zhi-Jie; Vysotski, Eugene S.

    2016-09-01

    The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

  14. Filtering and deconvolution for bioluminescence imaging of small animals

    International Nuclear Information System (INIS)

    Akkoul, S.

    2010-01-01

    This thesis is devoted to analysis of bioluminescence images applied to the small animal. This kind of imaging modality is used in cancerology studies. Nevertheless, some problems are related to the diffusion and the absorption of the tissues of the light of internal bioluminescent sources. In addition, system noise and the cosmic rays noise are present. This influences the quality of the images and makes it difficult to analyze. The purpose of this thesis is to overcome these disturbing effects. We first have proposed an image formation model for the bioluminescence images. The processing chain is constituted by a filtering stage followed by a deconvolution stage. We have proposed a new median filter to suppress the random value impulsive noise which corrupts the acquired images; this filter represents the first block of the proposed chain. For the deconvolution stage, we have performed a comparative study of various deconvolution algorithms. It allowed us to choose a blind deconvolution algorithm initialized with the estimated point spread function of the acquisition system. At first, we have validated our global approach by comparing our obtained results with the ground truth. Through various clinical tests, we have shown that the processing chain allows a significant improvement of the spatial resolution and a better distinction of very close tumor sources, what represents considerable contribution for the users of bioluminescence images. (author)

  15. Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca

    NARCIS (Netherlands)

    Eremeeva, Elena V.; Bartsev, Sergey I.; Berkel, van Willem J.H.; Vysotski, Eugene S.

    2017-01-01

    Upon binding their metal ion cofactors, Ca2+-regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca2+-regulated

  16. Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

    Science.gov (United States)

    2010-09-30

    photodiode array are simultaneously burst sampled through integrating transimpedance amplifiers with an integration period of 0.1 s. 64-bursts are...clear acrylic test section to reduce the optical path length and reducing integrator capacitance to increase transimpedance gain. A later improvement...gain, higher resolution analog to digital conversion, and greater transimpedance gain. Figure 5 Bioluminescence intensities from Pyrocystis

  17. REVIEW ARTICLE: Bioluminescent signals and the role of reflectors

    Science.gov (United States)

    Herring, Peter J.

    2000-11-01

    Organisms in a well lit environment use optical signals derived from the selective reflection of ambient light. In a dim or dark environment it is very difficult (because of low photon numbers) to detect the contrast between light reflected from the organism and that from the background, and many organisms use bioluminescent signals instead. The use of such signals on land is largely restricted to sexual signalling by the luminous beetles, but in the deep ocean their use is widespread, involving both many different organisms and a range of uses which parallel those of reflective signals on land. Some bioluminescent signals rely almost entirely on an optically unmodified light source (e.g. a secretion) but others depend upon complex optical structures, particularly reflectors, in the light-emitting organs. Reflectors in the light organs of many shrimp, squid and fish are based on constructive interference systems but employ different biological materials. They and other structures modify the angular, spectral and intensity distributions of bioluminescent signals. The ready availability of highly efficient biological reflectors has been a formative influence in the evolution of bioluminescent signalling in the sea.

  18. Structure of fungal oxyluciferin, the product of the bioluminescence reaction.

    Science.gov (United States)

    Purtov, K V; Osipova, Z M; Petushkov, V N; Rodionova, N S; Tsarkova, A S; Kotlobay, A A; Chepurnykh, T V; Gorokhovatsky, A Yu; Yampolsky, I V; Gitelson, J I

    2017-11-01

    The structure of fungal oxyluciferin was determined, the enzymatic bioluminescence reaction under substrate saturation conditions with discrete monitoring of formed products was conducted, and the structures of the end products of the reaction were established. On the basis of these studies, the scheme of oxyluciferin degradation to the end products was developed. The structure of fungal oxyluciferin was confirmed by counter synthesis.

  19. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

    OpenAIRE

    Buckley, SM; Delhove, JM; Perocheau, DP; Karda, R; Rahim, AA; Howe, SJ; Ward, NJ; Birrell, MA; Belvisi, MG; Arbuthnot, P; Johnson, MR; Waddington, SN; McKay, TR

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruse...

  20. Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

    Science.gov (United States)

    Song, Qinxin; Wei, Guijiang; Zhou, Guohua

    2014-07-01

    A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a dual cassette reporter system capable of assessing target gene translation and target gene product folding. The present invention further relates to vectors and host cells comprising the dual cassette reporter system. In addition the invention relates to the use...... of the dual cassette reporter system for assessing target gene translation and target gene product folding....

  2. Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria.

    Science.gov (United States)

    Thouand, Gérald; Daniel, Philippe; Horry, Habib; Picart, Pascal; Durand, Marie José; Killham, Ken; Knox, Oliver G G; DuBow, Michael S; Rousseau, Michel

    2003-01-01

    The purpose of the present paper was to study the influence of bacteria harbouring the luciferase-encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch-culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram-negative Escherichia coli::luxAB strains and a Gram-positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491-500 nm (+/- 5 nm) and a second peak at 585-595 (+/- 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550-650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram-positive and Gram-negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright 2003 John Wiley & Sons, Ltd.

  3. Bioluminescence imaging in a medium-sized animal by local injection of d-luciferin

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Sung Min; Min, Jung Joon; Kim, Sung Mi; Bom, Hee Seung [Chonnam National University Medical School, Gwangju (Korea, Republic of); Oh, Suk Jung; Kang, Han Saem; Kim, Kwang Yoon [ECOBIO INC., Gwangju (Korea, Republic of); Kim, Young Ho [Chosun University, Gwangju (Korea, Republic of)

    2005-07-01

    Luciferase is one of the most commonly used reporter enzymes in the field of molecular imaging. D-luciferin is known as the substrate for luciferase enzyme and its cost is very expensive. Therefore, the bioluminescence molecular imaging study has been allowed in small animals such as mice and rats. In this current study, we validated local injection of D-luciferin in articular capsule for bioluminescence imaging in rabbits. Chondrocytes were cultured and infected by replication-defective adenoviral vector encoding firefly luciferase. And then was performed different method of chondrocyte cell injection and transplantation into the knee of rabbits. The rabbits underwent imaging by cooled CCD camera after local injection of D-luciferin (3mg) into experimental knee joint as well as contralateral normal knee joint on days 1, 5, 7, 9. We sought whether optimal imaging signal was acquired by using cooled CCD camera after local injection of D-luciferin. We successfully visualized injected or transplanted cells in knee joint by local injection of D-luciferin. Total photon flux (7.86E+08 p/s/cm{sup 2}/sr) from the knee joint transplanted with cells approximately increased 10-fold more than (9.43E+07p/s/cm{sup 2}/sr) that from injected knee joints until 7 day. Imaging signal was observed in transplanted joints until day 9 after surgery while signal from injected knee was observed by day 7 after injection. We successfully carried out bioluminescence imaging study with medium sized animal by local injection of small amount of D-luciferin. Survival of chondrocytes were prolonged when surgically transplanted in joints than when directly injected in joint space.

  4. Filtering and deconvolution for bioluminescence imaging of small animals; Filtrage et deconvolution en imagerie de bioluminescence chez le petit animal

    Energy Technology Data Exchange (ETDEWEB)

    Akkoul, S.

    2010-06-22

    This thesis is devoted to analysis of bioluminescence images applied to the small animal. This kind of imaging modality is used in cancerology studies. Nevertheless, some problems are related to the diffusion and the absorption of the tissues of the light of internal bioluminescent sources. In addition, system noise and the cosmic rays noise are present. This influences the quality of the images and makes it difficult to analyze. The purpose of this thesis is to overcome these disturbing effects. We first have proposed an image formation model for the bioluminescence images. The processing chain is constituted by a filtering stage followed by a deconvolution stage. We have proposed a new median filter to suppress the random value impulsive noise which corrupts the acquired images; this filter represents the first block of the proposed chain. For the deconvolution stage, we have performed a comparative study of various deconvolution algorithms. It allowed us to choose a blind deconvolution algorithm initialized with the estimated point spread function of the acquisition system. At first, we have validated our global approach by comparing our obtained results with the ground truth. Through various clinical tests, we have shown that the processing chain allows a significant improvement of the spatial resolution and a better distinction of very close tumor sources, what represents considerable contribution for the users of bioluminescence images. (author)

  5. The progress of PET based reporter gene imaging

    International Nuclear Information System (INIS)

    Zhao Wei; Zhang Xiuli

    2005-01-01

    More than two decades of intense research have allowed gene therapy to move from the laboratory to the clinical setting, where its use for the treatment of human pathologies has been considerably increased in the last years. However, many crucial questions remain to be solved in this challenging field. In vivo imaging with positron emission tomography (PET) by combination of the appropriate PET reporter gene and PET reporter probe could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy. PET imaging could be used to define parameters not available by other techniques that are of substantial interest not only for the proper understanding of the gene therapy process, but also for its future development and clinical application in humans. (authors)

  6. Sensory Control and Function of Bacterial Bioluminescence

    National Research Council Canada - National Science Library

    Silverman, Michael

    1998-01-01

    ...) and for the synthesis of signal receptors (sensor kinases and response regulators). The genes and proteins defined by this study resemble elements of the phosphorelay paradigm known as two-component signal transduction...

  7. Bioluminescent Antibodies for Point-of-Care Diagnostics.

    Science.gov (United States)

    Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto; Johnsson, Kai

    2017-06-12

    We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  8. Advances of reporter gene monitoring stem cell therapy

    International Nuclear Information System (INIS)

    Zhou Xiang; Yin Hongyan; Zhang Yifan

    2010-01-01

    Stem cell therapy research has made great progress, demonstrating a broad application prospects. However, stem cell therapy as a new disease treatment, there are still many problems to be solved. Reporter gene imaging is a rapid development in recent years, a non-invasive, sensitive method of monitoring of stem cells, in particular radionuclide reporter gene imaging has high sensitivity and specificity of the advantages of strong and can carry out imaging of deep tissue and repeat imaging, is a tracer in vivo conditions, the most promising stem cell transplantation technique, showing good prospects for development. (authors)

  9. Bioluminescence determination of active caspase-3 in single apoptotic cells

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Klepárník, Karel; Matalová, Eva; Hegrová, Jitka; Přikryl, Jan; Švandová, Eva; Foret, František

    2013-01-01

    Roč. 34, č. 12 (2013), s. 1772-1777 ISSN 0173-0835 R&D Projects: GA ČR GAP206/11/2377 Grant - others:GA ČR(CZ) GAP502/12/1285 Program:GA Institutional support: RVO:68081715 ; RVO:67985904 Keywords : apoptosis * bioluminescence * caspase-3 Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.161, year: 2013

  10. Hyperspectral and multispectral bioluminescence optical tomography for small animal imaging

    International Nuclear Information System (INIS)

    Chaudhari, Abhijit J; Darvas, Felix; Bading, James R; Moats, Rex A; Conti, Peter S; Smith, Desmond J; Cherry, Simon R; Leahy, Richard M

    2005-01-01

    For bioluminescence imaging studies in small animals, it is important to be able to accurately localize the three-dimensional (3D) distribution of the underlying bioluminescent source. The spectrum of light produced by the source that escapes the subject varies with the depth of the emission source because of the wavelength-dependence of the optical properties of tissue. Consequently, multispectral or hyperspectral data acquisition should help in the 3D localization of deep sources. In this paper, we describe a framework for fully 3D bioluminescence tomographic image acquisition and reconstruction that exploits spectral information. We describe regularized tomographic reconstruction techniques that use semi-infinite slab or FEM-based diffusion approximations of photon transport through turbid media. Singular value decomposition analysis was used for data dimensionality reduction and to illustrate the advantage of using hyperspectral rather than achromatic data. Simulation studies in an atlas-mouse geometry indicated that sub-millimeter resolution may be attainable given accurate knowledge of the optical properties of the animal. A fixed arrangement of mirrors and a single CCD camera were used for simultaneous acquisition of multispectral imaging data over most of the surface of the animal. Phantom studies conducted using this system demonstrated our ability to accurately localize deep point-like sources and show that a resolution of 1.5 to 2.2 mm for depths up to 6 mm can be achieved. We also include an in vivo study of a mouse with a brain tumour expressing firefly luciferase. Co-registration of the reconstructed 3D bioluminescent image with magnetic resonance images indicated good anatomical localization of the tumour

  11. Image Reconstruction For Bioluminescence Tomography From Partial Measurement

    OpenAIRE

    Jiang, M.; Zhou, T.; Cheng, J. T.; Cong, W. X.; Wang, Ge

    2007-01-01

    The bioluminescence tomography is a novel molecular imaging technology for small animal studies. Known reconstruction methods require the completely measured data on the external surface, although only partially measured data is available in practice. In this work, we formulate a mathematical model for BLT from partial data and generalize our previous results on the solution uniqueness to the partial data case. Then we extend two of our reconstruction methods for BLT to this case. The first m...

  12. Repetitive Imaging of Reporter Gene Expression in the Lung

    Directory of Open Access Journals (Sweden)

    Jean-Christophe Richard

    2003-10-01

    Full Text Available Positron emission tomographic imaging is emerging as a powerful technology to monitor reporter transgene expression in the lungs and other organs. However, little information is available about its usefulness for studying gene expression over time. Therefore, we infected 20 rats with a replication-deficient adenovirus containing a fusion gene encoding for a mutant Herpes simplex virus type-1 thymidine kinase and an enhanced green fluorescent protein. Five additional rats were infected with a control virus. Pulmonary gene transfer was performed via intratracheal administration of vector using a surfactant-based method. Imaging was performed 4–6 hr, and 4, 7, and 10 days after gene transfer, using 9-(4-[18F]-fluoro-3-hydroxymethylbutylguanine, an imaging substrate for the mutant kinase. Lung tracer uptake assessed with imaging was moderately but significantly increased 4–6 hr after gene transfer, was maximal after 4 days, and was no longer detectable by 10 days. The temporal pattern of transgene expression measured ex vivo with in vitro assays of thymidine kinase activity and green fluorescent protein was similar to imaging. In conclusion, positron emission tomography is a reliable new tool to evaluate the onset and duration of reporter gene expression noninvasively in the lungs of intact animals.

  13. Quantitative and Functional Requirements for Bioluminescent Cancer Models.

    Science.gov (United States)

    Feys, Lynn; Descamps, Benedicte; Vanhove, Christian; Vermeulen, Stefan; Vandesompele, J O; Vanderheyden, Katrien; Messens, Kathy; Bracke, Marc; De Wever, Olivier

    2016-01-01

    Bioluminescent cancer models are widely used but detailed quantification of the luciferase signal and functional comparison with a non-transfected control cell line are generally lacking. In the present study, we provide quantitative and functional tests for luciferase-transfected cells. We quantified the luciferase expression in BLM and HCT8/E11 transfected cancer cells, and examined the effect of long-term luciferin exposure. The present study also investigated functional differences between parental and transfected cancer cells. Our results showed that quantification of different single-cell-derived populations are superior with droplet digital polymerase chain reaction. Quantification of luciferase protein level and luciferase bioluminescent activity is only useful when there is a significant difference in copy number. Continuous exposure of cell cultures to luciferin leads to inhibitory effects on mitochondrial activity, cell growth and bioluminescence. These inhibitory effects correlate with luciferase copy number. Cell culture and mouse xenograft assays showed no significant functional differences between luciferase-transfected and parental cells. Luciferase-transfected cells should be validated by quantitative and functional assays before starting large-scale experiments. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  14. Investigation of Processes and Factors Regulating the Generation, Maintenance and Breakdown of Bioluminescent Thin Layers

    National Research Council Canada - National Science Library

    Widder, Edith

    2001-01-01

    .... Katz's submersible holographic camera mounted on the upper work platform. Thin layers were located using real-time sensor feedback from intensified video recordings of stimulated bioluminescence...

  15. Bioluminescence in the Ocean: Origins of Biological, Chemical, and Ecological Diversity

    Science.gov (United States)

    Widder, E. A.

    2010-05-01

    From bacteria to fish, a remarkable variety of marine life depends on bioluminescence (the chemical generation of light) for finding food, attracting mates, and evading predators. Disparate biochemical systems and diverse phylogenetic distribution patterns of light-emitting organisms highlight the ecological benefits of bioluminescence, with biochemical and genetic analyses providing new insights into the mechanisms of its evolution. The origins and functions of some bioluminescent systems, however, remain obscure. Here, I review recent advances in understanding bioluminescence in the ocean and highlight future research efforts that will unite molecular details with ecological and evolutionary relationships.

  16. Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data

    DEFF Research Database (Denmark)

    Kannan, Soumya; Sams, Thomas; Maury, Jérôme

    2018-01-01

    activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system......Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds......, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter...

  17. Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice

    Directory of Open Access Journals (Sweden)

    Liu Zhihui

    2008-08-01

    Full Text Available Abstract Background Interleukin 1 beta (IL-1β plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. Results In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. Conclusion Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

  18. An adaptive regularization parameter choice strategy for multispectral bioluminescence tomography

    Energy Technology Data Exchange (ETDEWEB)

    Feng Jinchao; Qin Chenghu; Jia Kebin; Han Dong; Liu Kai; Zhu Shouping; Yang Xin; Tian Jie [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, P. O. Box 2728, Beijing 100190 (China); College of Electronic Information and Control Engineering, Beijing University of Technology, Beijing 100124 (China); Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, P. O. Box 2728, Beijing 100190 (China); Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, P. O. Box 2728, Beijing 100190 (China) and School of Life Sciences and Technology, Xidian University, Xi' an 710071 (China)

    2011-11-15

    Purpose: Bioluminescence tomography (BLT) provides an effective tool for monitoring physiological and pathological activities in vivo. However, the measured data in bioluminescence imaging are corrupted by noise. Therefore, regularization methods are commonly used to find a regularized solution. Nevertheless, for the quality of the reconstructed bioluminescent source obtained by regularization methods, the choice of the regularization parameters is crucial. To date, the selection of regularization parameters remains challenging. With regards to the above problems, the authors proposed a BLT reconstruction algorithm with an adaptive parameter choice rule. Methods: The proposed reconstruction algorithm uses a diffusion equation for modeling the bioluminescent photon transport. The diffusion equation is solved with a finite element method. Computed tomography (CT) images provide anatomical information regarding the geometry of the small animal and its internal organs. To reduce the ill-posedness of BLT, spectral information and the optimal permissible source region are employed. Then, the relationship between the unknown source distribution and multiview and multispectral boundary measurements is established based on the finite element method and the optimal permissible source region. Since the measured data are noisy, the BLT reconstruction is formulated as l{sub 2} data fidelity and a general regularization term. When choosing the regularization parameters for BLT, an efficient model function approach is proposed, which does not require knowledge of the noise level. This approach only requests the computation of the residual and regularized solution norm. With this knowledge, we construct the model function to approximate the objective function, and the regularization parameter is updated iteratively. Results: First, the micro-CT based mouse phantom was used for simulation verification. Simulation experiments were used to illustrate why multispectral data were used

  19. In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression.

    Science.gov (United States)

    Carlin, Sean; Pugachev, Andrei; Sun, Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C Clifton; Humm, John L

    2009-10-01

    To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the

  20. In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression

    International Nuclear Information System (INIS)

    Carlin, Sean; Pugachev, Andrei; Sun Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C. Clifton; Humm, John L.

    2009-01-01

    Purpose: To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer 18 F-fluoromisonidazole ( 18 F-FMISO). Methods: Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe 124 I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil ( 124 I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between 124 I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe 18 F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with 124 I-FIAU (3 h before sacrifice) and 18 F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between 18 F-FMISO and 124 I-FIAU on a pixel-by-pixel basis was performed. Results: Correlation coefficients between 124 I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between 18 F-FMISO and 124 I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. Conclusions: We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of

  1. Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

    Science.gov (United States)

    Cheng, Guyue; Dong, Xiaobing; Wang, Yulian; Peng, Dapeng; Wang, Xu; Hao, Haihong; Xie, Shuyu; Qu, Wei; Liu, Zhenli; Yuan, Zonghui

    2014-12-01

    Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100 μg kg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120 %, and the interassay coefficients of variation were less than 30 %. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known.

  2. SPECT imaging of cardiac reporter gene expression in living rabbits

    International Nuclear Information System (INIS)

    Liu Ying; Lan Xiaoli; Zhang Liang; Wu Tao; Jiang Rifeng; Zhang Yongxue

    2009-01-01

    This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine kinase (HSVI-tk) reporter gene in rabbits myocardium by using the reporter probe 131 I-2'-fluoro-2'-deoxy-l-β-D-arabinofuranosyl-5-iodouracil ( 131 I-FIAU) and SPECT. Rabbits of the study group received intramyocardial injection of Ad5-tk and control group received aseptic saline injection. Two sets of experiments were performed on the study group. Rabbits of the 1st set were injected with 131 I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1xl0 9 , 5x10 8 , 1x10 8 , 5x10 7 and 1x10 7 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1x10 9 , 5x10 8 , 1x10 8 , 5x10 7 , 1x10 7 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131 I-FIAU was injected and heart SPECT imaging was performed at 6, 24 and 48 h, before killing them for gamma counting of the hearts. Reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSVI-tk gene expression. Semi-quantitative analysis derived of region of interest (ROI) of SPECT images and RT-PCR images was performed and the relationship of SPECT images with ex vivo gamma counting and mRNA level were evaluated. SPECT images conformed 131 I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images quality was obtained at 24-48 h for different viral titers. The highest radioactivity in the focal myocardium was seen at 6 h, and then declined with time. The threshold was 5x10 7 pfu of virus titer. The result could be set better in 1-5x10 8 pfu by SPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECT images correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The HSVI-tk/ 131 I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter

  3. The pivotal role of multimodality reporter sensors in drug discovery: from cell based assays to real time molecular imaging.

    Science.gov (United States)

    Ray, Pritha

    2011-04-01

    Development and marketing of new drugs require stringent validation that are expensive and time consuming. Non-invasive multimodality molecular imaging using reporter genes holds great potential to expedite these processes at reduced cost. New generations of smarter molecular imaging strategies such as Split reporter, Bioluminescence resonance energy transfer, Multimodality fusion reporter technologies will further assist to streamline and shorten the drug discovery and developmental process. This review illustrates the importance and potential of molecular imaging using multimodality reporter genes in drug development at preclinical phases.

  4. Bioluminescence Tomography–Guided Radiation Therapy for Preclinical Research

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bin [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland (United States); Wang, Ken Kang-Hsin, E-mail: kwang27@jhmi.edu [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland (United States); Yu, Jingjing [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland (United States); School of Physics and Information Technology, Shaanxi Normal University, Shaanxi (China); Eslami, Sohrab; Iordachita, Iulian [Laboratory for Computational Sensing and Robotics, Johns Hopkins University, Baltimore, Maryland (United States); Reyes, Juvenal; Malek, Reem [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland (United States); Tran, Phuoc T. [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland (United States); Department of Oncology and Urology, Brady Urological Institute, Johns Hopkins University, Baltimore, Maryland (United States); Patterson, Michael S. [Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario (Canada); Wong, John W. [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland (United States)

    2016-04-01

    Purpose: In preclinical radiation research, it is challenging to localize soft tissue targets based on cone beam computed tomography (CBCT) guidance. As a more effective method to localize soft tissue targets, we developed an online bioluminescence tomography (BLT) system for small-animal radiation research platform (SARRP). We demonstrated BLT-guided radiation therapy and validated targeting accuracy based on a newly developed reconstruction algorithm. Methods and Materials: The BLT system was designed to dock with the SARRP for image acquisition and to be detached before radiation delivery. A 3-mirror system was devised to reflect the bioluminescence emitted from the subject to a stationary charge-coupled device (CCD) camera. Multispectral BLT and the incomplete variables truncated conjugate gradient method with a permissible region shrinking strategy were used as the optimization scheme to reconstruct bioluminescent source distributions. To validate BLT targeting accuracy, a small cylindrical light source with high CBCT contrast was placed in a phantom and also in the abdomen of a mouse carcass. The center of mass (CoM) of the source was recovered from BLT and used to guide radiation delivery. The accuracy of the BLT-guided targeting was validated with films and compared with the CBCT-guided delivery. In vivo experiments were conducted to demonstrate BLT localization capability for various source geometries. Results: Online BLT was able to recover the CoM of the embedded light source with an average accuracy of 1 mm compared to that with CBCT localization. Differences between BLT- and CBCT-guided irradiation shown on the films were consistent with the source localization revealed in the BLT and CBCT images. In vivo results demonstrated that our BLT system could potentially be applied for multiple targets and tumors. Conclusions: The online BLT/CBCT/SARRP system provides an effective solution for soft tissue targeting, particularly for small, nonpalpable, or

  5. Bioluminescence Tomography–Guided Radiation Therapy for Preclinical Research

    International Nuclear Information System (INIS)

    Zhang, Bin; Wang, Ken Kang-Hsin; Yu, Jingjing; Eslami, Sohrab; Iordachita, Iulian; Reyes, Juvenal; Malek, Reem; Tran, Phuoc T.; Patterson, Michael S.; Wong, John W.

    2016-01-01

    Purpose: In preclinical radiation research, it is challenging to localize soft tissue targets based on cone beam computed tomography (CBCT) guidance. As a more effective method to localize soft tissue targets, we developed an online bioluminescence tomography (BLT) system for small-animal radiation research platform (SARRP). We demonstrated BLT-guided radiation therapy and validated targeting accuracy based on a newly developed reconstruction algorithm. Methods and Materials: The BLT system was designed to dock with the SARRP for image acquisition and to be detached before radiation delivery. A 3-mirror system was devised to reflect the bioluminescence emitted from the subject to a stationary charge-coupled device (CCD) camera. Multispectral BLT and the incomplete variables truncated conjugate gradient method with a permissible region shrinking strategy were used as the optimization scheme to reconstruct bioluminescent source distributions. To validate BLT targeting accuracy, a small cylindrical light source with high CBCT contrast was placed in a phantom and also in the abdomen of a mouse carcass. The center of mass (CoM) of the source was recovered from BLT and used to guide radiation delivery. The accuracy of the BLT-guided targeting was validated with films and compared with the CBCT-guided delivery. In vivo experiments were conducted to demonstrate BLT localization capability for various source geometries. Results: Online BLT was able to recover the CoM of the embedded light source with an average accuracy of 1 mm compared to that with CBCT localization. Differences between BLT- and CBCT-guided irradiation shown on the films were consistent with the source localization revealed in the BLT and CBCT images. In vivo results demonstrated that our BLT system could potentially be applied for multiple targets and tumors. Conclusions: The online BLT/CBCT/SARRP system provides an effective solution for soft tissue targeting, particularly for small, nonpalpable, or

  6. Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2002-01-01

    Full Text Available Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD along with an optical reporter gene (luciferase. Following intratumoral injection of the vector into orthotopic 9L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging

  7. Experimental Study on Bioluminescence Tomography with Multimodality Fusion

    Directory of Open Access Journals (Sweden)

    Yujie Lv

    2007-01-01

    Full Text Available To verify the influence of a priori information on the nonuniqueness problem of bioluminescence tomography (BLT, the multimodality imaging fusion based BLT experiment is performed by multiview noncontact detection mode, which incorporates the anatomical information obtained by the microCT scanner and the background optical properties based on diffuse reflectance measurements. In the reconstruction procedure, the utilization of adaptive finite element methods (FEMs and a priori permissible source region refines the reconstructed results and improves numerical robustness and efficiency. The comparison between the absence and employment of a priori information shows that multimodality imaging fusion is essential to quantitative BLT reconstruction.

  8. Toxicity assessment of Hanford Site wastes by bacterial bioluminescence

    International Nuclear Information System (INIS)

    Rebagay, T.V.; Dodd, D.A.; Voogd, J.A.

    1991-09-01

    This paper examines the toxicity of the nonradioactive component of low-level wastes stored in tanks on the Hanford reservation. The use of a faster, cheaper bioassay to replace the 96 hour fish acute toxicity test is examined. The new bioassay is based on loss of bioluminescence of Photobacter phosphoreum (commonly called Microtox) following exposure to toxic materials. This bioassay is calibrated and compares well to the standard fish acute toxicity test for characterization of Hanford Wastes. 4 refs., 11 figs., 11 tabs

  9. DNA Nanoparticles: Detection of Long-Term Transgene Activity in Brain using Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    David M. Yurek

    2011-09-01

    Full Text Available In this study, we used bioluminescence imaging (BLI to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.

  10. Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

    Science.gov (United States)

    Kannan, Soumya; Sams, Thomas; Maury, Jérôme; Workman, Christopher T

    2018-03-16

    Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

  11. Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

    Science.gov (United States)

    Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

    2004-06-01

    The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

  12. hNIS-IRES-eGFP Dual Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2005-04-01

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

  13. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  14. Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kadonosono

    Full Text Available An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1 is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg mice that carry HRE/ODD-luciferase (HOL gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

  15. In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals

    International Nuclear Information System (INIS)

    Inoue, Yusuke; Okubo, Toshiyuki; Tojo, Arinobu; Sekine, Rieko; Soda, Yasushi; Kobayashi, Seiichiro; Nomura, Akiko; Izawa, Kiyoko; Kitamura, Toshio; Ohtomo, Kuni

    2006-01-01

    The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity. (orig.)

  16. Development of a Novel Preclinical Pancreatic Cancer Research Model: Bioluminescence Image-Guided Focal Irradiation and Tumor Monitoring of Orthotopic Xenografts1

    OpenAIRE

    Tuli, Richard; Surmak, Andrew; Reyes, Juvenal; Hacker-Prietz, Amy; Armour, Michael; Leubner, Ashley; Blackford, Amanda; Tryggestad, Erik; Jaffee, Elizabeth M; Wong, John; DeWeese, Theodore L; Herman, Joseph M

    2012-01-01

    PURPOSE: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. MATERIALS AND METHODS: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. B...

  17. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Gruenhagen, Jason Alan [Iowa State Univ., Ames, IA (United States)

    2003-01-01

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  18. Advances in study of perpes simplex virus type 1-thymidine kinase reporter gene imaging

    International Nuclear Information System (INIS)

    Liu Ying; Lan Xiaoli; Zhang Yongxue

    2007-01-01

    Radionuclide reporter gene imaging is an effect way to provide qualitative and quantitative information for gene therapy. There are three systems of reporter gene including kinase reporter gene. perpes simplex virus type 1-thymidine kinase (HSV1-tk) has perfect physical and chemical characteristic which is suit for imaging as reporter gene. It has been widely investigated and intensively researched. Two substrates of HSV1-tk are purine nucleosite derivant and acyclovir derivant, which can also be used as reporter probes of HSV1-tk. (authors)

  19. Validating tyrosinase homologue MelA as a photoacoustic reporter gene for imaging Escherichia coli

    Science.gov (United States)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert; Zemp, Roger

    2015-03-01

    Antibiotic drug resistance is a major worldwide issue. Development of new therapies against pathogenic bacteria requires appropriate research tools for replicating and characterizing infections. Previously fluorescence and bioluminescence modalities have been used to image infectious burden in animal models but scattering significantly limits imaging depth and resolution. We hypothesize that photoacoustic imaging, which has improved depth-toresolution ratio, could be useful for visualizing MelA-expressing bacteria since MelA is a bacterial tyrosinase homologue involved in melanin production. Using an inducible expression system, E. coli expressing MelA were visibly black in liquid culture. Phosphate buffered saline (PBS), MelA-expressing bacteria (at different dilutions in PBS), and chicken embryo blood were injected in plastic tubes which were imaged using a VisualSonics Vevo LAZR system. Photoacoustic imaging at 6 different wavelengths (680, 700, 750, 800, 850 and 900nm) enabled spectral de-mixing to distinguish melanin signals from blood. The signal to noise ratio of 9x diluted MelA bacteria was 55, suggesting that ~20 bacteria cells could be detected with our system. When MelA bacteria were injected as a 100 μL bolus into a chicken embryo, photoacoustic signals from deoxy- and oxy- hemoglobin as well as MelA-expressing bacteria could be separated and overlaid on an ultrasound image, allowing visualization of the bacterial location. Photoacoustic imaging may be a useful tool for visualizing bacterial infections and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  20. In vivo SPECT reporter gene imaging of regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Ehsan Sharif-Paghaleh

    Full Text Available Regulatory T cells (Tregs were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+CD25(+FoxP3(+ cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99mTcO(4(- and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6 mice by SPECT/CT using (99mTcO(4(-. After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.

  1. Rapid Analysis of Eukaryotic Bioluminescence to Assess Potential Groundwater Contamination Events

    Directory of Open Access Journals (Sweden)

    Zacariah L. Hildenbrand

    2015-01-01

    Full Text Available Here we present data using a bioluminescent dinoflagellate, Pyrocystis lunula, in a toxicological bioassay to rapidly assess potential instances of groundwater contamination associated with natural gas extraction. P. lunula bioluminescence can be quantified using spectrophotometry as a measurement of organismal viability, with normal bioluminescent output declining with increasing concentration(s of aqueous toxicants. Glutaraldehyde and hydrochloric acid (HCl, components used in hydraulic fracturing and shale acidization, triggered significant toxicological responses in as little as 4 h. Conversely, P. lunula was not affected by the presence of arsenic, selenium, barium, and strontium, naturally occurring heavy metal ions potentially associated with unconventional drilling activities. If exogenous compounds, such as glutaraldehyde and HCl, are thought to have been introduced into groundwater, quantification of P. lunula bioluminescence after exposure to water samples can serve as a cost-effective detection and risk assessment tool to rapidly assess the impact of putative contamination events attributed to unconventional drilling activity.

  2. Dive Activities for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Information about dive activities were recorded by personnel during the "Bioluminescence 2009" expedition, July 20 through 31, 2009. Additional information was...

  3. Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes.

    Science.gov (United States)

    Girotti, S; Ferri, E; Cascione, M L; Comuzio, S; Mazzuca, A; Orlandini, A; Breccia, A

    1989-07-01

    Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.

  4. Effect of irradiation on detection of bacteria in dehydrated vegetables with ATP bioluminescence assay

    International Nuclear Information System (INIS)

    Xiao Huan; Luo Shishi; Wang Zegang; Feng Min; Zhu Jiating; Chen Xiulan; Zhai Jianqing

    2011-01-01

    ATP bioluminescence intensity of 4 kinds of irradiated dehydrated vegetables was inconsistent with the bacteria number, the reasons were investigated in this paper. Results showed that irradiation had little effect on background luminescence, and there was no effect on luciferase-luminous system. When irradiation killed the bacteria, the ATPase activity also decreased. As a result, the ATP content in bacteria didn't decreased with the killed of bacteria, which contributed to the increase of free ATP in ATP extract and finally led to the disagreement between the bioluminescence intensity and the actual number of bacteria. When the free ATP in the dehydrated vegetable was removed, the bioluminescence intensity of ATP extract was consistent with the actual number of bacteria in irradiated dehydrated vegetable and ATP bioluminescence technology could be used in bacteria detection of irradiated samples. (authors)

  5. U-SPECT-BioFluo : An integrated radionuclide, bioluminescence, and fluorescence imaging platform

    NARCIS (Netherlands)

    Van Oosterom, M.N.; Kreuger, R.; Buckle, T.; Mahn, W.A.; Bunschoten, A.; Josephson, L.; Van Leeuwen, F.W.B.; Beekman, F.J.

    2014-01-01

    Background: In vivo bioluminescence, fluorescence, and single-photon emission computed tomography (SPECT) imaging provide complementary information about biological processes. However, to date these signatures are evaluated separately on individual preclinical systems. In this paper, we introduce a

  6. An Experimental-Numerical Study of Small Scale Flow Interaction with Bioluminescent Plankton

    National Research Council Canada - National Science Library

    Latz, Michael

    1998-01-01

    Numerical and experimental approaches were used to investigate the effects of quantified flow stimuli on bioluminescence sUmulatidn at the small length and time scales appropriate for individual plankton...

  7. Ship Sensor Observations for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Hourly measurements made by selected ship sensors on the R/V Seward Johnson during the "Bioluminescence 2009" expedition sponsored by the National Oceanic and...

  8. Submersible Data (Dive Waypoints) for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data and information collected by the submersible Johnson Sea-Link II at waypoints along its track during seventeen dives of the 2009 "Bioluminescence" expedition...

  9. Ship track for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Ship track of the R/V Seward Johnson during the "Bioluminescence 2009" expedition sponsored by the National Oceanic and Atmospheric Administration (NOAA) Office of...

  10. PET/CT imaging of human somatostatin receptor 2 (hsstr2) as reporter gene for gene therapy

    International Nuclear Information System (INIS)

    Hofmann, M.; Gazdhar, A.; Weitzel, T.; Schmid, R.; Krause, T.

    2006-01-01

    Localized information on region-selective gene expression in small animals is widely obtained by use of reporter genes inducing light emission. Using these reporter genes for imaging deep inside the human body fluorescent probes are hindered by attenuation, scattering and possible fluorescence quenching. This can be overcome by use of radio-peptide receptors as reporter genes. Therefore, the feasibility of the somatostatin receptor 2 expression vector system for expression imaging was checked against a control vector containing luciferase gene. For in vivo transduction of vector DNA into the rat forelimb muscles the in vivo electroporation technique was chosen because of its high regio-selectivity. The gene expression was imaged by high-sensitive CCD camera (luciferase activity) and by PET/CT using a Ga-68-DOTATOC as radio peptide probe. The relative sstr2 expression was enhanced by gene transduction at maximum to a factor of 15. The PET/CT images could be fully quantified. The above demonstrated feasibility of radio-peptide PET/CT reporter gene imaging may serve in the future as a tool for full quantitative understanding of regional gene expression, especially in large animals and humans

  11. PET/CT imaging of human somatostatin receptor 2 (hsstr2) as reporter gene for gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, M. [Molecular Imaging and Therapy Group (MIT-Bern), Clinic of Nuclear Medicine, Inselspital, Medical School Bern (Switzerland)]. E-mail: Michael.Hofmann@insel.ch; Gazdhar, A. [Division of Pulmonary Medicine, University Hospital Bern (Switzerland); Weitzel, T. [Molecular Imaging and Therapy Group (MIT-Bern), Clinic of Nuclear Medicine, Inselspital, Medical School Bern (Switzerland); Schmid, R. [Division of Thoracic Surgery, University Hospital Bern (Switzerland); Krause, T. [Molecular Imaging and Therapy Group (MIT-Bern), Clinic of Nuclear Medicine, Inselspital, Medical School Bern (Switzerland)

    2006-12-20

    Localized information on region-selective gene expression in small animals is widely obtained by use of reporter genes inducing light emission. Using these reporter genes for imaging deep inside the human body fluorescent probes are hindered by attenuation, scattering and possible fluorescence quenching. This can be overcome by use of radio-peptide receptors as reporter genes. Therefore, the feasibility of the somatostatin receptor 2 expression vector system for expression imaging was checked against a control vector containing luciferase gene. For in vivo transduction of vector DNA into the rat forelimb muscles the in vivo electroporation technique was chosen because of its high regio-selectivity. The gene expression was imaged by high-sensitive CCD camera (luciferase activity) and by PET/CT using a Ga-68-DOTATOC as radio peptide probe. The relative sstr2 expression was enhanced by gene transduction at maximum to a factor of 15. The PET/CT images could be fully quantified. The above demonstrated feasibility of radio-peptide PET/CT reporter gene imaging may serve in the future as a tool for full quantitative understanding of regional gene expression, especially in large animals and human000.

  12. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

    Directory of Open Access Journals (Sweden)

    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  13. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

    Science.gov (United States)

    Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

    2009-03-13

    Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  14. New bioreactor for in situ simultaneous measurement of bioluminescence and cell density

    Science.gov (United States)

    Picart, Pascal; Bendriaa, Loubna; Daniel, Philippe; Horry, Habib; Durand, Marie-José; Jouvanneau, Laurent; Thouand, Gérald

    2004-03-01

    This article presents a new device devoted to the simultaneous measurement of bioluminescence and optical density of a bioluminescent bacterial culture. It features an optoelectronic bioreactor with a fully autoclavable module, in which the bioluminescent bacteria are cultivated, a modulated laser diode dedicated to optical density measurement, and a detection head for the acquisition of both bioluminescence and optical density signals. Light is detected through a bifurcated fiber bundle. This setup allows the simultaneous estimation of the bioluminescence and the cell density of the culture medium without any sampling. The bioluminescence is measured through a highly sensitive photomultiplier unit which has been photometrically calibrated to allow light flux measurements. This was achieved by considering the bioluminescence spectrum and the full optical transmission of the device. The instrument makes it possible to measure a very weak light flux of only a few pW. The optical density is determined through the laser diode and a photodiode using numerical synchronous detection which is based on the power spectrum density of the recorded signal. The detection was calibrated to measure optical density up to 2.5. The device was validated using the Vibrio fischeri bacterium which was cultivated under continuous culture conditions. A very good correlation between manual and automatic measurements processed with this instrument has been demonstrated. Furthermore, the optoelectronic bioreactor enables determination of the luminance of the bioluminescent bacteria which is estimated to be 6×10-5 W sr-1 m-2 for optical density=0.3. Experimental results are presented and discussed.

  15. Bacterial bioluminescence onset and quenching: a dynamical model for a quorum sensing-mediated property

    OpenAIRE

    Side, Domenico Delle; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Di Salvo, Marco; Talà, Adelfia; Chechkin, Aleksei; Seno, Flavio; Trovato, Antonio

    2017-01-01

    We present an effective dynamical model for the onset of bacterial bioluminescence, one of the most studied quorum sensing-mediated traits. Our model is built upon simple equations that describe the growth of the bacterial colony, the production and accumulation of autoinducer signal molecules, their sensing within bacterial cells, and the ensuing quorum activation mechanism that triggers bioluminescent emission. The model is directly tested to quantitatively reproduce the experimental distri...

  16. Effect of Naphthalene and Salicylate Analogues on the Bioluminescence of Bioreporter Pseudomonas Fluorescens HK44.

    Czech Academy of Sciences Publication Activity Database

    Trögl, Josef; Kuncová, Gabriela; Kubicová, L.; Pařík, P.; Hálová, Jaroslava; Demnerová, K.; Ripp, S.; Sayler, G. S.

    2007-01-01

    Roč. 52, 1 (2007) , s. 3-14 ISSN 0015-5632 R&D Projects: GA ČR(CZ) GA104/05/2637; GA ČR(CZ) GA203/06/1244 Institutional research plan: CEZ:AV0Z40720504; CEZ:AV0Z40320502 Keywords : pseudomonas fluorescens HK44 * bioluminescence * bioluminescence Subject RIV: CE - Biochemistry Impact factor: 0.989, year: 2007

  17. Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: a case for bolus injections.

    Science.gov (United States)

    Sanches, Pedro Gomes; Mühlmeister, Mareike; Seip, Ralf; Kaijzel, Eric; Löwik, Clemens; Böhmer, Marcel; Tiemann, Klaus; Grüll, Holger

    2014-12-10

    Localized gene delivery has many potential clinical applications. However, the nucleic acids (e.g. pDNA and siRNA) are incapable of passively crossing the endothelium, cell membranes and other biological barriers which must be crossed to reach their intracellular targets. A possible solution is the use of ultrasound to burst circulating microbubbles inducing transient permeabilization of surrounding tissues which mediates nucleic acid extravasation and cellular uptake. In this study we report on an optimization of the ultrasound gene delivery technique. Naked pDNA (200 μg) encoding luciferase and SonoVue® microbubbles were co-injected intravenously in mice. The hindlimb skeletal muscles were exposed to ultrasound from a non-focused transducer (1 MHz, 1.25 MPa, PRI 30s) and injection protocols and total amounts as well as ultrasound parameters were systemically varied. Gene expression was quantified relative to a control using a bioluminescence camera system at day 7 after sonication. Bioluminescence ratios in sonicated/control muscles of up to 101× were obtained. In conclusion, we were able to specifically deliver genetic material to the selected skeletal muscles and overall, the use of bolus injections and high microbubble numbers resulted in increased gene expression reflected by stronger bioluminescence signals. Based on our data, bolus injections seem to be required in order to achieve transient highly concentrated levels of nucleic acids and microbubbles at the tissue of interest which upon ultrasound exposure should lead to increased levels of gene delivery. Thus, ultrasound mediated gene delivery is a promising technique for the clinical translation of localized drug delivery. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Evaluation of monkeypox virus infection of prairie dogs (Cynomys ludovicianus) using in vivo bioluminescent imaging

    Science.gov (United States)

    Falendysz, Elizabeth A.; Londoño-Navas, Angela M.; Meteyer, Carol U.; Pussini, Nicola; Lopera, Juan G.; Osorio, Jorge E.; Rocke, Tonie E.

    2014-01-01

    Monkeypox (MPX) is a re-emerging zoonotic disease that is endemic in Central and West Africa, where it can cause a smallpox-like disease in humans. Despite many epidemiologic and field investigations of MPX, no definitive reservoir species has been identified. Using recombinant viruses expressing the firefly luciferase (luc) gene, we previously demonstrated the suitability of in vivo bioluminescent imaging (BLI) to study the pathogenesis of MPX in animal models. Here, we evaluated BLI as a novel approach for tracking MPX virus infection in black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs were affected during a multistate outbreak of MPX in the US in 2003 and have since been used as an animal model of this disease. Our BLI results were compared with PCR and virus isolation from tissues collected postmortem. Virus was easily detected and quantified in skin and superficial tissues by BLI before and during clinical phases, as well as in subclinical secondary cases, but was not reliably detected in deep tissues such as the lung. Although there are limitations to viral detection in larger wild rodent species, BLI can enhance the use of prairie dogs as an animal model of MPX and can be used for the study of infection, disease progression, and transmission in potential wild rodent reservoirs.

  19. Detection and quantitation of circulating tumor cell dynamics by bioluminescence imaging in an orthotopic mammary carcinoma model.

    Directory of Open Access Journals (Sweden)

    Laura Sarah Sasportas

    Full Text Available Circulating tumor cells (CTCs have been detected in the bloodstream of both early-stage and advanced cancer patients. However, very little is know about the dynamics of CTCs during cancer progression and the clinical relevance of longitudinal CTC enumeration. To address this, we developed a simple bioluminescence imaging assay to detect CTCs in mouse models of metastasis. In a 4T1 orthotopic metastatic mammary carcinoma mouse model, we demonstrated that this quantitative method offers sensitivity down to 2 CTCs in 0.1-1mL blood samples and high specificity for CTCs originating from the primary tumor, independently of their epithelial status. In this model, we simultaneously monitored blood CTC dynamics, primary tumor growth, and lung metastasis progression over the course of 24 days. Early in tumor development, we observed low numbers of CTCs in blood samples (10-15 cells/100 µL and demonstrated that CTC dynamics correlate with viable primary tumor growth. To our knowledge, these data represent the first reported use of bioluminescence imaging to detect CTCs and quantify their dynamics in any cancer mouse model. This new assay is opening the door to the study of CTC dynamics in a variety of animal models. These studies may inform clinical decision on the appropriate timing of blood sampling and value of longitudinal CTC enumeration in cancer patients.

  20. Image analyzing method to evaluate in situ bioluminescence from an obligate anaerobe cultivated under various dissolved oxygen concentrations.

    Science.gov (United States)

    Ninomiya, Kazuaki; Yamada, Ryuji; Matsumoto, Masami; Fukiya, Satoru; Katayama, Takane; Ogino, Chiaki; Shimizu, Nobuaki

    2013-02-01

    An image analyzing method was developed to evaluate in situ bioluminescence expression, without exposing the culture sample to the ambient oxygen atmosphere. Using this method, we investigated the effect of dissolved oxygen concentration on bioluminescence from an obligate anaerobe Bifidobacterium longum expressing bacterial luciferase which catalyzes an oxygen-requiring bioluminescent reaction. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose

    Directory of Open Access Journals (Sweden)

    Madoka eYonekura

    2013-03-01

    Full Text Available Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter–luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light–dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both POsSPS1::LUC and POsSPS11::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the 2 OsSPS genes.

  2. Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

    Science.gov (United States)

    Luke, Garry A; Ryan, Martin D

    2018-01-01

    To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

  3. The origins of marine bioluminescence: turning oxygen defence mechanisms into deep-sea communication tools.

    Science.gov (United States)

    Rees, J F; de Wergifosse, B; Noiset, O; Dubuisson, M; Janssens, B; Thompson, E M

    1998-04-01

    Bioluminescence, the emission of ecologically functional light by living organisms, emerged independently on several occasions, yet the evolutionary origins of most bioluminescent systems remain obscure. We propose that the luminescent substrates of the luminous reactions (luciferins) are the evolutionary core of most systems, while luciferases, the enzymes catalysing the photogenic oxidation of the luciferin, serve to optimise the expression of the endogenous chemiluminescent properties of the luciferin. Coelenterazine, a luciferin occurring in many marine bioluminescent groups, has strong antioxidative properties as it is highly reactive with reactive oxygen species such as the superoxide anion or peroxides. We suggest that the primary function of coelenterazine was originally the detoxification of the deleterious oxygen derivatives. The functional shift from its antioxidative to its light-emitting function might have occurred when the strength of selection for antioxidative defence mechanisms decreased. This might have been made possible when marine organisms began colonising deeper layers of the oceans, where exposure to oxidative stress is considerably reduced because of reduced light irradiance and lower oxygen levels. A reduction in metabolic activity with increasing depth would also have decreased the endogenous production of reactive oxygen species. Therefore, in these organisms, mechanisms for harnessing the chemiluminescence of coelenterazine in specialised organs could have developed, while the beneficial antioxidative properties were maintained in other tissues. The full range of graded irradiance in the mesopelagic zone, where the majority of organisms are bioluminescent, would have provided a continuum for the selection and improvement of proto-bioluminescence. Although the requirement for oxygen or reactive oxygen species observed in bioluminescent systems reflects the high energy required to produce visible light, it may suggest that oxygen

  4. Feasibility of sodium/iodide symporter gene as a new imaging reporter gene: comparison with HSV1-tk

    International Nuclear Information System (INIS)

    Shin, Jae Hoon; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il; Kang, Joo Hyun; Jeong, Jae Min; Lee, Dong Soo; Kim, Chul Woo; Lee, Myung Chul

    2004-01-01

    Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D 2 receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium/iodide symporter (NIS) gene can be easily monitored with radioiodines and technetium-99m using a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([ 125 I]IVDU) and 125 I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [ 125 I]IVDU and 131 I, and 131 I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of 125 I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [ 125 I]IVDU than CT-26 and CTN. NIS gene expression and 125 I accumulation were found to be directly correlated (R 2 =0.923), as were HSV1-tk gene expression and [ 125 I]IVDU accumulation (R 2 =0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,240±3,755 cpm and 34,039±5,346 cpm, respectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of 131 I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [ 125 I]IVDU than CT-26 and CTN. Scintigraphy using 131 I easily visualised CTN and CMN tumours. In conclusion, the NIS gene may be viewed as an imaging

  5. Rapid drug susceptibility test of mycobacterium tuberculosis by bioluminescence sensor

    Science.gov (United States)

    Lu, Bin; Xu, Shunqing; Chen, Zifei; Zhou, Yikai

    2001-09-01

    With the persisting increase of drug-resistant stains of M. Tuberculosis around the world, rapid and sensitive detection of antibiotic of M. Tuberculosis is becoming more and more important. In the present study, drug susceptibility of M. tuberculosis were detected by recombination mycobacteriophage combined with bioluminescence sensor. It is based on the use of recombination mycobacteriophage which can express firefly luciferase when it infects viable mycobacteria, and can effectively produce quantifiable photon. Meanwhile, in mycobacterium cells treated with active antibiotic, no light is observed. The emitted light is recorded by a bioluminscence sensor, so the result of drug-resistant test can be determined by the naked eye. 159 stains of M. tuberculosis were applied to this test on their resistant to rifampin, streptomycin and isoniazid. It is found that the agreement of this assay with Liewenstein- Jensen slat is: rifampin 95.60 percent, isoniazid 91.82 percent, streptomycin 88.68 percent, which showed that it is a fast and practical method to scene and detect drug resistant of mycobacterium stains.

  6. Validating Bioluminescence Imaging as a High-Throughput, Quantitative Modality for Assessing Tumor Burden

    Directory of Open Access Journals (Sweden)

    Zain Paroo

    2004-04-01

    Full Text Available Bioluminescence imaging (BLI is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r > .8, p < .001 between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.

  7. Rapid and Quantitative Assessment of Cancer Treatment Response Using In Vivo Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2000-01-01

    Full Text Available Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies [1 ]. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal [2], subcutaneous, and intravascular [3] cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeuticinduced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI, respectively. There was excellent correlation (r=0.91 between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl-1-nitrosourea (BCNU treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951. These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

  8. Real-time bioluminescence imaging of macroencapsulated fibroblasts reveals allograft protection in rhesus monkeys (Macaca mulatta).

    Science.gov (United States)

    Tarantal, Alice F; Lee, C Chang I; Itkin-Ansari, Pamela

    2009-07-15

    Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte device was shown to provide long-term immunoprotection of murine islets in a mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner. Encapsulated cells were transplanted subcutaneously (n=2), or cells were injected without encapsulation (n=1) and outcomes compared. BLI was performed to monitor cell survival. The BLI signal from the encapsulated cells remained robust postinsertion and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days. These data demonstrate that TheraCyte encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys.

  9. Bioluminescence imaging of chondrocytes in rabbits by intraarticular injection of D-luciferin

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Sung Min; Min, Jung Joon; Kim, Sung Mi; Bom, Hee Seung [Chonnam National University Medical School, Gwangju (Korea, Republic of); Oh, Suk Jung; Kang, Han Saem; Kim, Kwang Yoon [ECOBIO INC., Gwangju (Korea, Republic of); Kim, Young Ho [College of Natural Science, Chosun University, Gwangju (Korea, Republic of)

    2007-02-15

    Luciferase is one of the most commonly used reporter enzymes in the field of in vivo optical imaging. D-luciferin, the substrate for firefly luciferase has very high cost that allows this kind of experiment limited to small animals such as mice and rats. In this current study, we validated local injection of D-luciferin in the articular capsule for bioluminescence imaging in rabbits. Chondrocytes were cultured and infected by replication-defective adenoviral vector encoding firefly luciferase (Fluc). Chondrocytes expressing Fluc were injected or implanted in the left knee joint. The rabbits underwent optical imaging studies after local injection of D-luciferin at 1, 5, 7, 9 days after cellular administration. We sought whether optimal imaging signals was could be by a cooled CCD camera after local injection of D-luciferin. Imaging signal was not observed from the left knee joint after intraperitoneal injection of D-luciferin (15 mg/kg), whereas it was observed after intraarticular injection. Photon intensity from the left knee joint of rabbits was compared between cell injected and implanted groups after intraarticular injection of D-luciferin. During the period of imaging studies, photon intensity of the cell implanted group was 5-10 times higher than that of the cell injected group. We successfully imaged chondrocytes expressing Fluc after intraarticular injection of D-luciferin. This technique may be further applied to develop new drugs for knee joint disease.

  10. Molecular MR imaging of cancer gene therapy. Ferritin transgene reporter takes the stage

    International Nuclear Information System (INIS)

    Hasegawa, Sumitaka; Furukawa, Takako; Saga, Tsuneo

    2010-01-01

    Molecular imaging using magnetic resonance (MR) imaging has been actively investigated and made rapid progress in the past decade. Applied to cancer gene therapy, the technique's high spatial resolution allows evaluation of gene delivery into target tissues. Because noninvasive monitoring of the duration, location, and magnitude of transgene expression in tumor tissues or cells provides useful information for assessing therapeutic efficacy and optimizing protocols, molecular imaging is expected to become a critical step in the success of cancer gene therapy in the near future. We present a brief overview of the current status of molecular MR imaging, especially in vivo reporter gene imaging using ferritin and other reporters, discuss its application to cancer gene therapy, and present our research of MR imaging detection of electroporation-mediated cancer gene therapy using the ferritin reporter gene. (author)

  11. Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

    Science.gov (United States)

    Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

    2011-01-01

    Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

  12. Visualization of ecdysteroid activity using a reporter gene in the crustacean, Daphnia.

    Science.gov (United States)

    Asada, Miki; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2014-02-01

    Ecdysone is a hormone known to play a pivotal role in crustaceans and insects. In order to evaluate the ecdysone activities in the environment and within the organism, we have developed a biomonitoring Daphnia strain by introducing a reporter gene. In this study, the ecdysone response element was inserted in the upstream region of a reporter gene, and the DNA construct was injected into Daphnia eggs. The expression of the reporter gene was detected during the early embryonic development stage. In addition, when the eggs expressing the reporter gene were exposed to ecdysone, there was enhanced expression of the reporter gene at detectable levels, while the presence of an antagonist led to its downregulation. These results suggested that this system could be potentially developed for monitoring ecdysone activities in media. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Photon hunting in the twilight zone: visual features of mesopelagic bioluminescent sharks.

    Directory of Open Access Journals (Sweden)

    Julien M Claes

    Full Text Available The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai and one dalatiid species (Squaliolus aliae]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax fall within the range 484-491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent

  14. Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

    Science.gov (United States)

    Affi, Mahmoud; Solliec, Camille; Legentilhomme, Patrick; Comiti, Jacques; Legrand, Jack; Jouanneau, Sulivan; Thouand, Gérald

    2016-12-01

    Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a

  15. Photon hunting in the twilight zone: visual features of mesopelagic bioluminescent sharks.

    Science.gov (United States)

    Claes, Julien M; Partridge, Julian C; Hart, Nathan S; Garza-Gisholt, Eduardo; Ho, Hsuan-Ching; Mallefet, Jérôme; Collin, Shaun P

    2014-01-01

    The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax) fall within the range 484-491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep

  16. The application of superweak bioluminescence on freshness degree of chicken egg

    International Nuclear Information System (INIS)

    Zhao Hongxia; Li Guochen; Li Qiangzheng; Li Juan

    2007-01-01

    The luminescence of chicken egg in storage is studied by a detection system of superweak bioluminescence. The results show that egg has the strongest vigour on the third day after it is laid, subsequently the luminescence presents decay with oscillation. These eggs, which have been stored for 3 days, are most suitable for hatching. Different eggs have different luminescence intensities depending on the vigour of the egg. The stronger the vigour of the egg is, the more intensive the luminescence is. Superweak bioluminescence as a comprehensive index of biology and biochemistry response can be used for inspecting the freshness degree of the egg, and the test is nondestructive and sensitive

  17. Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications.

    Science.gov (United States)

    Zampini, Massimiliano; Mur, Luis A J; Rees Stevens, Pauline; Pachebat, Justin A; Newbold, C James; Hayes, Finbarr; Kingston-Smith, Alison

    2016-05-25

    Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology.

  18. Transient expression of β-glucuronidase reporter gene in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... Therefore, special attention must be given to the use of genetic manipulation for .... of gus gene was used as a visual marker in transformation. Bacteria ... selective culture (SC) medium containing 200 mg/l carbenicillin and. 50 mg/l .... clones had caused the difference of host-plant response to bacterial ...

  19. Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas.

    Science.gov (United States)

    Abdelwahab, Mohammed G; Sankar, Tejas; Preul, Mark C; Scheck, Adrienne C

    2011-11-06

    The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are

  20. Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

    Directory of Open Access Journals (Sweden)

    Nan Wu

    Full Text Available BACKGROUND: Fluorescence and bioluminescence resonance energy transfer (F/BRET are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now. METHODOLOGY: We used a thrombin bioprobe based on a form of BRET (BRET(H, which uses the BRET(1 substrate, native coelenterazine, with the typical BRET(2 donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay. PRINCIPAL FINDINGS: The BRET(H thrombin bioprobe has a lower limit of detection (LOD than previously reported for the equivalent BRET(1-based version but it is substantially brighter than the BRET(2 version. The normalised BRET(H ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays. CONCLUSIONS: These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

  1. Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Schalén, Martin; Anyaogu, Diana Chinyere; Hoof, Jakob Blæsbjerg

    2016-01-01

    roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans....... The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. Results: Fourteen protein secretion...... pathway related genes were overexpressed with a tet-ON promoter in the RFP-secreting reporter strain and macromorphology, physiology and protein secretion were monitored when the secretory genes were induced. Overexpression of several of the chosen genes was shown to cause anomalies on growth, micro...

  2. Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter.

    Science.gov (United States)

    Kathiria, Palak; Sidler, Corinne; Woycicki, Rafal; Yao, Youli; Kovalchuk, Igor

    2013-07-01

    The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding β-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA.

  3. Transient expression of β-glucuronidase reporter gene in ...

    African Journals Online (AJOL)

    Agrobacterium tumefaciens strain LBA4404 carrying the pBI.121 binary plasmid was used in transformation to introduce the gus (ß-glucuronidase/GUS) gene into teak shoot-tissues. In vitro regenerated shoots from various teak clones, i.e. the ITB, GT, P97, P96, P75, P20, and P108 clones were vacuum-infiltrated for 5 min in ...

  4. Detection of antibodies in blood plasma using bioluminescent sensor proteins and a smartphone

    NARCIS (Netherlands)

    Arts, R.; den Hartog, I.; Zijlema, S.E.; Thijssen, V.; van der Beelen, S.H.E.; Merkx, M.

    2016-01-01

    Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies

  5. Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals

    NARCIS (Netherlands)

    Klerk, Clara P. W.; Overmeer, Renée M.; Niers, Tatjana M. H.; Versteeg, Henri H.; Richel, Dick J.; Buckle, Tessa; van Noorden, Cornelis J. F.; van Tellingen, Olaf

    2007-01-01

    A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLI for quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are

  6. Fast in vivo bioluminescence tomography using a novel pure optical imaging technique

    Directory of Open Access Journals (Sweden)

    Shuang Zhang

    2017-05-01

    Full Text Available Bioluminescence tomography (BLT is a novel optical molecular imaging technique that advanced the conventional planar bioluminescence imaging (BLI into a quantifiable three-dimensional (3D approach in preclinical living animal studies in oncology. In order to solve the inverse problem and reconstruct tumor lesions inside animal body accurately, the prior structural information is commonly obtained from X-ray computed tomography (CT. This strategy requires a complicated hybrid imaging system, extensive post imaging analysis and involvement of ionizing radiation. Moreover, the overall robustness highly depends on the fusion accuracy between the optical and structural information. Here, we present a pure optical bioluminescence tomographic (POBT system and a novel BLT workflow based on multi-view projection acquisition and 3D surface reconstruction. This method can reconstruct the 3D surface of an imaging subject based on a sparse set of planar white-light and bioluminescent images, so that the prior structural information can be offered for 3D tumor lesion reconstruction without the involvement of CT. The performance of this novel technique was evaluated through the comparison with a conventional dual-modality tomographic (DMT system and a commercialized optical imaging system (IVIS Spectrum using three breast cancer xenografts. The results revealed that the new technique offered comparable in vivo tomographic accuracy with the DMT system (P>0.05 in much shorter data analysis time. It also offered significantly better accuracy comparing with the IVIS system (P<0.04 without sacrificing too much time.

  7. Nucleic acid detection using BRET-beacons based on bioluminescent protein-DNA hybrids

    NARCIS (Netherlands)

    Engelen, W.; van de Wiel, K.M.; Meijer, L.H.H.; Saha, B.; Merkx, M.

    2017-01-01

    Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM

  8. Dual-color bioluminescent sensor proteins for therapeutic drug monitoring of antitumor antibodies

    NARCIS (Netherlands)

    van Rosmalen, M.; Ni, Y.; Vervoort, D.F.M.; Arts, R.; Ludwig, S.K.J.; Merkx, M.

    2018-01-01

    Monitoring the levels of therapeutic antibodies in individual patients would allow patient-specific dose optimization, with the potential for major therapeutic and financial benefits. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor)

  9. Integrated visualization of multi-angle bioluminescence imaging and micro CT

    NARCIS (Netherlands)

    Kok, P.; Dijkstra, J.; Botha, C.P.; Post, F.H.; Kaijzel, E.; Que, I.; Löwik, C.W.G.M.; Reiber, J.H.C.; Lelieveldt, B.P.F.

    2007-01-01

    This paper explores new methods to visualize and fuse multi-2D bioluminescence imaging (BLI) data with structural imaging modalities such as micro CT and MR. A geometric, back-projection-based 3D reconstruction for superficial lesions from multi-2D BLI data is presented, enabling a coarse estimate

  10. Modeling bioluminescent photon transport in tissue based on Radiosity-diffusion model

    Science.gov (United States)

    Sun, Li; Wang, Pu; Tian, Jie; Zhang, Bo; Han, Dong; Yang, Xin

    2010-03-01

    Bioluminescence tomography (BLT) is one of the most important non-invasive optical molecular imaging modalities. The model for the bioluminescent photon propagation plays a significant role in the bioluminescence tomography study. Due to the high computational efficiency, diffusion approximation (DA) is generally applied in the bioluminescence tomography. But the diffusion equation is valid only in highly scattering and weakly absorbing regions and fails in non-scattering or low-scattering tissues, such as a cyst in the breast, the cerebrospinal fluid (CSF) layer of the brain and synovial fluid layer in the joints. A hybrid Radiosity-diffusion model is proposed for dealing with the non-scattering regions within diffusing domains in this paper. This hybrid method incorporates a priori information of the geometry of non-scattering regions, which can be acquired by magnetic resonance imaging (MRI) or x-ray computed tomography (CT). Then the model is implemented using a finite element method (FEM) to ensure the high computational efficiency. Finally, we demonstrate that the method is comparable with Mont Carlo (MC) method which is regarded as a 'gold standard' for photon transportation simulation.

  11. Application de la bioluminescence au dénombrement des microorganismes vivants dans les vins

    Directory of Open Access Journals (Sweden)

    Aline Lonvaud-Funel

    1982-12-01

    Bioluminescence was applied to enumerate the microorganisms present in wine. An excellent correlation is obtained by counting colonies grown in Petri dishes. The simplicity of the manipulations and the rapid obtention of results are the principal benefits of this method. Research is still necessary both in the differentiation of yeasts and bacteria and the reduction of the threshold of detection.

  12. Observations and Measurements of Planktonic Bioluminescence in and Around a Milky Sea

    Science.gov (United States)

    1988-03-01

    malticharnel analysers operating in the multiscaler mode. The details of both the onboard underway system and the LPTC systems have been published (Lapota...the Arabian Sea during the southwest monsoon. No nutrient data was collected during our study, yet phosphates, nitrates , and trace BIOLUMINESCENCE IN

  13. Total evidence phylogeny and the evolution of adult bioluminescence in fireflies (Coleoptera: Lampyridae).

    Science.gov (United States)

    Martin, Gavin J; Branham, Marc A; Whiting, Michael F; Bybee, Seth M

    2017-02-01

    Fireflies are some of the most captivating organisms on the planet. They have a rich history as subjects of scientific study, especially in relation to their bioluminescent behavior. Yet, the phylogenetic relationships of fireflies are still poorly understood. Here, we present the first total evidence approach to reconstruct lampyrid phylogeny using both a molecular matrix from six loci and an extensive morphological matrix. Using this phylogeny we test the hypothesis that adult bioluminescence evolved after the origin of the firefly clade. The ancestral state of adult bioluminescence is recovered as non-bioluminescent with one to six gains and five to ten subsequent losses. The monophyly of the family, as well as the subfamilies is also tested. Ototretinae, Cyphonocerinae, Luciolinae (incl. Pristolycus), Amydetinae, "cheguevarinae" sensu Jeng 2008, and Photurinae are highly supported as monophyletic. With the exception of four taxa, Lampyrinae is also recovered as monophyletic with high support. Based on phylogenetic and morphological data Lamprohiza, Phausis, and Lamprigera are transferred to Lampyridae incertae sedis. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    Science.gov (United States)

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  15. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

    Directory of Open Access Journals (Sweden)

    Hackett Perry B

    2006-06-01

    Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

  16. The Use of Stimulable Bioluminescence From Dinoflagellates as a Means of Detecting Toxicity in the Marine Environment

    Science.gov (United States)

    1993-03-01

    AND SSTýTL FUNDINCG NUMI)) W, TIHE USE OF STIMt LABILE BIOLUMINESCENCE FROM DI NOIFLAGELLATk. PH: M1E69 AS A MEAN’S OF DETrECTING ToxicITY IN THE...bioluminescence dinoflagellates for asseossmnent of toxic effects when exposed to a single tox~icant or mixture. Successful use of this type of bioassav... tributyltin chloride (TFITCI), Copper (11) Sulfate (CuSO 4 I. zinc sulfate (ZnSO4 ), or storm drain effluent. Stimulable bioluminescence was measured at

  17. Bioluminescence Detection of Cells Having Stabilized p53 in Response to a Genotoxic Event

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2004-01-01

    Full Text Available Inactivation of p53 is one of the most frequent molecular events in neoplastic transformation. Approximately 60% of all human tumors have mutations in both p53 alleles. Wild-type p53 activity is regulated in large part by the proteosome-dependent degradation of p53, resulting in a short p53 half-life in unstressed and untransformed cells. Activation of p53 by a variety of stimuli, including DNA damage induced by genotoxic drugs or radiation, is accomplished by stabilization of wild-type p53. The stabilized and active p53 can result in either cell-cycle arrest or apoptosis. Surprisingly, the majority of tumor-associated, inactivating p53 mutations also result in p53 accumulation. Thus, constitutive elevation of p53 levels in cells is a reliable measure of p53 inactivation, whereas transiently increased p53 levels reflect a recent genotoxic stress. In order to facilitate noninvasive imaging of p53 accumulation, we here describe the construction of a p53-luciferase fusion protein. Induction of DNA damage in cells expressing the fusion protein resulted in a time-dependent accumulation of the fusion that was noninvasively detected using bioluminescence imaging and validated by Western blot analysis. The p53-Luc protein retains p53 function because its expression in HCT116 cells lacking functional p53 resulted in activation of p21 expression as well as induction of apoptosis in response to a DNA damaging event. Employed in a transgenic animal model, the proposed p53-reporter fusion protein will be useful for studying p53 activation in response to exposure to DNA-damaging carcinogenic agents. It could also be used to study p53 stabilization as a result of inactivating p53 mutations. Such studies will further our understanding of p53's role as the “guardian of the genome” and its function in tumorigenesis.

  18. Development of a Novel Preclinical Pancreatic Cancer Research Model: Bioluminescence Image-Guided Focal Irradiation and Tumor Monitoring of Orthotopic Xenografts1

    Science.gov (United States)

    Tuli, Richard; Surmak, Andrew; Reyes, Juvenal; Hacker-Prietz, Amy; Armour, Michael; Leubner, Ashley; Blackford, Amanda; Tryggestad, Erik; Jaffee, Elizabeth M; Wong, John; DeWeese, Theodore L; Herman, Joseph M

    2012-01-01

    PURPOSE: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. MATERIALS AND METHODS: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response. RESULTS: Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls. CONCLUSIONS: We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response. PMID:22496923

  19. Development of a novel preclinical pancreatic cancer research model: bioluminescence image-guided focal irradiation and tumor monitoring of orthotopic xenografts.

    Science.gov (United States)

    Tuli, Richard; Surmak, Andrew; Reyes, Juvenal; Hacker-Prietz, Amy; Armour, Michael; Leubner, Ashley; Blackford, Amanda; Tryggestad, Erik; Jaffee, Elizabeth M; Wong, John; Deweese, Theodore L; Herman, Joseph M

    2012-04-01

    We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response. Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls. We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response.

  20. Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

    Science.gov (United States)

    In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

    1997-03-01

    Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

  1. Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

    Science.gov (United States)

    Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

    2017-09-18

    Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

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

    Directory of Open Access Journals (Sweden)

    Shu-Ye Jiang

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

  3. Non-invasive imaging using reporter genes altering cellular water permeability

    Science.gov (United States)

    Mukherjee, Arnab; Wu, Di; Davis, Hunter C.; Shapiro, Mikhail G.

    2016-12-01

    Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

  4. The Repetitive Detection of Toluene with Bioluminescence Bioreporter Pseudomonas putida TVA8 Encapsulated in Silica Hydrogel on an Optical Fiber.

    Czech Academy of Sciences Publication Activity Database

    Kuncová, Gabriela; Ishizaki, Takayuki; Solovyev, Andrey; Trögl, J.; Ripp, S.

    2016-01-01

    Roč. 9, č. 6 (2016), s. 467 ISSN 1996-1944 Institutional support: RVO:67985858 Keywords : bioluminescent biosensor * silica gel * encapsulation Subject RIV: CC - Organic Chemistry Impact factor: 2.654, year: 2016

  5. Ship track for Islands in the Stream 2002 - Pharmaceutical Discovery, Vision, and Bioluminescence - Office of Ocean Exploration

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Ship track of the R/V Seward Johnson during the 2002 "Islands in the Stream - Pharmaceutical Discovery, Vision, and Bioluminescence" expedition sponsored by the...

  6. Biological Education of IVFRU and FIAU for HSV1-TK Reporter Gene Monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Su Hee; Kim, Eun Jung; Lee, Eun Ah; Lee, Jong Chan; Choi, Tae Hyun; Lee, Kyo Chul; An, Gwang Il; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    The Herpes Simplex Virus Type1-thymidine kinase (HSV1-TK) system is a useful gene therapy monitoring method. HSV1-TK is one of the most widely used effector gene systems used for imaging gene expression, in association with its use as a reporter gene. It has resulted the development of a number of radiolabeled HSV1-TK substrates for the non-invasive detection of HSV1-TK expression. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. Prodrug activation or sucide gene therapy has been shown to be successful in potentiating the therapeutic index by sensitizing genetically modified tumor cells to various prodrugs or enhancing the action of commonly used chemotherapeutic agents. The most studied prodrug activation approaches involve transfection of tumors with HSV1-TK gene. (Z)-5-(2-iodovinyl)-2'-fluoro- 2'-deoxyuridine (IVFRU) possesses a 2'-fluoro substituent in the ribose configuration, is considered to protect IVFRU from enzyme mediated degradation in vivo. It is obviously potential substrates for HSV1-TK imaging. 2'-Fiuoro-2'-deoxy-1-{beta}-D-arabinofuranosyl- 5-iodo-uridine (FIAU), an anticancer drug widely used in clinical practice, is an analogue of thymidine. In a series of studies using adenovirus vector for gene transfer described the appropriate combination of exogenously introduced HSV1-TK as a 'marker/reporter gene' and radiolabelled FIAU as a 'marker substrate/reporter probe' for monitoring gene therapy and gene expression.

  7. Biological Education of IVFRU and FIAU for HSV1-TK Reporter Gene Monitoring

    International Nuclear Information System (INIS)

    Hong, Su Hee; Kim, Eun Jung; Lee, Eun Ah; Lee, Jong Chan; Choi, Tae Hyun; Lee, Kyo Chul; An, Gwang Il; Cheon, Gi Jeong

    2006-01-01

    The Herpes Simplex Virus Type1-thymidine kinase (HSV1-TK) system is a useful gene therapy monitoring method. HSV1-TK is one of the most widely used effector gene systems used for imaging gene expression, in association with its use as a reporter gene. It has resulted the development of a number of radiolabeled HSV1-TK substrates for the non-invasive detection of HSV1-TK expression. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. Prodrug activation or sucide gene therapy has been shown to be successful in potentiating the therapeutic index by sensitizing genetically modified tumor cells to various prodrugs or enhancing the action of commonly used chemotherapeutic agents. The most studied prodrug activation approaches involve transfection of tumors with HSV1-TK gene. (Z)-5-(2-iodovinyl)-2'-fluoro- 2'-deoxyuridine (IVFRU) possesses a 2'-fluoro substituent in the ribose configuration, is considered to protect IVFRU from enzyme mediated degradation in vivo. It is obviously potential substrates for HSV1-TK imaging. 2'-Fiuoro-2'-deoxy-1-β-D-arabinofuranosyl- 5-iodo-uridine (FIAU), an anticancer drug widely used in clinical practice, is an analogue of thymidine. In a series of studies using adenovirus vector for gene transfer described the appropriate combination of exogenously introduced HSV1-TK as a 'marker/reporter gene' and radiolabelled FIAU as a 'marker substrate/reporter probe' for monitoring gene therapy and gene expression

  8. In vivo bioluminescence imaging for leptomeningeal dissemination of medulloblastoma in mouse models

    International Nuclear Information System (INIS)

    Choi, Seung Ah; Kwak, Pil Ae; Kim, Seung-Ki; Park, Sung-Hye; Lee, Ji Yeoun; Wang, Kyu-Chang; Oh, Hyun Jeong; Kim, Kyuwan; Lee, Dong Soo; Hwang, Do Won; Phi, Ji Hoon

    2016-01-01

    The primary cause of treatment failure in medulloblastomas (MB) is the development of leptomeningeal dissemination (seeding). For translational research on MB seeding, one of the major challenges is the development of reliable experimental models that simulate the seeding and growth characteristics of MBs. To overcome this obstacle, we improved an experimental mouse model by intracisternal inoculation of human MB cells and monitoring with in vivo live images. Human MB cells (UW426, D283 and MED8A) were transfected with a firefly luciferase gene and a Thy1.1 (CD90.1) marker linked with IRES under the control of the CMV promoter in a retroviral DNA backbone (effLuc). The MB-effLuc cells were injected into the cisterna magna using an intrathecal catheter, and bioluminescence images were captured. We performed histopathological analysis to confirm the extent of tumor seeding. The luciferase activity of MB-effLuc cells displayed a gradually increasing pattern, which correlated with a quantitative luminometric assay. Live imaging showed that the MB-effLuc cells were diffusely distributed in the cervical spinal cord and the lumbosacral area. All mice injected with UW426-effLuc, D283-effLuc and MED8A-effLuc died within 51 days. The median survival was 22, 41 and 12 days after injection of 1.2 × 10 6 UW426-effLuc, D283-effLuc and MED8A-effLuc cells, respectively. The histopathological studies revealed that the MB-effLuc cells spread extensively and diffusely along the leptomeninges of the brain and spinal cord, forming tumor cell-coated layers. The tumor cells in the subarachnoid space expressed a human nuclei marker and Ki-67. Compared with the intracerebellar injection method in which the subfrontal area and distal spinal cord were spared by tumor cell seeding in some mice, the intracisternal injection model more closely resembled the widespread leptomeningeal seeding observed in MB patients. The results and described method are valuable resources for further

  9. In vivo bioluminescence imaging validation of a human biopsy-derived orthotopic mouse model of glioblastoma multiforme.

    Science.gov (United States)

    Jarzabek, Monika A; Huszthy, Peter C; Skaftnesmo, Kai O; McCormack, Emmet; Dicker, Patrick; Prehn, Jochen H M; Bjerkvig, Rolf; Byrne, Annette T

    2013-05-01

    Glioblastoma multiforme (GBM), the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI). A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI) were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.

  10. In Vivo Bioluminescence Imaging Validation of a Human Biopsy–Derived Orthotopic Mouse Model of Glioblastoma Multiforme

    Directory of Open Access Journals (Sweden)

    Monika A. Jarzabek

    2013-05-01

    Full Text Available Glioblastoma multiforme (GBM, the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI. A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.

  11. Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports.

    Science.gov (United States)

    Yoshimura, Hidekane; Oshikawa, Chie; Nakayama, Jun; Moteki, Hideaki; Usami, Shin-Ichi

    2015-05-01

    This study examines the CLRN1 gene mutation analysis in Japanese patients who were diagnosed with Usher syndrome type 3 (USH3) on the basis of clinical findings. Genetic analysis using massively parallel DNA sequencing (MPS) was conducted to search for 9 causative USH genes in 2 USH3 patients. We identified the novel pathogenic mutation in the CLRN1 gene in 2 patients. The missense mutation was confirmed by functional prediction software and segregation analysis. Both patients were diagnosed as having USH3 caused by the CLRN1 gene mutation. This is the first report of USH3 with a CLRN1 gene mutation in Asian populations. Validating the presence of clinical findings is imperative for properly differentiating among USH subtypes. In addition, mutation screening using MPS enables the identification of causative mutations in USH. The clinical diagnosis of this phenotypically variable disease can then be confirmed. © The Author(s) 2015.

  12. WellReader: a MATLAB program for the analysis of fluorescence and luminescence reporter gene data.

    Science.gov (United States)

    Boyer, Frédéric; Besson, Bruno; Baptist, Guillaume; Izard, Jérôme; Pinel, Corinne; Ropers, Delphine; Geiselmann, Johannes; de Jong, Hidde

    2010-05-01

    Fluorescent and luminescent reporter gene systems in combination with automated microplate readers allow real-time monitoring of gene expression on the population level at high precision and sampling density. This generates large amounts of data for the analysis of which computer tools are missing to date. We have developed WellReader, a MATLAB program for the analysis of fluorescent and luminescent reporter gene data. WellReader allows the user to load the output files of microplate readers, remove outliers, correct for background effects and smooth and fit the data. Moreover, it computes biologically relevant quantities from the measured signals, notably promoter activities and protein concentrations, and compares the resulting expression profiles of different genes under different conditions. WellReader is available under a LGPL licence at http://prabi1.inrialpes.fr/trac/wellreader.

  13. High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.

    Science.gov (United States)

    Keegan, Alissa; Plath, Kathrin; Damoiseaux, Robert

    2018-01-01

    Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.

  14. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma.

    Science.gov (United States)

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-07-21

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

  15. Photon Counting System for High-Sensitivity Detection of Bioluminescence at Optical Fiber End.

    Science.gov (United States)

    Iinuma, Masataka; Kadoya, Yutaka; Kuroda, Akio

    2016-01-01

    The technique of photon counting is widely used for various fields and also applicable to a high-sensitivity detection of luminescence. Thanks to recent development of single photon detectors with avalanche photodiodes (APDs), the photon counting system with an optical fiber has become powerful for a detection of bioluminescence at an optical fiber end, because it allows us to fully use the merits of compactness, simple operation, highly quantum efficiency of the APD detectors. This optical fiber-based system also has a possibility of improving the sensitivity to a local detection of Adenosine triphosphate (ATP) by high-sensitivity detection of the bioluminescence. In this chapter, we are introducing a basic concept of the optical fiber-based system and explaining how to construct and use this system.

  16. Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

    Science.gov (United States)

    Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

    2015-07-15

    Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

  17. The first report of prion-related protein gene (PRNT) polymorphisms in goat.

    Science.gov (United States)

    Kim, Yong-Chan; Jeong, Byung-Hoon

    2017-06-01

    Prion protein is encoded by the prion protein gene (PRNP). Polymorphisms of several members of the prion gene family have shown association with prion diseases in several species. Recent studies on a novel member of the prion gene family in rams have shown that prion-related protein gene (PRNT) has a linkage with codon 26 of prion-like protein (PRND). In a previous study, codon 26 polymorphism of PRND has shown connection with PRNP haplotype which is strongly associated with scrapie vulnerability. In addition, the genotype of a single nucleotide polymorphism (SNP) at codon 26 of PRND is related to fertilisation capacity. These findings necessitate studies on the SNP of PRNT gene which is connected with PRND. In goat, several polymorphism studies have been performed for PRNP, PRND, and shadow of prion protein gene (SPRN). However, polymorphism on PRNT has not been reported. Hence, the objective of this study was to determine the genotype and allelic distribution of SNPs of PRNT in 238 Korean native goats and compare PRNT DNA sequences between Korean native goats and several ruminant species. A total of five SNPs, including PRNT c.-114G > T, PRNT c.-58A > G in the upstream of PRNT gene, PRNT c.71C > T (p.Ala24Val) and PRNT c.102G > A in the open reading frame (ORF) and c.321C > T in the downstream of PRNT gene, were found in this study. All five SNPs of caprine PRNT gene in Korean native goat are in complete linkage disequilibrium (LD) with a D' value of 1.0. Interestingly, comparative sequence analysis of the PRNT gene revealed five mismatches between DNA sequences of Korean native goats and those of goats deposited in the GenBank. Korean native black goats also showed 5 mismatches in PRNT ORF with cattle. To the best of our knowledge, this is the first genetic research of the PRNT gene in goat.

  18. [Myocardial single photon emission tomography imaging of reporter gene expression in rabbits].

    Science.gov (United States)

    Liu, Ying; Lan, Xiao-li; Zhang, Liang; Wu, Tao; Jiang, Ri-feng; Zhang, Yong-xue

    2009-06-01

    To explore the feasibility of single photon emission computed tomography (SPECT) detection of heart reporter gene expression and observed the optimal transfecting titer and imaging time by using herpes simplex virus 1-thymidine kinase (HSV1-tk) as reporter gene and 131I-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (131I-FIAU) as reporter probe in rabbit myocardium. The recombinant Ad-tk carrying HSV1-tk gene and adenovirus (Ad) as vector was constructed and intramyocardially injected to rabbits at various concentrations (1 x 10(9) pfu, 5 x 10(8) pfu, 1 x 10(8) pfu, 5 x 10(7) pfu, 1 x 10(7) pfu). Two days later, rabbits were injected with 600 microCi 131I-FIAU in ear-margin vein and then underwent SPECT myocardium imaging for detection of HSV1-tk expression at 6 h, 24 h, 48 h and 72 h after injection, rabbits with 1 x 10(9) pfu Ad-tk injection were imaged at 96 h and 120 h. Rabbits were sacrificed after imaging and the total myocardial 131I-FIAU accumulation was quantified in percent of injected dose per gram myocardium (% ID/g). The myocardial Ad-tk expression was determined with RT-PCR. Reporter gene was detected by SPECT imaging in the injection site while not detected in the control myocardium and site remote from injection. RT-PCR results also evidenced HSV1-tk express in the injection site. The SPECT target/nontarget ratio was correlated with ex vivo gamma-counting (r2 = 0.933, Ppfu by SPECT imaging. The cardiac SPECT reporter gene imaging with HSV1-tk as reporter gene and 131I-FIAU as reporter probe is feasible.

  19. Deep-sea bioluminescence blooms after dense water formation at the ocean surface.

    Directory of Open Access Journals (Sweden)

    Christian Tamburini

    Full Text Available The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as "open-sea convection". It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts.

  20. Hybrid light transport model based bioluminescence tomography reconstruction for early gastric cancer detection

    Science.gov (United States)

    Chen, Xueli; Liang, Jimin; Hu, Hao; Qu, Xiaochao; Yang, Defu; Chen, Duofang; Zhu, Shouping; Tian, Jie

    2012-03-01

    Gastric cancer is the second cause of cancer-related death in the world, and it remains difficult to cure because it has been in late-stage once that is found. Early gastric cancer detection becomes an effective approach to decrease the gastric cancer mortality. Bioluminescence tomography (BLT) has been applied to detect early liver cancer and prostate cancer metastasis. However, the gastric cancer commonly originates from the gastric mucosa and grows outwards. The bioluminescent light will pass through a non-scattering region constructed by gastric pouch when it transports in tissues. Thus, the current BLT reconstruction algorithms based on the approximation model of radiative transfer equation are not optimal to handle this problem. To address the gastric cancer specific problem, this paper presents a novel reconstruction algorithm that uses a hybrid light transport model to describe the bioluminescent light propagation in tissues. The radiosity theory integrated with the diffusion equation to form the hybrid light transport model is utilized to describe light propagation in the non-scattering region. After the finite element discretization, the hybrid light transport model is converted into a minimization problem which fuses an l1 norm based regularization term to reveal the sparsity of bioluminescent source distribution. The performance of the reconstruction algorithm is first demonstrated with a digital mouse based simulation with the reconstruction error less than 1mm. An in situ gastric cancer-bearing nude mouse based experiment is then conducted. The primary result reveals the ability of the novel BLT reconstruction algorithm in early gastric cancer detection.

  1. ATP bioluminescence: Surface hygiene monitoring in milk preparation room of neonatal intensive care unit

    Science.gov (United States)

    Mohamad, Mahirah; Ishak, Shareena; Jaafar, Rohana; Sani, Norrakiah Abdullah

    2018-04-01

    ATP Bioluminescence application and standard microbiological analyses were used to evaluate the cleanliness of milk contact surfaces and non-milk contact surfaces in milk preparation room of neonatal intensive care unit (NICU) of Universiti Kebangsaan Malaysia Medical Centre (UKMMC). A total of 44 samples including the breast pump, milk bottle, milk bottle screw top and screw ring, teats, measuring cups, waterless warmer, refrigerator, dishwasher and pasteurizer inner wall were tested on May 2017. 3M Clean and Trace Hygiene Monitoring (UXL100 ATP Test swabs) and the bioluminescence reader Clean-Trace NG Luminometer (3M) were used to measure the Relative Light Unit (RLU) and microbiological analysis using 3M Quick Swab and 3MTM PetrifilmTM for enumeration of aerobic count, Staphylococcus aureus, Enterobacteriaceae, coliform and detection of Escherichia coli (CFU /100cm2 or utensil/item). The RLU values were from 11 to 194 and passed the ATP benchmark for intensive care unit (ICU), < 250 RLU as recommended. Aerobic colony count was only found in waterless warmer (0.05±0.01 mean log CFU/warmer). None of S. aureus, Enterobacteriaceae, E. coli and coliform was detected in all samples. A weak correlation was found between bioluminescence measurements RLU and the microbiological analysis (CFU). However, the use of ATP bioluminescence in monitoring milk preparation room cleanliness can be a useful method for assessing rapidly the surface hygiene as well as to verify the Sanitation Standard Operating Procedure (SSOP) prior to implementation of Hazard Analysis and Critical Control Points (HACCP) in milk preparation room.

  2. Deep-sea bioluminescence blooms after dense water formation at the ocean surface.

    Science.gov (United States)

    Tamburini, Christian; Canals, Miquel; Durrieu de Madron, Xavier; Houpert, Loïc; Lefèvre, Dominique; Martini, Séverine; D'Ortenzio, Fabrizio; Robert, Anne; Testor, Pierre; Aguilar, Juan Antonio; Samarai, Imen Al; Albert, Arnaud; André, Michel; Anghinolfi, Marco; Anton, Gisela; Anvar, Shebli; Ardid, Miguel; Jesus, Ana Carolina Assis; Astraatmadja, Tri L; Aubert, Jean-Jacques; Baret, Bruny; Basa, Stéphane; Bertin, Vincent; Biagi, Simone; Bigi, Armando; Bigongiari, Ciro; Bogazzi, Claudio; Bou-Cabo, Manuel; Bouhou, Boutayeb; Bouwhuis, Mieke C; Brunner, Jurgen; Busto, José; Camarena, Francisco; Capone, Antonio; Cârloganu, Christina; Carminati, Giada; Carr, John; Cecchini, Stefano; Charif, Ziad; Charvis, Philippe; Chiarusi, Tommaso; Circella, Marco; Coniglione, Rosa; Costantini, Heide; Coyle, Paschal; Curtil, Christian; Decowski, Patrick; Dekeyser, Ivan; Deschamps, Anne; Donzaud, Corinne; Dornic, Damien; Dorosti, Hasankiadeh Q; Drouhin, Doriane; Eberl, Thomas; Emanuele, Umberto; Ernenwein, Jean-Pierre; Escoffier, Stéphanie; Fermani, Paolo; Ferri, Marcelino; Flaminio, Vincenzo; Folger, Florian; Fritsch, Ulf; Fuda, Jean-Luc; Galatà, Salvatore; Gay, Pascal; Giacomelli, Giorgio; Giordano, Valentina; Gómez-González, Juan-Pablo; Graf, Kay; Guillard, Goulven; Halladjian, Garadeb; Hallewell, Gregory; van Haren, Hans; Hartman, Joris; Heijboer, Aart J; Hello, Yann; Hernández-Rey, Juan Jose; Herold, Bjoern; Hößl, Jurgen; Hsu, Ching-Cheng; de Jong, Marteen; Kadler, Matthias; Kalekin, Oleg; Kappes, Alexander; Katz, Uli; Kavatsyuk, Oksana; Kooijman, Paul; Kopper, Claudio; Kouchner, Antoine; Kreykenbohm, Ingo; Kulikovskiy, Vladimir; Lahmann, Robert; Lamare, Patrick; Larosa, Giuseppina; Lattuada, Dario; Lim, Gordon; Presti, Domenico Lo; Loehner, Herbert; Loucatos, Sotiris; Mangano, Salvatore; Marcelin, Michel; Margiotta, Annarita; Martinez-Mora, Juan Antonio; Meli, Athina; Montaruli, Teresa; Moscoso, Luciano; Motz, Holger; Neff, Max; Nezri, Emma Nuel; Palioselitis, Dimitris; Păvălaş, Gabriela E; Payet, Kevin; Payre, Patrice; Petrovic, Jelena; Piattelli, Paolo; Picot-Clemente, Nicolas; Popa, Vlad; Pradier, Thierry; Presani, Eleonora; Racca, Chantal; Reed, Corey; Riccobene, Giorgio; Richardt, Carsten; Richter, Roland; Rivière, Colas; Roensch, Kathrin; Rostovtsev, Andrei; Ruiz-Rivas, Joaquin; Rujoiu, Marius; Russo, Valerio G; Salesa, Francisco; Sánchez-Losa, Augustin; Sapienza, Piera; Schöck, Friederike; Schuller, Jean-Pierre; Schussler, Fabian; Shanidze, Rezo; Simeone, Francesco; Spies, Andreas; Spurio, Maurizio; Steijger, Jos J M; Stolarczyk, Thierry; Taiuti, Mauro G F; Toscano, Simona; Vallage, Bertrand; Van Elewyck, Véronique; Vannoni, Giulia; Vecchi, Manuela; Vernin, Pascal; Wijnker, Guus; Wilms, Jorn; de Wolf, Els; Yepes, Harold; Zaborov, Dmitry; De Dios Zornoza, Juan; Zúñiga, Juan

    2013-01-01

    The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as "open-sea convection". It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts.

  3. Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees.

    Science.gov (United States)

    Chabot, Adrien; Shrit, Ralla A; Blekhman, Ran; Gilad, Yoav

    2007-08-01

    Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.

  4. Assessment of tumor energy and oxygenation status by bioluminescence, nuclear magnetic resonance spectroscopy, and cryospectrophotometry.

    Science.gov (United States)

    Mueller-Klieser, W; Schaefer, C; Walenta, S; Rofstad, E K; Fenton, B M; Sutherland, R M

    1990-03-15

    The energy and oxygenation status of tumors from two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) were assessed using three independent techniques. Tumor energy metabolism was investigated in vivo by 31P nuclear magnetic resonance spectroscopy. After nuclear magnetic resonance measurements, tumors were frozen in liquid nitrogen to determine the tissue ATP concentration by imaging bioluminescence and to register the intracapillary oxyhemoglobin (HbO2) saturation using the cryospectrophotometric method. There was a positive correlation between the nucleoside triphosphate beta/total resonance ratio or a negative correlation between the Pi/total resonance ratio and the model ATP concentration obtained by bioluminescence, respectively. This was true for small tumors with no extended necrosis irrespective of tumor type. Moreover, a positive correlation was obtained between the HbO2 saturations and the ATP concentration measured with bioluminescence. The results demonstrate the potential of combined studies using noninvasive, integrating methods and high-resolution imaging techniques for characterizing the metabolic milieu in tumors.

  5. Distribution of bioluminescent fungi across old-growth and secondary tropical rain forest in Costa Rica

    Directory of Open Access Journals (Sweden)

    Carolina Seas-Carvajal

    2013-06-01

    Full Text Available Most research on bioluminescent fungi is concentrated on their taxonomic relationships, while the basics of their natural history and ecological relationships are poorly understood. In this study, we compared the distribution of bioluminescent fungi between old-growth and secondary forest as related to four different soil types at the tropical rainforest of La Selva Biological Station in Costa Rica. The study was conducted during the wet season of 2009. Bioluminescent fungi were sought following eight different transects distributed evenly in old-growth and secondary forests across four different soil types, covering an area of 9 420m². We found fungi in four different substrates: litter, fallen branches, dead trunks, and roots, for a total of 61 samples. Correspondence analysis showed that the occurrence of fungi and soil types were related (inertia=0.21, p=0.071. We found a significant relationship between the presence of fungi and the distribution of soil types (X²=18.89, df=9, p=0.026. We found only three samples with fruiting bodies, two of which had Mycena and the other had one fungus of the order Xylariales (possibly Hypoxylon sp., Kretzschmariella sp., Xylaria sp.. Future work will concentrate on exploring other aspects of their ecology, such as their dispersal and substrate preference. This information will facilitate field identification and will foster more research on the distribution, seasonality, reproductive phenology and ecological requirements of this group of Fungi.

  6. A two-hour antibiotic susceptibility test by ATP-bioluminescence.

    Science.gov (United States)

    March Rosselló, Gabriel Alberto; García-Loygorri Jordán de Urries, María Cristina; Gutiérrez Rodríguez, María Purificación; Simarro Grande, María; Orduña Domingo, Antonio; Bratos Pérez, Miguel Ángel

    2016-01-01

    The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  7. Factors Influencing Quantification of in Vivo Bioluminescence Imaging: Application to Assessment of Pancreatic Islet Transplants

    Directory of Open Access Journals (Sweden)

    John Virostko

    2004-10-01

    Full Text Available The aim of this study is to determine and characterize factors influencing in vivo bioluminescence imaging (BLI and apply them to the specific application of imaging transplanted pancreatic islets. Noninvasive quantitative assessment of transplanted pancreatic islets poses a formidable challenge. Murine pancreatic islets expressing firefly luciferase were transplanted under the renal capsule or into the portal vein of nonobese diabetic–severe combined immunodeficiency mice and the bioluminescence was quantified with a cooled charge coupled device camera and digital photon image analysis. The important, but often neglected, effects of wound healing, mouse positioning, and transplantation site on bioluminescence measurements were investigated by imaging a constant emission, isotropic light-emitting bead (λ = 600 implanted at the renal or hepatic site. The renal beads emitted nearly four times more light than hepatic beads with a smaller spot size, indicating that light absorption and scatter are greatly influenced by the transplant site and must be accounted for in BLI measurements. Detected luminescence decreased with increasing angle between the mouse surface normal and optical axis. By defining imaging parameters such as postsurgical effects, animal positioning, and light attenuation as a function of transplant site, this study develops BLI as a useful imaging modality for quantitative assessment of islets post-transplantation.

  8. An ebCMOS camera system for marine bioluminescence observation: The LuSEApher prototype

    Energy Technology Data Exchange (ETDEWEB)

    Dominjon, A., E-mail: a.dominjon@ipnl.in2p3.fr [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Ageron, M. [CNRS/IN2P3, Centre de Physique des Particules de Marseille, Marseille, F-13288 (France); Barbier, R. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Universite de Lyon, Universite Lyon 1, Lyon F-69003 (France); Billault, M.; Brunner, J. [CNRS/IN2P3, Centre de Physique des Particules de Marseille, Marseille, F-13288 (France); Cajgfinger, T. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Universite de Lyon, Universite Lyon 1, Lyon F-69003 (France); Calabria, P. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Chabanat, E. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Universite de Lyon, Universite Lyon 1, Lyon F-69003 (France); Chaize, D.; Doan, Q.T.; Guerin, C.; Houles, J.; Vagneron, L. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France)

    2012-12-11

    The ebCMOS camera, called LuSEApher, is a marine bioluminescence recorder device adapted to extreme low light level. This prototype is based on the skeleton of the LUSIPHER camera system originally developed for fluorescence imaging. It has been installed at 2500 m depth off the Mediterranean shore on the site of the ANTARES neutrino telescope. The LuSEApher camera is mounted on the Instrumented Interface Module connected to the ANTARES network for environmental science purposes (European Seas Observatory Network). The LuSEApher is a self-triggered photo detection system with photon counting ability. The presentation of the device is given and its performances such as the single photon reconstruction, noise performances and trigger strategy are presented. The first recorded movies of bioluminescence are analyzed. To our knowledge, those types of events have never been obtained with such a sensitivity and such a frame rate. We believe that this camera concept could open a new window on bioluminescence studies in the deep sea.

  9. Disposable bioluminescence-based biosensor for detection of bacterial count in food.

    Science.gov (United States)

    Luo, Jinping; Liu, Xiaohong; Tian, Qing; Yue, Weiwei; Zeng, Jing; Chen, Guangquan; Cai, Xinxia

    2009-11-01

    A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10(3) to 10(8) colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n=22) within 5min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.

  10. Early Infantile Leigh-like Gene Defects Have a Poor Prognosis: Report and Review

    Directory of Open Access Journals (Sweden)

    Majid Alfadhel

    2017-10-01

    Full Text Available Solute carrier family 19 (thiamine transporter, member 3 ( SCL19A3 gene defect produces an autosomal recessive neurodegenerative disorder associated with different phenotypes and acronyms. One of the common presentations is early infantile lethal Leigh-like syndrome. We report a case of early infantile Leigh-like SLC19A3 gene defects of patients who died at 4 months of age with no response to a high dose of biotin and thiamine. In addition, we report a novel mutation that was not reported previously. Finally, we review the literature regarding early infantile Leigh-like SLC19A3 gene defects and compare the literature with our patient.

  11. Environmental Application of Reporter-Genes Based Biosensors for Chemical Contamination Screening

    Directory of Open Access Journals (Sweden)

    Matejczyk Marzena

    2014-12-01

    Full Text Available The paper presents results of research concerning possibilities of applications of reporter-genes based microorganisms, including the selective presentation of defects and advantages of different new scientific achievements of methodical solutions in genetic system constructions of biosensing elements for environmental research. The most robust and popular genetic fusion and new trends in reporter genes technology – such as LacZ (β-galactosidase, xylE (catechol 2,3-dioxygenase, gfp (green fluorescent proteins and its mutated forms, lux (prokaryotic luciferase, luc (eukaryotic luciferase, phoA (alkaline phosphatase, gusA and gurA (β-glucuronidase, antibiotics and heavy metals resistance are described. Reporter-genes based biosensors with use of genetically modified bacteria and yeast successfully work for genotoxicity, bioavailability and oxidative stress assessment for detection and monitoring of toxic compounds in drinking water and different environmental samples, surface water, soil, sediments.

  12. A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains

    Energy Technology Data Exchange (ETDEWEB)

    Charrier, Thomas; Durand, Marie-Jose; Jouanneau, Sulivan; Thouand, Gerald [UMR CNRS 6144 GEPEA, CBAC, Nantes University, PRES UNAM, Campus de la Courtaisiere-IUT, La Roche-sur-Yon cedex (France); Dion, Michel [UMR CNRS 6204, Nantes University, PRES UNAM, Biotechnologie, Biocatalyse, Bioregulation, 2, Rue de la Houssiniere, BP 92208, Nantes cedex 3 (France); Pernetti, Mimma; Poncelet, Denis [ONIRIS-ENITIAA, UMR CNRS GEPEA, Rue de la Geraudiere, BP 82225, Nantes cedex 3 (France)

    2011-05-15

    This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria-Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor. (orig.)

  13. Registration of planar bioluminescence to magnetic resonance and x-ray computed tomography images as a platform for the development of bioluminescence tomography reconstruction algorithms.

    Science.gov (United States)

    Beattie, Bradley J; Klose, Alexander D; Le, Carl H; Longo, Valerie A; Dobrenkov, Konstantine; Vider, Jelena; Koutcher, Jason A; Blasberg, Ronald G

    2009-01-01

    The procedures we propose make possible the mapping of two-dimensional (2-D) bioluminescence image (BLI) data onto a skin surface derived from a three-dimensional (3-D) anatomical modality [magnetic resonance (MR) or computed tomography (CT)] dataset. This mapping allows anatomical information to be incorporated into bioluminescence tomography (BLT) reconstruction procedures and, when applied using sources visible to both optical and anatomical modalities, can be used to evaluate the accuracy of those reconstructions. Our procedures, based on immobilization of the animal and a priori determined fixed projective transforms, should be more robust and accurate than previously described efforts, which rely on a poorly constrained retrospectively determined warping of the 3-D anatomical information. Experiments conducted to measure the accuracy of the proposed registration procedure found it to have a mean error of 0.36+/-0.23 mm. Additional experiments highlight some of the confounds that are often overlooked in the BLT reconstruction process, and for two of these confounds, simple corrections are proposed.

  14. Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

    International Nuclear Information System (INIS)

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung; Kim, Yeon Soo

    2004-01-01

    For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells

  15. Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays.

    Science.gov (United States)

    Houtman, Corine J; Sterk, Saskia S; van de Heijning, Monique P M; Brouwer, Abraham; Stephany, Rainer W; van der Burg, Bart; Sonneveld, Edwin

    2009-04-01

    Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical-analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical-analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical-analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.

  16. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

    NARCIS (Netherlands)

    Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

    2002-01-01

    Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The

  17. A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes

    DEFF Research Database (Denmark)

    Seefried, Lothar; Mueller-Deubert, Sigrid; Wentzer, Thomas Schwarz

    2010-01-01

    of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching...

  18. New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment

    Czech Academy of Sciences Publication Activity Database

    Svobodová, Kateřina; Cajthaml, Tomáš

    2010-01-01

    Roč. 88, č. 4 (2010), s. 839-847 ISSN 0175-7598 R&D Projects: GA ČR GAP503/10/0408 Institutional research plan: CEZ:AV0Z50200510 Keywords : endocrine disruptors * in vitro bioassays * reporter gene assays Subject RIV: EE - Microbiology, Virology Impact factor: 3.280, year: 2010

  19. A case report on ATP6V0A4 gene mutation: Forecast of familial ...

    African Journals Online (AJOL)

    The patient does not have hearing impairment. ATP6V0A4 gene ... This case report emphasizes the significance of early diagnosis and genetic screening of recessive forms of ... Buffer defect /low buffer type distal RTA also involves the loss of ... weight of 4 Kg with no emergency treatment at the time of birth. Afterwards, his ...

  20. Bioluminescence resonance energy transfer (BRET) imaging of protein–protein interactions within deep tissues of living subjects

    Science.gov (United States)

    Dragulescu-Andrasi, Anca; Chan, Carmel T.; Massoud, Tarik F.; Gambhir, Sanjiv S.

    2011-01-01

    Identifying protein–protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications. PMID:21730157

  1. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  2. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  3. In vivo quantitative imaging of point-like bioluminescent and fluorescent sources: Validation studies in phantoms and small animals post mortem

    Science.gov (United States)

    Comsa, Daria Craita

    2008-10-01

    There is a real need for improved small animal imaging techniques to enhance the development of therapies in which animal models of disease are used. Optical methods for imaging have been extensively studied in recent years, due to their high sensitivity and specificity. Methods like bioluminescence and fluorescence tomography report promising results for 3D reconstructions of source distributions in vivo. However, no standard methodology exists for optical tomography, and various groups are pursuing different approaches. In a number of studies on small animals, the bioluminescent or fluorescent sources can be reasonably approximated as point or line sources. Examples include images of bone metastases confined to the bone marrow. Starting with this premise, we propose a simpler, faster, and inexpensive technique to quantify optical images of point-like sources. The technique avoids the computational burden of a tomographic method by using planar images and a mathematical model based on diffusion theory. The model employs in situ optical properties estimated from video reflectometry measurements. Modeled and measured images are compared iteratively using a Levenberg-Marquardt algorithm to improve estimates of the depth and strength of the bioluminescent or fluorescent inclusion. The performance of the technique to quantify bioluminescence images was first evaluated on Monte Carlo simulated data. Simulated data also facilitated a methodical investigation of the effect of errors in tissue optical properties on the retrieved source depth and strength. It was found that, for example, an error of 4 % in the effective attenuation coefficient led to 4 % error in the retrieved depth for source depths of up to 12mm, while the error in the retrieved source strength increased from 5.5 % at 2mm depth, to 18 % at 12mm depth. Experiments conducted on images from homogeneous tissue-simulating phantoms showed that depths up to 10mm could be estimated within 8 %, and the relative

  4. Splicing defect in FKBP10 gene causes autosomal recessive osteogenesis imperfecta disease: a case report.

    Science.gov (United States)

    Maghami, Fatemeh; Tabei, Seyed Mohammad Bagher; Moravej, Hossein; Dastsooz, Hassan; Modarresi, Farzaneh; Silawi, Mohammad; Faghihi, Mohammad Ali

    2018-05-25

    Osteogenesis imperfecta (OI) is a group of connective tissue disorder caused by mutations of genes involved in the production of collagen and its supporting proteins. Although the majority of reported OI variants are in COL1A1 and COL1A2 genes, recent reports have shown problems in other non-collagenous genes involved in the post translational modifications, folding and transport, transcription and proliferation of osteoblasts, bone mineralization, and cell signaling. Up to now, 17 types of OI have been reported in which types I to IV are the most frequent cases with autosomal dominant pattern of inheritance. Here we report an 8- year- old boy with OI who has had multiple fractures since birth and now he is wheelchair-dependent. To identify genetic cause of OI in our patient, whole exome sequencing (WES) was carried out and it revealed a novel deleterious homozygote splice acceptor site mutation (c.1257-2A > G, IVS7-2A > G) in FKBP10 gene in the patient. Then, the identified mutation was confirmed using Sanger sequencing in the proband as homozygous and in his parents as heterozygous, indicating its autosomal recessive pattern of inheritance. In addition, we performed RT-PCR on RNA transcripts originated from skin fibroblast of the proband to analyze the functional effect of the mutation on splicing pattern of FKBP10 gene and it showed skipping of the exon 8 of this gene. Moreover, Real-Time PCR was carried out to quantify the expression level of FKBP10 in the proband and his family members in which it revealed nearly the full decrease in the level of FKBP10 expression in the proband and around 75% decrease in its level in the carriers of the mutation, strongly suggesting the pathogenicity of the mutation. Our study identified, for the first time, a private pathogenic splice site mutation in FKBP10 gene and further prove the involvement of this gene in the rare cases of autosomal recessive OI type XI with distinguished clinical manifestations.

  5. Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils.

    Science.gov (United States)

    Cheng, Hsiao-Ying; Masiello, Caroline A; Del Valle, Ilenne; Gao, Xiaodong; Bennett, George N; Silberg, Jonathan J

    2018-03-16

    Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, C 2 H 4 is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CH 3 Br is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.

  6. Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

    Science.gov (United States)

    Viviani, Vadim R; Neves, Deimison Rodrigues; Amaral, Danilo Trabuco; Prado, Rogilene A; Matsuhashi, Takuto; Hirano, Takashi

    2014-08-19

    Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar

  7. [Determination of minimal concentrations of biocorrosion inhibitors by a bioluminescence method in relation to bacteria, participating in biocorrosion].

    Science.gov (United States)

    Efremenko, E N; Azizov, R E; Makhlis, T A; Abbasov, V M; Varfolomeev, S D

    2005-01-01

    By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Katon, Khazar, VFIKS-82, Nitro-1, Kaspii-2, and Kaspii-4) suppressing most common microbial corrosion agents: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only dividing cells but also their dormant living counterparts, are two- to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.

  8. A novel mutation in the SH3BP2 gene causes cherubism: case report

    Directory of Open Access Journals (Sweden)

    Yu Shi-Feng

    2006-12-01

    Full Text Available Abstract Background Cherubism is a rare hereditary multi-cystic disease of the jaws, characterized by its typical appearance in early childhood, and stabilization and remission after puberty. It is genetically transmitted in an autosomal dominant fashion and the gene coding for SH3-binding protein 2 (SH3BP2 may be involved. Case presentation We investigated a family consisting of 21 members with 3 female affected individuals with cherubism from Northern China. Of these 21 family members, 17 were recruited for the genetic analysis. We conducted the direct sequence analysis of the SH3BP2 gene among these 17 family members. A disease-causing mutation was identified in exon 9 of the gene. It was an A1517G base change, which leads to a D419G amino acid substitution. Conclusion To our knowledge, the A1517G mutation has not been reported previously in cherubism. This finding is novel.

  9. Case report of novel CACNA1A gene mutation causing episodic ataxia type 2

    Directory of Open Access Journals (Sweden)

    David Alan Isaacs

    2017-05-01

    Full Text Available Background: Episodic ataxia type 2 (OMIM 108500 is an autosomal dominant channelopathy characterized by paroxysms of ataxia, vertigo, nausea, and other neurologic symptoms. More than 50 mutations of the CACNA1A gene have been discovered in families with episodic ataxia type 2, although 30%–50% of all patients with typical episodic ataxia type 2 phenotype have no detectable mutation of the CACNA1A gene. Case: A 46-year-old Caucasian man, with a long history of bouts of imbalance, vertigo, and nausea, presented to our hospital with 2 weeks of ataxia and headache. Subsequent evaluation revealed a novel mutation in the CACNA1A gene: c.1364 G > A Arg455Gln. Acetazolamide was initiated with symptomatic improvement. Conclusion: This case report expands the list of known CACNA1A mutations associated with episodic ataxia type 2.

  10. Adenylate kinase amplification of ATP bioluminescence for hygiene monitoring in the food and beverage industry.

    Science.gov (United States)

    Corbitt, A J; Bennion, N; Forsythe, S J

    2000-06-01

    Fourteen food residues, Escherichia coli O157:H7 and Staphylococcus aureus on stainless steel surfaces were detected using a combined assay with adenylate kinase as a cellular marker and ATP bioluminescence. The limit of sensitivity ranged from 0.02 to 708 microg for minced meat and broccoli, respectively. Both methods gave the same detection limit (105 cfu) for E. coli and Staph. aureus on stainless steel surfaces. The combined adenylate kinase-ATP assay is applicable to monitor the hygiene of work surfaces, especially those prone to contamination by meat and vegetable residues.

  11. Effect of Iron Fe (II and Fe (III in a Binary System Evaluated Bioluminescent Method

    Directory of Open Access Journals (Sweden)

    Elena Sorokina

    2013-01-01

    Full Text Available The effect of iron ions Fe2+ and Fe3+ on the bioluminescent recombinant strain of Escherichia coli in a single-component and binary system. Found that for the bacteria E. coli Fe3+ ions are more toxic than Fe2+. Under the combined effect of iron toxicity increases, the percentage of luminescence quenching increases, but the value is much less than the sum of the indicator for the Fe2+ and Fe3+. The biological effect of insertion of iron is not proportional to their content in the mixture.

  12. Use of reporter-gene based bacteria to quantify phenanthrene biodegradation and toxicity in soil

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Doyun [Department of Civil and Environmental Engineering, Seoul National University, Gwanakno 599, Seoul 151-742 (Korea, Republic of); Moon, Hee Sun [School of Earth and Environmental Science, Seoul National University, Gwanakno 599, Seoul 151-742 (Korea, Republic of); Lin, Chu-Ching; Barkay, Tamar [Department of Biochemistry and Microbiology, Rutgers University, 76 Lipman Drive, New Brunswick, NJ 08901 (United States); Nam, Kyoungphile, E-mail: kpnam@snu.ac.k [Department of Civil and Environmental Engineering, Seoul National University, Gwanakno 599, Seoul 151-742 (Korea, Republic of)

    2011-02-15

    A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 {+-} 0.72) and strain S (from 1 to 0.40 {+-} 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed. - Research highlights: A novel reporter bacterial strain has been developed. The bacterium can quantitatively determine the change in fluorescence intensity. The intensity can represent the bioavailable phenanthrene in solid matrix. - A cell-killing gene harboring reporter bacterium shows phenanthrene toxicity.

  13. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  14. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    International Nuclear Information System (INIS)

    VanEtten, H.

    1997-01-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes

  15. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    Energy Technology Data Exchange (ETDEWEB)

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  16. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper

    2011-01-01

    A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...... relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means...

  17. Late onset bipolar disorder and frontotemporal dementia with mutation in progranulin gene: a case report.

    Science.gov (United States)

    Rubino, Elisa; Vacca, Alessandro; Gallone, Salvatore; Govone, Flora; Zucca, Milena; Gai, Annalisa; Ferrero, Patrizia; Fenoglio, Pierpaola; Giordana, Maria Teresa; Rainero, Innocenzo

    2017-11-01

    Bipolar disorder is a chronic psychiatric illness characterised by fluctuation in mood state, with a relapsing and remitting course. Frontotemporal dementia (FTD) is a clinically and genetically heterogeneous syndrome, with the most frequent phenotype being behavioural variant frontotemporal dementia (bvFTD). Here, we report the case of an Italian male presenting with late-onset bipolar disorder that developed into bvFTD over time, carrying a mutation in the GRN gene. Interestingly, the patient carried the c.1639 C > T variant in the GRN gene, resulting in a R547C substitution. Our case report further corroborates the notion that, in addition to FTD, progranulin may be involved in the neurobiology of bipolar disorder type 1, and suggests to screen patients with late-onset bipolar disorder for GRN mutations.

  18. Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report

    International Nuclear Information System (INIS)

    Martinez-Cadenas, Conrado; Lozoya, Rafael; Boldó, Enrique; Bosch, Nuria; Peñas, Lucas; Flores-Couce, Esther; Ochoa, Enrique; Munárriz, Javier; Aracil, Juan P; Tajahuerce, Marcos; Royo, Ramón

    2011-01-01

    Melanoma of the anal region is a very uncommon disease, accounting for only 0.2-0.3% of all melanoma cases. Mutations of the BRAF gene are usually absent in melanomas occurring in this region as well as in other sun-protected regions. The development of a tumour in a longstanding perianal fistula is also extremely rare. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process, although we have found only two cases of fistula-generating melanomas reported in the literature. Here we report the case of a 38-year-old male who presented with a perianal fistula of four years of evolution. Histopathological examination of the fistulous tract confirmed the presence of malignant melanoma. Due to the small size and the central location of the melanoma inside the fistulous tract, we believe the melanoma reported here developed in the epithelium of the fistula once the latter was already formed. Resected sentinel lymph nodes were negative and the patient, after going through a wide local excision, remains disease-free nine years after diagnosis. DNA obtained from melanoma tissue was analysed by automated direct sequencing and the V600E (T1799A) mutation was detected in exon 15 of the BRAF gene. Since fistulae experience persistent inflammation, the fact that this melanoma harbours a BRAF mutation strengthens the view that oxidative stress caused by inflammatory processes plays an important role in the genesis of BRAF gene mutations

  19. Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

    DEFF Research Database (Denmark)

    Larashati, Sekar; Schyth, Brian Dall; Lorenzen, Niels

    2011-01-01

    be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human...... coinjection and the assay is important in order to detect knock down by siRNA. Our experiment reveal in vivo knock down at 72 hours post injection of reporter gene and siRNA, but further dose-response experiments are required to confirm specifity....

  20. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  1. Detection of Heteromers Formed by Cannabinoid CB1, Dopamine D2, and Adenosine A2A G-Protein-Coupled Receptors by Combining Bimolecular Fluorescence Complementation and Bioluminescence Energy Transfer

    Science.gov (United States)

    Navarro, Gemma; Carriba, Paulina; Gandí, Jorge; Ciruela, Francisco; Casadó, Vicent; Cortés, Antoni; Mallol, Josefa; Canela, Enric I.; Lluis, Carmen; Franco, Rafael

    2008-01-01

    Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells. PMID:18956124

  2. Detection of Heteromers Formed by Cannabinoid CB1, Dopamine D2, and Adenosine A2A G-Protein-Coupled Receptors by Combining Bimolecular Fluorescence Complementation and Bioluminescence Energy Transfer

    Directory of Open Access Journals (Sweden)

    Gemma Navarro

    2008-01-01

    Full Text Available Functional interactions in signaling occur between dopamine D2 (D2R and cannabinoid CB1 (CB1R receptors, between CB1R and adenosine A2A (A2AR receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells.

  3. Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

    Science.gov (United States)

    Arts, Remco; den Hartog, Ilona; Zijlema, Stefan E; Thijssen, Vito; van der Beelen, Stan H E; Merkx, Maarten

    2016-04-19

    Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

  4. The effect of radiation on bioluminescent bacteria: possible use of luminescent bacteria as a biological dosemeter

    International Nuclear Information System (INIS)

    Mantel, J.; Freidin, M.; Perry, H.

    1983-01-01

    The purpose of the study was to investigate the response of the bioluminescent Photobacterium phosphoreum to radiation, and the possible use of the bacteria as a biological radiation dosemeter, i.e. a water-equivalent biological system that will compare beams not merely on the basis of absorbed dose, but also have intrinsic RBE values for different radiation beams. Samples were irradiated by a 12 MeV electron beam at a dose rate of 3.0 Gy min -1 , by 60 Co gamma rays at 2.85 Gy min -1 , and by 100 kVsub(p) x-rays at a dose rate of 2.13 Gy min -1 . To study dose-rate dependence, the survival fraction was obtained for a 12 MeV electron beam at 0.50 and 12 Gy min -1 for 20.0 Gy. The survival fraction proved to be independent of dose rate in this range. The results presented in this work indicate that by using bioluminescent bacteria, RBE measurements can be markedly simplified and the results interpreted unequivocally. (U.K.)

  5. Bioluminescence in a complex coastal environment: 1. Temporal dynamics of nighttime water-leaving radiance

    Science.gov (United States)

    Moline, Mark A.; Oliver, Matthew J.; Mobley, Curtis D.; Sundman, Lydia; Bensky, Thomas; Bergmann, Trisha; Bissett, W. Paul; Case, James; Raymond, Erika H.; Schofield, Oscar M. E.

    2007-11-01

    Nighttime water-leaving radiance is a function of the depth-dependent distribution of both the in situ bioluminescence emissions and the absorption and scattering properties of the water. The vertical distributions of these parameters were used as inputs for a modified one-dimensional radiative transfer model to solve for spectral bioluminescence water-leaving radiance from prescribed depths of the water column. Variation in the water-leaving radiance was consistent with local episodic physical forcing events, with tidal forcing, terrestrial runoff, particulate accumulation, and biological responses influencing the shorter timescale dynamics. There was a >90 nm shift in the peak water-leaving radiance from blue (˜474 nm) to green as light propagated to the surface. In addition to clues in ecosystem responses to physical forcing, the temporal dynamics in intensity and spectral quality of water-leaving radiance provide suitable ranges for assessing detection. This may provide the information needed to estimate the depth of internal light sources in the ocean, which is discussed in part 2 of this paper.

  6. A look at some systemic properties of self-bioluminescent emission

    Science.gov (United States)

    Creath, Katherine

    2008-08-01

    Self-bioluminescent emission (SBE) is a type of biological chemiluminescence where photons are emitted as part of chemical reactions occurring during metabolic processes. This emission is also known as biophoton emission, ultraweak photon emission and ultraweak bioluminescence. This paper outlines research over the past century on some systemic properties of SBE as measured with biological detectors, photomultiplier detectors and ultra-sensitive imaging arrays. There is an apparent consensus in the literature that emission in the deep blue and ultraviolet (150-450nm) is related to DNA / RNA processes while emission in the red and near infrared (600-1000nm) is related to mitochondria and oxidative metabolisms involving reactive oxygen species, singlet oxygen and free radicals in plant, animal and human cells along with chlorophyll fluorescent decay in plants. Additionally, there are trends showing that healthy, unstressed and uninjured samples have less emission than samples that are unhealthy, stressed or injured. Mechanisms producing this emission can be narrowed down by isolating the wavelength region of interest and waiting for short-term fluorescence to decay leaving the ultraweak long-term metabolic emission. Examples of imaging this emission in healthy versus unhealthy, stressed versus unstressed, and injured versus uninjured plant parts are shown. Further discussion poses questions still to be answered related to properties such as coherence, photon statistics, and methodological means of isolating mechanisms.

  7. Using ATP-driven bioluminescence assay to monitor microbial safety in a contemporary human cadaver laboratory.

    Science.gov (United States)

    Benninger, Brion; Maier, Thomas

    2015-03-01

    The objective of this study was to utilize a cost-effective method for assessing the levels of bacterial, yeast, and mold activity during a human dissection laboratory course. Nowadays, compliance with safety regulations is policed by institutions at higher standards than ever before. Fear of acquiring an unknown infection is one of the top concerns of professional healthcare students, and it provokes anti-laboratory anxiety. Human cadavers are not routinely tested for bacteria and viruses prior to embalming. Human anatomy dissecting rooms that house embalmed cadavers are normally cleaned after the dissected cadavers have been removed. There is no evidence that investigators have ever assessed bacterial and fungal activities using adenosine triphosphate (ATP)-driven bioluminescence assays. A literature search was conducted on texts, journals, and websites regarding bacterial, yeast, and mold activities in an active cadaver laboratory. Midway into a clinical anatomy course, ATP bioluminescence assays were used to swab various sites within the dissection room, including entrance and exiting door handles, water taps, cadaver tables, counter tops, imaging material, X-ray box switches, and the cadaver surfaces. The results demonstrated very low activities on cadaver tables, washing up areas, and exiting door handles. There was low activity on counter tops and X-ray boxes. There was medium activity on the entrance door handles. These findings suggest an inexpensive and accurate method for monitoring safety compliance and microbial activity. Students can feel confident and safe in the environment in which they work. © 2014 Wiley Periodicals, Inc.

  8. Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

    International Nuclear Information System (INIS)

    Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

    1988-01-01

    Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria

  9. Survival of bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in soft cheeses.

    Science.gov (United States)

    Ramsaran, H; Chen, J; Brunke, B; Hill, A; Griffiths, M W

    1998-07-01

    Pasteurized and raw milks that had been inoculated at 10(4) cfu/ml with bioluminescent strains of Listeria monocytogenes and Escherichia coli O157:H7 were used in the manufacture of Camembert and Feta cheeses with or without nisin-producing starter culture. Survival of both organisms was determined during the manufacture and storage of Camembert and Feta cheeses at 2 +/- 1 degree C for 65 and 75 d, respectively. Bacterial bioluminescence was used as an indicator to enumerate the colonies plated on selective Listeria agar and on MacConkey agar. Escherichia coli O157:H7 survived the manufacturing process of both cheeses and was present at the end of the storage period in greater numbers than in the initial inoculum. At the end of 75 d of storage, E. coli O157:H7 was found in the brine of Feta cheese. The counts of L. monocytogenes increased as the pH of the Camembert cheese increased, and there were significant differences between the counts from samples taken from the inside and the counts from samples obtained near the surface of the cheese. The Feta cheese that contained nisin was the only cheese in which L. monocytogenes was at the level of the initial inoculum after 75 d of storage.

  10. Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

    Science.gov (United States)

    Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

    2001-06-01

    A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

  11. The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

    Directory of Open Access Journals (Sweden)

    Mingyue Sun

    2014-09-01

    Full Text Available The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

  12. The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

    Directory of Open Access Journals (Sweden)

    Dashuai Mu

    Full Text Available Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3 was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%. To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.

  13. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    Science.gov (United States)

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  14. A miniaturized device for bioluminescence analysis of caspase-3/7 5 activity in a single apoptotic cell

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Lišková, Marcela; Matalová, E.; Klepárník, Karel

    Roč. 406 , č. 22 (2014), s. 5389-5394 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : apoptosis * bioluminescence * single-cell analysis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.436, year: 2014

  15. Bioluminescence-Based Method for Measuring Assimilable Organic Carbon in Pretreatment Water for Reverse Osmosis Membrane Desalination ▿

    Science.gov (United States)

    Weinrich, Lauren A.; Schneider, Orren D.; LeChevallier, Mark W.

    2011-01-01

    A bioluminescence-based assimilable organic carbon (AOC) test was developed for determining the biological growth potential of seawater within the reverse osmosis desalination pretreatment process. The test uses Vibrio harveyi, a marine organism that exhibits constitutive luminescence and is nutritionally robust. AOC was measured in both a pilot plant and a full-scale desalination plant pretreatment. PMID:21148685

  16. Bioluminescence : the potential of a non-invasive bio-optical imaging technique and improvement of animal research

    NARCIS (Netherlands)

    Hesselink, J. W.; van Dam, G. M.

    2007-01-01

    Bioluminescence is an optical imaging technique that exploits the emission of photons at specific wavelengths based on energy-dependent reactions catalysed by luciferases. The technique makes it possible to monitor measure, and track biological processes in living animals. A short review is

  17. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    -induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations...

  18. Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real-time bioluminescence imaging

    DEFF Research Database (Denmark)

    Bartkova, Simona; Kokotovic, Branko; Dalsgaard, Inger

    2017-01-01

    Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here...

  19. A miniaturized device for bioluminescence analysis of caspase-3/7 5 activity in a single apoptotic cell

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Lišková, Marcela; Matalová, E.; Klepárník, Karel

    2014-01-01

    Roč. 406, č. 22 (2014), s. 5389-5394 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : apoptosis * bioluminescence * single-cell analysis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.436, year: 2014

  20. Fast monitoring of indoor bioaerosol concentrations with ATP bioluminescence assay using an electrostatic rod-type sampler.

    Directory of Open Access Journals (Sweden)

    Ji-Woon Park

    Full Text Available A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: < 1 min of indoor bioaerosol concentrations with ATP bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations.

  1. Spatiotemporal expression of heme oxygenase-1 detected by in vivo bioluminescence after hepatic ischemia in HO-1/luc mice

    NARCIS (Netherlands)

    Su, Huawei; van Dam, Gooitzen M.; Buis, Carlijn I.; Visser, Dorien S.; Hesselink, Jan Willem; Schuurs, Theo A.; Leuvenink, Henri G. D.; Contag, Christopher H.; Porte, Robert J.

    Upregulation of heme oxygenase-1 (HO-1) has been proposed as a critical mechanism protecting against cellular stress during liver transplantation, providing a potential target for new therapeutic interventions. We investigated the feasibility of in vivo bioluminescence imaging (BLI) to noninvasively

  2. Synthetic strategies for controlling inter- and intramolecular interactions: Applications in single-molecule fluorescence imaging, bioluminescence imaging, and palladium catalysis

    Science.gov (United States)

    Conley, Nicholas R.

    proximity of the Cy3 and Cy5 fluorophores, behaves as an optical photoswitch in the presence of a thiol reagent. This unique property was employed to achieve sub-diffraction-limited imaging of the stalks of Caulobacter crescentus cells with 30-nm resolution using STORM (stochastic optical reconstruction microscopy). Lastly, the synthesis of the first selenium analogue of firefly luciferin is described, and this analogue is shown to be a competent substrate for firefly luciferase (fLuc). Remarkably, it exhibits red-shifted bioluminescence emission relative to the native sulfur analogue. The in vivo performance of the selenium and sulfur analogues in imaging are compared by tail-vein injection into nude mice bearing subcutaneous tumor xenografts of a human breast cancer cell line that was stably transduced to express fLuc. Part II of this thesis begins by addressing design considerations in the development of palladium catalysts that effect oxidative transformations under mild conditions (i.e., 1 atm air, room temperature) using molecular oxygen as the terminal oxidant. A newly synthesized cationic palladium complex, [(2,9-dimethylphenanthroline)Pd(OAc)]2[OTf]2, is shown to catalyze aerobic alcohol oxidation under such conditions with an unprecedented initial turnover frequency, but the presence of partially reduced oxygen species results in competitive ligand oxidation with concomitant decrease in catalyst activity. To remedy this, oxidatively resistant ligands, which are essential for the development of next-generation, high-turnover-frequency palladium catalysts that utilize oxygen as a terminal oxidant, have been prepared and effectively employed. In addition, the first general palladium-catalyzed route to the carbonylation of diols is reported. In this system, carbon monoxide (1 atm) serves the carbonyl source, (2,9-dimethylphenanthroline)Pd(OAc) 2 acts as the catalyst, and N-chlorosuccinimide and iodosobenzene are the oxidants for 1,2- and 1,3-diols, respectively. This

  3. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging.

    Science.gov (United States)

    Boutier, Maxime; Ronsmans, Maygane; Ouyang, Ping; Fournier, Guillaume; Reschner, Anca; Rakus, Krzysztof; Wilkie, Gavin S; Farnir, Frédéric; Bayrou, Calixte; Lieffrig, François; Li, Hong; Desmecht, Daniel; Davison, Andrew J; Vanderplasschen, Alain

    2015-02-01

    Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

  4. Use of L-Glutamic Acid in a New Enrichment Broth (R-TATP Broth) for Detecting the Presence or Absence of Molds in Raw Ingredients/Personal Care Product Formulations by Using an ATP Bioluminescence Assay.

    Science.gov (United States)

    Yang, Youjun; English, Donald J

    The present study reports the effects of adding L-glutamic acid to a new enrichment broth designated as R-TATP broth, to promote the growth of slow-growing mold microorganisms such as Aspergillus brasiliensis and Aspergillus oryzae , without interfering in the growth of other types of microorganisms. This L-glutamic acid containing enrichment broth would be particularly valuable in a rapid microbial detection assay such as an adenosine triphosphate (ATP) bioluminescence assay. By using this new enrichment broth, the amount of ATP (represented as relative light unit ratio after normalized with the negative test control) from mold growth was significantly increased by reducing the time of detection of microbial contamination in a raw ingredient or personal care product formulation from an incubation period of 48-18 h. By using L-glutamic acid in this enrichment broth, the lag phase of the mold growth cycle was shortened. In response to various concentrations of L-glutamic acid in R-TATP broth, there was an increased amount of ATP that had been produced by mold metabolism in an ATP bioluminescence assay. By using L-glutamic acid in R-TATP broth in an ATP bioluminescence assay, the presence of mold could be detected in 18 h as well as other types of microorganisms that may or may not be present in a test sample. By detecting the presence or absence of microbial contamination in 18 h, it is superior in comparison to a 48-96 h incubation period by using either a standard or rapid detection method.

  5. Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

    Science.gov (United States)

    Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

    2006-11-15

    Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

  6. Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report

    Directory of Open Access Journals (Sweden)

    Tajahuerce Marcos

    2011-08-01

    Full Text Available Abstract Background Melanoma of the anal region is a very uncommon disease, accounting for only 0.2-0.3% of all melanoma cases. Mutations of the BRAF gene are usually absent in melanomas occurring in this region as well as in other sun-protected regions. The development of a tumour in a longstanding perianal fistula is also extremely rare. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process, although we have found only two cases of fistula-generating melanomas reported in the literature. Case Presentation Here we report the case of a 38-year-old male who presented with a perianal fistula of four years of evolution. Histopathological examination of the fistulous tract confirmed the presence of malignant melanoma. Due to the small size and the central location of the melanoma inside the fistulous tract, we believe the melanoma reported here developed in the epithelium of the fistula once the latter was already formed. Resected sentinel lymph nodes were negative and the patient, after going through a wide local excision, remains disease-free nine years after diagnosis. DNA obtained from melanoma tissue was analysed by automated direct sequencing and the V600E (T1799A mutation was detected in exon 15 of the BRAF gene. Conclusion Since fistulae experience persistent inflammation, the fact that this melanoma harbours a BRAF mutation strengthens the view that oxidative stress caused by inflammatory processes plays an important role in the genesis of BRAF gene mutations.

  7. Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: LXL730724@hotmail.com; Liu Ying; He Yong; Wu Tao; Zhang Binqing; Gao Zairong; An Rui [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: zhyx1229@163.com

    2010-04-15

    Aim: To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with {sup 131}I-2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil ({sup 131}I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models. Methods: HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with {sup 131}I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1x10{sup 8} pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5x10{sup 8}, 1x10{sup 8}, 5x10{sup 7} and 1x10{sup 7} pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models. Results: From the ARG images, rats injected with Ad5-tk showed significant {sup 131}I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of {sup 131}I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5x10{sup 7} pfu, and the optimal Ad titer was 1x10{sup 8} pfu

  8. Development of a codon optimization strategy using the efor RED reporter gene as a test case

    Science.gov (United States)

    Yip, Chee-Hoo; Yarkoni, Orr; Ajioka, James; Wan, Kiew-Lian; Nathan, Sheila

    2018-04-01

    Synthetic biology is a platform that enables high-level synthesis of useful products such as pharmaceutically related drugs, bioplastics and green fuels from synthetic DNA constructs. Large-scale expression of these products can be achieved in an industrial compliant host such as Escherichia coli. To maximise the production of recombinant proteins in a heterologous host, the genes of interest are usually codon optimized based on the codon usage of the host. However, the bioinformatics freeware available for standard codon optimization might not be ideal in determining the best sequence for the synthesis of synthetic DNA. Synthesis of incorrect sequences can prove to be a costly error and to avoid this, a codon optimization strategy was developed based on the E. coli codon usage using the efor RED reporter gene as a test case. This strategy replaces codons encoding for serine, leucine, proline and threonine with the most frequently used codons in E. coli. Furthermore, codons encoding for valine and glycine are substituted with the second highly used codons in E. coli. Both the optimized and original efor RED genes were ligated to the pJS209 plasmid backbone using Gibson Assembly and the recombinant DNAs were transformed into E. coli E. cloni 10G strain. The fluorescence intensity per cell density of the optimized sequence was improved by 20% compared to the original sequence. Hence, the developed codon optimization strategy is proposed when designing an optimal sequence for heterologous protein production in E. coli.

  9. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  10. The construction and identification of hypoxia-regulated recombinant plasmid with reporter gene hNIS

    International Nuclear Information System (INIS)

    Hu Qunchao; Wu Jinchang; Zhou Jundong; Gu Ke

    2011-01-01

    Objective: To construct pShuttle-5 × HRE-CMV-NIS recombinant plasmid regulated by hypoxia-responsive element, which can possibly by used to detect the expression of hypoxia induced factor-α (HIF-1α) gene under hypoxia condition. Methods: Artificially synthesize the nucleotide sequences of five copies of hypoxia response elements (HREs) were cloned into pGL3-promoter vector to construct pGL3-promoter-5 × HRE vector. Human sodium/iodide symporter (hNIS) gene cDNA was amplified from human genome by RT-PCR, and subcloned into pGL3-promoter-5 × HRE vector then was sequenced. After treated with CoCl 2 as hypoxia mimic, HEK293 cells were transfected with recombinant plasmid with hNIS gene, while cells treated with DMSO as the control. Meanwhile, pcDNA3.1-HIF-1α and recombinant hNIS gene vectors were transfected into HEK293 cells at the ratio of 3 to 1, while co-transfection with pcDNA3.1 and pShuttle-NIS vectors cells were taken as the control. NIS mRNA expression was analyzed by qRT-PCR while function of NIS protein was tested by 99m TcO 4 - -uptake. Results: The sequence data of hNIS gene in recombinant plasmid were in accordance with those reported in the literatures. Compared with control groups, HEK293 cells co-transfected with both pShuttle-5 × HRE-CMV-NIS and HIF-1α gene vectors and CoCl 2 -treated after pShuttle-NIS transfecting presented higher mRNA expressions of NIS and 99m TcO 4 - uptake (P<0.01). Conclusion: HIF-1α can be bound to and activate pShuttle-5 × HRE-CMV-NIS in cells to accumulate radioactive nuclide 99m TcO 4 - and this technique is potential for detection of expression and activity of HIF-1α, the indicator of cell hypoxia. (authors)

  11. Seasonal variation of deep-sea bioluminescence in the Ionian Sea

    International Nuclear Information System (INIS)

    Craig, Jessica; Jamieson, Alan J.; Bagley, Philip M.; Priede, Imants G.

    2011-01-01

    The ICDeep (Image Intensified Charge Coupled Device for Deep sea research) profiler was used to measure the density of deep bioluminescent animals (BL) through the water column in the east, west and mid-Ionian Sea and in the Algerian Basin. A west to east decrease in BL density was found. Generalized additive modelling was used to investigate seasonal variation in the east and west Ionian Sea (NESTOR and NEMO neutrino telescope sites, respectively) from BL measurements in autumn 2008 and spring 2009. A significant seasonal effect was found in the west Ionian Sea (p<0.001), where a deep autumnal peak in BL density occurred between 500 and 2400 m. No significant seasonal variation in BL density was found in the east Ionian Sea (p=0.07). In both spring and autumn, significant differences in BL density were found through the water column between the east and west Ionian Sea (p<0.001).

  12. Seasonal variation of deep-sea bioluminescence in the Ionian Sea

    Energy Technology Data Exchange (ETDEWEB)

    Craig, Jessica, E-mail: j.craig@abdn.ac.u [University of Aberdeen, Oceanlab, Main Street, Newburgh, Aberdeenshire, AB41 6AA (United Kingdom); Jamieson, Alan J.; Bagley, Philip M.; Priede, Imants G. [University of Aberdeen, Oceanlab, Main Street, Newburgh, Aberdeenshire, AB41 6AA (United Kingdom)

    2011-01-21

    The ICDeep (Image Intensified Charge Coupled Device for Deep sea research) profiler was used to measure the density of deep bioluminescent animals (BL) through the water column in the east, west and mid-Ionian Sea and in the Algerian Basin. A west to east decrease in BL density was found. Generalized additive modelling was used to investigate seasonal variation in the east and west Ionian Sea (NESTOR and NEMO neutrino telescope sites, respectively) from BL measurements in autumn 2008 and spring 2009. A significant seasonal effect was found in the west Ionian Sea (p<0.001), where a deep autumnal peak in BL density occurred between 500 and 2400 m. No significant seasonal variation in BL density was found in the east Ionian Sea (p=0.07). In both spring and autumn, significant differences in BL density were found through the water column between the east and west Ionian Sea (p<0.001).

  13. Sparse reconstruction for quantitative bioluminescence tomography based on the incomplete variables truncated conjugate gradient method.

    Science.gov (United States)

    He, Xiaowei; Liang, Jimin; Wang, Xiaorui; Yu, Jingjing; Qu, Xiaochao; Wang, Xiaodong; Hou, Yanbin; Chen, Duofang; Liu, Fang; Tian, Jie

    2010-11-22

    In this paper, we present an incomplete variables truncated conjugate gradient (IVTCG) method for bioluminescence tomography (BLT). Considering the sparse characteristic of the light source and insufficient surface measurement in the BLT scenarios, we combine a sparseness-inducing (ℓ1 norm) regularization term with a quadratic error term in the IVTCG-based framework for solving the inverse problem. By limiting the number of variables updated at each iterative and combining a variable splitting strategy to find the search direction more efficiently, it obtains fast and stable source reconstruction, even without a priori information of the permissible source region and multispectral measurements. Numerical experiments on a mouse atlas validate the effectiveness of the method. In vivo mouse experimental results further indicate its potential for a practical BLT system.

  14. Noninvasive imaging of transplanted living functional cells transfected with a reporter estrogen receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Takamatsu, Shinji [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan)]. E-mail: shinjit@fmsrsa.fukui-med.ac.jp; Furukawa, Takako [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Yonekura, Yoshiharu [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan)

    2005-11-01

    The transplantation of functional cells such as dopaminergic cells into damaged tissue is now clinically ongoing, but at present the population of surviving cells at the transplantation site mostly cannot be noninvasively examined. To visualize surviving transplanted functional cells using a noninvasive method, we chose the estrogen receptor ligand binding domain (ERL) as a reporter molecule and 16{alpha}-[{sup 18}F]-fluoro-17{beta}-estradiol (FES) for its ligand. We used a mouse embryonic stem (ES) cell line for recipient cells as a model. To obtain ES cells that constitutively or inducibly express ERL, we transfected two types of expression vectors into EB5 parental ES cell line using the lipofection method and obtained about 30 clones for each of the two types of transfectants. Then, to examine the expression level of ERL, we performed Western blotting analysis. Ligand uptake experiments were carried out using [{sup 3}H]-estradiol with or without excessive unlabeled estradiol for control cells and ERL transfectants. Each selected clone was also used for in vivo positron emission tomography (PET) imaging studies involving FES in nude mice transplanted with control cells and ERL transfectants. In some of the clones transfected with the inducible-type ERL gene, protein was expressed much higher than in the controls. However, constitutive-type ERL gene-transfected ES cells showed no protein production in spite of their gene expression activity being considerably high. All clones also expressed equal levels of the Oct-3/4 gene, a marker of pluripotency, in comparison with the parental cells. Also, the specific uptake of [{sup 3}H]-estradiol was over 30 times higher in inducer-treated ERL-expressing ES cells compared to untreated control cells. Finally, by performing dynamic PET imaging, we successfully visualized ERL-expressing teratomas using FES.

  15. Noninvasive imaging of transplanted living functional cells transfected with a reporter estrogen receptor gene

    International Nuclear Information System (INIS)

    Takamatsu, Shinji; Furukawa, Takako; Mori, Tetsuya; Yonekura, Yoshiharu; Fujibayashi, Yasuhisa

    2005-01-01

    The transplantation of functional cells such as dopaminergic cells into damaged tissue is now clinically ongoing, but at present the population of surviving cells at the transplantation site mostly cannot be noninvasively examined. To visualize surviving transplanted functional cells using a noninvasive method, we chose the estrogen receptor ligand binding domain (ERL) as a reporter molecule and 16α-[ 18 F]-fluoro-17β-estradiol (FES) for its ligand. We used a mouse embryonic stem (ES) cell line for recipient cells as a model. To obtain ES cells that constitutively or inducibly express ERL, we transfected two types of expression vectors into EB5 parental ES cell line using the lipofection method and obtained about 30 clones for each of the two types of transfectants. Then, to examine the expression level of ERL, we performed Western blotting analysis. Ligand uptake experiments were carried out using [ 3 H]-estradiol with or without excessive unlabeled estradiol for control cells and ERL transfectants. Each selected clone was also used for in vivo positron emission tomography (PET) imaging studies involving FES in nude mice transplanted with control cells and ERL transfectants. In some of the clones transfected with the inducible-type ERL gene, protein was expressed much higher than in the controls. However, constitutive-type ERL gene-transfected ES cells showed no protein production in spite of their gene expression activity being considerably high. All clones also expressed equal levels of the Oct-3/4 gene, a marker of pluripotency, in comparison with the parental cells. Also, the specific uptake of [ 3 H]-estradiol was over 30 times higher in inducer-treated ERL-expressing ES cells compared to untreated control cells. Finally, by performing dynamic PET imaging, we successfully visualized ERL-expressing teratomas using FES

  16. Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

    Science.gov (United States)

    Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

    2014-08-01

    We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. © 2014. Published by The Company of Biologists Ltd.

  17. Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

    LENUS (Irish Health Repository)

    Zagozdzon, Agnieszka M

    2012-05-30

    AbstractBackgroundNumerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study.ResultsA new mouse strain (MMTV-Luc2 mice) expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based in vivo imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled ex vivo at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal.ConclusionsWe have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and\\/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for in vivo imaging techniques.

  18. Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Toan Nham

    Full Text Available Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba, followed by a colonization of the draining inguinal lymph node(s, and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

  19. Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

    Directory of Open Access Journals (Sweden)

    Zagozdzon Agnieszka M

    2012-05-01

    Full Text Available Abstract Background Numerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study. Results A new mouse strain (MMTV-Luc2 mice expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based in vivo imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled ex vivo at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal. Conclusions We have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for in vivo imaging techniques.

  20. Imaging of Bubonic Plague Dynamics by In Vivo Tracking of Bioluminescent Yersinia pestis

    Science.gov (United States)

    Nham, Toan; Filali, Sofia; Danne, Camille; Derbise, Anne; Carniel, Elisabeth

    2012-01-01

    Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response. PMID:22496846

  1. Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi

    Energy Technology Data Exchange (ETDEWEB)

    Byers, D.M.

    1989-01-01

    Bioluminescent bacteria require myristic acid (C14:0) to produce the myristaldehyde substrate of the light-emitting luciferase reaction. Since both endogenous and exogenous C14:0 can be used for this purpose, the metabolism of exogenous fatty acids by luminescent bacteria has been investigated. Both Vibrio harveyi and Vibrio fischeri incorporated label from (1-14C)myristic acid (C14:0) into phospholipid acyl chains as well as into CO2. In contrast, Photobacterium phosphoreum did not exhibit phospholipid acylation or beta-oxidation using exogenous fatty acids. Unlike Escherichia coli, the two Vibrio species can directly elongate fatty acids such as octanoic (C8:0), lauric (C12:0), and myristic acid, as demonstrated by radio-gas liquid chromatography. The induction of bioluminescence in late exponential growth had little effect on the ability of V. harveyi to elongate fatty acids, but it did increase the amount of C14:0 relative to C16:0 labeled from (14C)C8:0. This was not observed in a dark mutant of V. harveyi that is incapable of supplying endogenous C14:0 for luminescence. Cerulenin preferentially decreased the labeling of C16:0 and of unsaturated fatty acids from all 14C-labeled fatty acid precursors as well as from (14C)acetate, suggesting that common mechanisms may be involved in elongation of fatty acids from endogenous and exogenous sources. Fatty acylation of the luminescence-related synthetase and reductase enzymes responsible for aldehyde synthesis exhibited a chain-length preference for C14:0, which also was indicated by reverse-phase thin-layer chromatography of the acyl groups attached to these enzymes. The ability of V. harveyi to activate and elongate exogenous fatty acids may be related to an adaptive requirement to metabolize intracellular C14:0 generated by the luciferase reaction during luminescence development.

  2. Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi

    International Nuclear Information System (INIS)

    Byers, D.M.

    1989-01-01

    Bioluminescent bacteria require myristic acid (C14:0) to produce the myristaldehyde substrate of the light-emitting luciferase reaction. Since both endogenous and exogenous C14:0 can be used for this purpose, the metabolism of exogenous fatty acids by luminescent bacteria has been investigated. Both Vibrio harveyi and Vibrio fischeri incorporated label from [1-14C]myristic acid (C14:0) into phospholipid acyl chains as well as into CO2. In contrast, Photobacterium phosphoreum did not exhibit phospholipid acylation or beta-oxidation using exogenous fatty acids. Unlike Escherichia coli, the two Vibrio species can directly elongate fatty acids such as octanoic (C8:0), lauric (C12:0), and myristic acid, as demonstrated by radio-gas liquid chromatography. The induction of bioluminescence in late exponential growth had little effect on the ability of V. harveyi to elongate fatty acids, but it did increase the amount of C14:0 relative to C16:0 labeled from [14C]C8:0. This was not observed in a dark mutant of V. harveyi that is incapable of supplying endogenous C14:0 for luminescence. Cerulenin preferentially decreased the labeling of C16:0 and of unsaturated fatty acids from all 14C-labeled fatty acid precursors as well as from [14C]acetate, suggesting that common mechanisms may be involved in elongation of fatty acids from endogenous and exogenous sources. Fatty acylation of the luminescence-related synthetase and reductase enzymes responsible for aldehyde synthesis exhibited a chain-length preference for C14:0, which also was indicated by reverse-phase thin-layer chromatography of the acyl groups attached to these enzymes. The ability of V. harveyi to activate and elongate exogenous fatty acids may be related to an adaptive requirement to metabolize intracellular C14:0 generated by the luciferase reaction during luminescence development

  3. Gene polymorphisms in association with self-reported stroke in US adults

    Directory of Open Access Journals (Sweden)

    Amy Z Fan

    2010-03-01

    Full Text Available Amy Z Fan1, Jing Fang1, Ajay Yesupriya2, Man-huei Chang2, Greta Kilmer1, Meaghan House3, Donald Hayes1, Renée M Ned2, Nicole F Dowling2, Ali H Mokdad1 1National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 2Office of Public Health Genomics, Coordinating Center for Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 3School of Public Health, Emory University, Atlanta, GA, USAPurpose: Epidemiologic studies suggest that several gene variants increase the risk of stroke, and population-based studies help provide further evidence. We identified polymorphisms associated with the prevalence of self-reported stroke in US populations using a representative sample.Methods: Our sample comprised US adults in the Third National Health and Nutrition Examination (NHANES III DNA bank. We examined nine candidate gene variants within ACE, F2, F5, ITGA2, MTHFR, and NOS3 for associations with self-reported stroke. We used multivariate regression and Cox proportional hazards models to test the association between these variants and history of stroke.Results: In regression models, the rs4646994 variant of ACE (I/I and I/D genotypes was associated with higher prevalence adjusted prevalence odds ratio [APOR] = 2.66 [1.28, 5.55] and 2.23 [1.30, 3.85], respectively compared with the D/D genotype. The heterozygous genotype of MTHFR rs1801131 (A/C was associated with lower prevalence of stroke (APOR = 0.48 [0.25, 0.92] compared with A/A and C/C genotypes. For rs2070744 of NOS3, both the C/T genotype (APOR = 1.91 [1.12, 3.27] and C/C genotype (APOR = 3.31 [1.66, 6.60] were associated with higher prevalence of stroke compared with the T/T genotype.Conclusion: Our findings suggest an association between the prevalence of self-reported stroke and polymorphisms in ACE, MTHFR, and NOS3 in a population-based sample. Keywords: stroke, gene, polymorphisms, NHANES III, gene

  4. The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

    Energy Technology Data Exchange (ETDEWEB)

    Plotan, Monika; Elliott, Christopher T. [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom); Scippo, Marie Louise [Department of Food Sciences, University of Liege, 4000 Liege (Belgium); Muller, Marc [Molecular Biology and Genetic Engineering GIGA-R, University of Liege, 4000 Liege (Belgium); Antignac, Jean-Philippe [LABERCA, ENVN, USC INRA 2013, BP 50707, 44 307, Nantes (France); Malone, Edward [The State Laboratory, Young' s Cross, Celbridge, Co. Kildare (Ireland); Bovee, Toine F.H. [RIKILT Institute of Food Safety, P.O. Box 230, AE Wageningen 6700 (Netherlands); Mitchell, Samuel [Agri-Food and Biosciences Institute, Belfast BT9 5PX (United Kingdom); Connolly, Lisa, E-mail: l.connolly@qub.ac.uk [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom)

    2011-08-26

    The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC{sub 50} of 0.01 ng mL{sup -1} and 0.16 ng mL{sup -1} respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally

  5. The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

    International Nuclear Information System (INIS)

    Plotan, Monika; Elliott, Christopher T.; Scippo, Marie Louise; Muller, Marc; Antignac, Jean-Philippe; Malone, Edward; Bovee, Toine F.H.; Mitchell, Samuel; Connolly, Lisa

    2011-01-01

    The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC 50 of 0.01 ng mL -1 and 0.16 ng mL -1 respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the

  6. Nance-Horan syndrome: a contiguous gene syndrome involving deletion of the amelogenin gene? A case report and molecular analysis.

    Science.gov (United States)

    Franco, E; Hodgson, S; Lench, N; Roberts, G J

    1995-03-01

    A case of Nance-Horan syndrome in a male is presented, with some features of the condition in his carrier mother and her mother. It is proposed that Nance-Horan syndrome might be a contiguous gene syndrome mapping to chromosome Xp21.2-p22.3. The proband had congenital cataract microphthalmia and dental abnormalities including screwdriver shaped incisors and evidence of enamel pitting hypoplasia. The region Xp21.2-p22.3 also contains the tooth enamel protein gene, amelogenin (AMGX). Using molecular genetic techniques, we have shown that there is no evidence that the AMGX gene is deleted in this case of the Nance-Horan syndrome.

  7. Report of space experiment project, 'Rad Gene', performed in the International Space Station Kibo

    International Nuclear Information System (INIS)

    Ohnishi, Takeo; Takahashi, Akihisa; Nagamatsu, Aiko

    2010-01-01

    This report summarizes results of the project in the title adopted by Japan Aerospace Exploration Agency (JAXA) (in 2000) aiming to elucidate the biological effect of space environment, and contains 3 major parts of the process of the experiment, and of findings by analysis after flight and in radioadaptive response. The process for the experiment includes training of the experimenter crew (Dr. S. Magnus) in JAXA, preparation of samples (frozen cells with normal and mutated p53 genes derived from human lymphoblast TK6) and their transfer to the Space Shuttle Endeavour STS-126 launched on Nov. 15, 2008 (Japanese time) for cell culturing in Feb., 2009. Analyses after flight back to the Kennedy Space Center on Mar. 29, 2009, done on the ground in Japan thereafter include the physical evaluation, confirmation of DNA damage, and phenotypic expression with DNA- and protein-arrays (genes induced for expression of p53-related phenotypes in those cells which were stored frozen in the space, thawed on the ground and then cultured, genes induced for expressing the phenotypes and p53-related proteins expressed in cells cultured in space). Physically, total absorbed dose and dose equivalent are found to be respectively 43.5 mGy and 71.2 mSv (0.5 mSv/day). Interestingly, the biologically estimated dose by DNA-double strand breaks detected by γH2AX staining, 94.5 mSv (0.7 mSv/day), in living, frozen cells in space, is close to the above physical dose. Expression experiments of p53-related phenotypes have revealed that expression of 750 or more genes in 41,000 genes in the array is changed: enhanced or suppressed by space radiation, micro-gravity and/or their mixed effects in space environment. In 642 protein antibodies in the array, 2 proteins are found enhanced and 8, suppressed whereas heat-shock protein is unchanged. Radioadaptive response is the acquisition of radio-resistance to acute exposure by previous irradiation of small dose (window width 20-100 mSv) in normal p53

  8. The first case report of a large deletion of the BRCA1 gene in Croatia: A case report.

    Science.gov (United States)

    Musani, Vesna; Sušac, Ilona; Ozretić, Petar; Eljuga, Domagoj; Levanat, Sonja

    2017-12-01

    Breast cancer is one of the most common cancers in women, and it is the leading cause of cancer related deaths in Croatia. BRCA1 and BRCA2 gene mutations are the most common cause of hereditary breast cancer. In this report we describe a Croatian patient with no apparent family history of cancer, who developed breast cancer first at 29, and again at 33. Due to the early development of first breast cancer and triple negative status of the second, the attending physician suspected a hereditary aspect. Patient was sent to BRCA1 genetic testing. Subsequently, her mother and sister were sent to check for the mutation found in the patient. BRCA1 exons 4-6 deletion was determined and sequencing confirmed the deletion as NG_005905.2:g.107648_117905del10257. Mother and sister were not affected, but since there were no available family members on the fathers' side, it was not possible to determine if this was a case of de novo mutation. Until now, only in three reports with the similar mutation the exact mutation borders were determined. The mutation in this case was not the same as previously reported and was more than twice in size. All large deletions should be described at the nucleotide level, so that in cases with missing family data it would be possible to deduce if the mutation is already known. If the mutation is already known, it is probably not a de novo event, since it is unlikely that the breakpoints would be exactly the same more than once.

  9. β-Globin gene sequencing of hemoglobin Austin revises the historically reported electrophoretic migration pattern.

    Science.gov (United States)

    Racsa, Lori D; Luu, Hung S; Park, Jason Y; Mitui, Midori; Timmons, Charles F

    2014-06-01

    Hemoglobin (Hb) Austin was defined in 1977, using amino acid sequencing of samples from 3 unrelated Mexican-Americans, as a substitution of serine for arginine at position 40 of the β-globin chain (Arg40Ser). Its electrophoretic migration on both cellulose acetate (pH 8.4) and citrate agar (pH 6.2) was reported between Hb F and Hb A, and this description persists in reference literature. OBJECTIVES.-To review the clinical features and redefine the diagnostic characteristics of Hb Austin. Eight samples from 6 unrelated individuals and 2 siblings, all with Hispanic surnames, were submitted for abnormal Hb identification between June 2010 and September 2011. High-performance liquid chromatography, isoelectric focusing (IEF), citrate agar electrophoresis, and bidirectional DNA sequencing of the entire β-globin gene were performed. DNA sequencing confirmed all 8 individuals to be heterozygous for Hb Austin (Arg40Ser). Retention time on high-performance liquid chromatography and migration on citrate agar electrophoresis were consistent with that identification. Migration on IEF, however, was not between Hb F and Hb A, as predicted from the report of cellulose acetate electrophoresis. By IEF, Hb Austin migrated anodal to ("faster than") Hb A. Hemoglobin Austin (Arg40Ser) appears on IEF as a "fast," anodally migrating, Hb variant, just as would be expected from its amino acid substitution. The cited historic report is, at best, not applicable to IEF and is probably erroneous. Our observation of 8 cases in 16 months suggests that this variant may be relatively common in some Hispanic populations, making its recognition important. Furthermore, gene sequencing is proving itself a powerful and reliable tool for definitive identification of Hb variants.

  10. Involvement of the BDNF Gene in Loneliness in Adolescence : A Report of Opposite Gene Effects in Boys and Girls

    NARCIS (Netherlands)

    Verhagen, Maaike; van Roekel, Eeske; Engels, Rutger C. M. E.

    2014-01-01

    Previous research has shown that loneliness has a heritable component and that genes within the serotonin-, dopamine-, and oxytocin systems are related to loneliness in adolescence. In the present study, the relation between the BDNF Val66Met polymorphism and loneliness in adolescent boys and girls

  11. Biolistic transformation of Schistosoma mansoni: Studies with modified reporter-gene constructs containing regulatory regions of protease genes

    Czech Academy of Sciences Publication Activity Database

    Dvořák, Jan; Beckmann, S.; Lim, K.-C.; Engel, J. C.; Grevelding, C. G.; McKerrow, J. H.; Caffrey, C. R.

    2010-01-01

    Roč. 170, č. 1 (2010), s. 37-40 ISSN 0166-6851 Institutional research plan: CEZ:AV0Z60220518 Keywords : Schistosoma * Protease * Transgene * Gene promoter * Biolistics * Electroporation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.875, year: 2010

  12. MR molecular imaging of tumours using ferritin heavy chain reporter gene expression mediated by the hTERT promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yan [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China); The First Affiliated Hospital of ChengDu Medical College, Department of Radiology, ChengDu (China); Gong, Ming-fu; Yang, Hua; Zhang, Song; Wang, Guang-xian; Su, Tong-sheng; Wen, Li; Zhang, Dong [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China)

    2016-11-15

    Using the human telomerase reverse transcriptase (hTERT) promoter and the modified ferritin heavy chain (Fth) reporter gene, reporter gene expression for MRI was examined in telomerase positive and negative tumour cells and xenografts. Activity of the reporter gene expression vector Lenti-hTERT-Fth1-3FLAG-Puro was compared to constitutive CMV-driven expression and to the untransfected parental control in five tumour cell lines: A549, SKOV3, 293T, U2OS and HPDLF. In vitro, transfected cells were evaluated for FLAG-tagged protein expression, iron accumulation and transverse relaxation. In vivo, tumours transduced by lentiviral vector injection were imaged using T2*WI. Changes in tumour signal intensity were validated by histology. Only telomerase positive tumour cells expressed FLAG-tagged Fth and displayed an increase in R2* above the parental control, with a corresponding change in T2*WI. In addition, only telomerase positive tumours, transduced by injection of the reporter gene expression construct, exhibited a change in signal intensity on T2*WI. Tumour histology verified the expression of FLAG-tagged Fth and iron accumulation in telomerase positive tissue. Reporter gene expression for MRI, using the Fth reporter and the hTERT promoter, may be a useful strategy for the non-invasive diagnosis of many types of cancer. (orig.)

  13. Case report: value of gene expression profiling in the diagnosis of atypical neuroblastoma.

    Science.gov (United States)

    Harttrampf, Anne C; Chen, Qingrong; Jüttner, Eva; Geiger, Julia; Vansant, Gordon; Khan, Javed; Kontny, Udo

    2017-08-17

    Nephroblastoma and neuroblastoma belong to the most common abdominal malignancies in childhood. Similarities in the initial presentation may provide difficulties in distinguishing between these two entities, especially if unusual variations to prevalent patterns of disease manifestation occur. Because of the risk of tumor rupture, European protocols do not require biopsy for diagnosis, which leads to misdiagnosis in some cases. We report on a 4½-year-old girl with a renal tumor displaying radiological and laboratory characteristics supporting the diagnosis of nephroblastoma. Imaging studies showed tumor extension into the inferior vena cava and bilateral lung metastases while urine catecholamines and MIBG-scintigraphy were negative. Preoperative chemotherapy with vincristine, actinomycine D and adriamycin according to the SIOP2001/GPOH protocol for the treatment of nephroblastoma was initiated and followed by surgical tumor resection. Histopathology revealed an undifferentiated tumor with expression of neuronal markers, suggestive of neuroblastoma. MYCN amplification could not be detected. DNA-microarray analysis was performed using Affymetrix genechip human genome U133 plus 2.0 and artificial neural network analysis. Results were confirmed by multiplex RT-PCR. Principal component analysis using 84 genes showed that the patient sample was clearly clustering with neuroblastoma tumors. This was confirmed by hierarchical clustering of the multiplex RT-PCR data. The patient underwent treatment for high-risk neuroblastoma comprising chemotherapy including cisplatin, etoposide, vindesine, dacarbacine, ifosfamide, vincristine, adriamycine and autologous stem cell transplantation followed by maintenance therapy with 13-cis retinoic acid (GPOH NB2004 High Risk Trial Protocol) and is in complete long-term remission. The use of gene expression profiling in an individual patient strongly contributed to clarification in a diagnostic dilemma which finally led to a change of

  14. Hybrid radiosity-SP3 equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    Science.gov (United States)

    Chen, Xueli; Zhang, Qitan; Yang, Defu; Liang, Jimin

    2014-01-01

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP3 equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP3) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

  15. Hybrid radiosity-SP3 equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    International Nuclear Information System (INIS)

    Chen, Xueli; Zhang, Qitan; Yang, Defu; Liang, Jimin

    2014-01-01

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP 3 equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP 3 ) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions

  16. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

    1999-01-01

    Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrog......Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential...... antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were...... calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands...

  17. The Use of Stimulable Bioluminescence from Marine Dinoflagellates as a Means of Detecting Toxicity in the Marine Environment

    Science.gov (United States)

    1993-04-01

    FROM MARINE PR: ME65 DINOFLAGELLATES AS A MEANS OF DETECTING TOXICITY IN THE PE: 060372N MARINE ENVIRONMENT WU: DN288604 6ý AUTHOR(S) Accesion For I...measure the acute and sublethal effects of heavy metals ( tributyltin , copper, and zinc) and storm drain effluent on the light output from marine...Grovhoug 3 THE USE OF STIM1ULABLE BIOLUMINESCENCE FROM MARINE DINOFLAGELLATES AS A MEANS OF DETECTING TOXICITY IN THE MARINE ENVIRONMENT. REFERENCE

  18. Bioluminescence and the Actin Cytoskeleton in the Dinoflagellate Pyrocystis fusiformis: An Examination of Organelle Transport and Mechanotransduction

    OpenAIRE

    McDougall, Carrie A.

    2002-01-01

    Bioluminescence (BL), light produced by organisms, is a diverse and widespread marine phenomenon. yet little studied by researchers. Major contributors to sea surface BL displays are dinoflagellates, which produce rapid BL flashes upon fluid motion; mechanical stimulation triggers a 200-ms flash within 20 ms, representing one of the most rapid sensor-effector transduction systems described. In some dinoflagellate species the sensor-effector link is not constant throughout a 24-hour period. Me...

  19. Developmental and Microbiological Analysis of the Inception of Bioluminescent Symbiosis in the Marine Fish Nuchequula nuchalis (Perciformes: Leiognathidae)▿

    OpenAIRE

    Dunlap, Paul V.; Davis, Kimberly M.; Tomiyama, Shinichi; Fujino, Misato; Fukui, Atsushi

    2008-01-01

    Many marine fish harbor luminous bacteria as bioluminescent symbionts. Despite the diversity, abundance, and ecological importance of these fish and their apparent dependence on luminous bacteria for survival and reproduction, little is known about developmental and microbiological events surrounding the inception of their symbioses. To gain insight on these issues, we examined wild-caught larvae of the leiognathid fish Nuchequula nuchalis, a species that harbors Photobacterium leiognathi as ...

  20. A novel mutation of WFS1 gene in a Chinese patient with Wolfram syndrome: a case report.

    Science.gov (United States)

    Li, Min; Liu, Jia; Yi, Huan; Xu, Li; Zhong, Xiufeng; Peng, Fuhua

    2018-03-17

    Wolfram syndrome (WS), caused by mutations of the Wolfram syndrome 1 (WFS1) gene on chromosome 4p16.1, is an autosomal recessive disorder characterized by diabetes insipidus (DI), neuro-psychiatric disorders, hearing deficit, and urinary tract anomalies. Here we report a 11-year-old Chinese boy who presented with visual loss, was suspected with optic neuritis (ON) or neuromyelitis optica (NMO) and referred to our department for further diagnosis. Finally he was diagnosed with WS because of diabetes mellitus (DM) and optic atrophy (OA). Eight exons and flanking introns of WFS1 gene were analyzed by sequencing. A novel mutation c.1760G > A in WFS1 gene of exon 8 was identified. This report reviews a case of WS associated with a novel mutation, c.1760G > A in WFS1 gene of exon 8, and emphasizes that WS should be taken into account for juveniles with visual loss and diabetes mellitus.

  1. Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

    Directory of Open Access Journals (Sweden)

    Orozco Javier I

    2011-10-01

    Full Text Available Abstract Introduction Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. Case presentation A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. Conclusion In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion.

  2. Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.

    Science.gov (United States)

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Aoki, Miku; Taguchi, Katsutoshi; Tanaka, Masaki

    2016-02-01

    The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

  3. Case reports of juvenile GM1 gangliosidosisis type II caused by mutation in GLB1 gene.

    Science.gov (United States)

    Karimzadeh, Parvaneh; Naderi, Samaneh; Modarresi, Farzaneh; Dastsooz, Hassan; Nemati, Hamid; Farokhashtiani, Tayebeh; Shamsian, Bibi Shahin; Inaloo, Soroor; Faghihi, Mohammad Ali

    2017-07-17

    Type II or juvenile GM1-gangliosidosis is an autosomal recessive lysosomal storage disorder, which is clinically distinct from infantile form of the disease by the lack of characteristic cherry-red spot and hepatosplenomegaly. The disease is characterized by slowly progressive neurodegeneration and mild skeletal changes. Due to the later age of onset and uncharacteristic presentation, diagnosis is frequently puzzled with other ataxic and purely neurological disorders. Up to now, 3-4 types of GM1-gangliosidosis have been reported and among them type I is the most common phenotype with the age of onset around 6 months. Various forms of GM1-gangliosidosis are caused by GLB1 gene mutations but severity of the disease and age of onset are directly related to the position and the nature of deleterious mutations. However, due to its unique genetic cause and overlapping clinical features, some researchers believe that GM1 gangliosidosis represents an overlapped disease spectrum instead of four distinct types. Here, we report a less frequent type of autosomal recessive GM1 gangliosidosis with perplexing clinical presentation in three families in the southwest part of Iran, who are unrelated but all from "Lurs" ethnic background. To identify disease-causing mutations, Whole Exome Sequencing (WES) utilizing next generation sequencing was performed. Four patients from three families were investigated with the age of onset around 3 years old. Clinical presentations were ataxia, gate disturbances and dystonia leading to wheelchair-dependent disability, regression of intellectual abilities, and general developmental regression. They all were born in consanguineous families with no previous documented similar disease in their parents. A homozygote missense mutation in GLB1 gene (c. 601 G > A, p.R201C) was found in all patients. Using Sanger sequencing this identified mutation was confirmed in the proband, their parents, grandparents, and extended family members, confirming

  4. Comparisons of hybrid radiosity-diffusion model and diffusion equation for bioluminescence tomography in cavity cancer detection

    Science.gov (United States)

    Chen, Xueli; Yang, Defu; Qu, Xiaochao; Hu, Hao; Liang, Jimin; Gao, Xinbo; Tian, Jie

    2012-06-01

    Bioluminescence tomography (BLT) has been successfully applied to the detection and therapeutic evaluation of solid cancers. However, the existing BLT reconstruction algorithms are not accurate enough for cavity cancer detection because of neglecting the void problem. Motivated by the ability of the hybrid radiosity-diffusion model (HRDM) in describing the light propagation in cavity organs, an HRDM-based BLT reconstruction algorithm was provided for the specific problem of cavity cancer detection. HRDM has been applied to optical tomography but is limited to simple and regular geometries because of the complexity in coupling the boundary between the scattering and void region. In the provided algorithm, HRDM was first applied to three-dimensional complicated and irregular geometries and then employed as the forward light transport model to describe the bioluminescent light propagation in tissues. Combining HRDM with the sparse reconstruction strategy, the cavity cancer cells labeled with bioluminescent probes can be more accurately reconstructed. Compared with the diffusion equation based reconstruction algorithm, the essentiality and superiority of the HRDM-based algorithm were demonstrated with simulation, phantom and animal studies. An in vivo gastric cancer-bearing nude mouse experiment was conducted, whose results revealed the ability and feasibility of the HRDM-based algorithm in the biomedical application of gastric cancer detection.

  5. Use of a highly sensitive two-dimensional luminescence imaging system to monitor endogenous bioluminescence in plant leaves

    Directory of Open Access Journals (Sweden)

    Flor-Henry Michel

    2004-11-01

    Full Text Available Abstract Background All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used. Results We developed a novel configuration of a cooled charge-coupled device (CCD for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures. Conclusions The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves.

  6. Distribution of bioluminescent fungi across old-growth and secondary tropical rain forest in Costa Rica

    Directory of Open Access Journals (Sweden)

    Carolina Seas-Carvajal

    2013-06-01

    Full Text Available Most research on bioluminescent fungi is concentrated on their taxonomic relationships, while the basics of their natural history and ecological relationships are poorly understood. In this study, we compared the distribution of bioluminescent fungi between old-growth and secondary forest as related to four different soil types at the tropical rainforest of La Selva Biological Station in Costa Rica. The study was conducted during the wet season of 2009. Bioluminescent fungi were sought following eight different transects distributed evenly in old-growth and secondary forests across four different soil types, covering an area of 9 420m². We found fungi in four different substrates: litter, fallen branches, dead trunks, and roots, for a total of 61 samples. Correspondence analysis showed that the occurrence of fungi and soil types were related (inertia=0.21, p=0.071. We found a significant relationship between the presence of fungi and the distribution of soil types (X²=18.89, df=9, p=0.026. We found only three samples with fruiting bodies, two of which had Mycena and the other had one fungus of the order Xylariales (possibly Hypoxylon sp., Kretzschmariella sp., Xylaria sp.. Future work will concentrate on exploring other aspects of their ecology, such as their dispersal and substrate preference. This information will facilitate field identification and will foster more research on the distribution, seasonality, reproductive phenology and ecological requirements of this group of Fungi.La mayoría de las investigaciones sobre los hongos bioluminiscentes se ha centrado en relaciones taxonómicas. Los aspectos básicos de la historia natural y relaciones ecológicas de este grupo son poco conocidos. En este estudio, comparamos la distribución de hongos bioluminiscentes entre el bosque primario y el secundario en la Estación Biológica La Selva, Costa Rica en relación con cuatro tipos de suelo. El estudio se realizó durante la estación lluviosa

  7. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

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    Olivier Sandre

    2017-02-01

    Full Text Available The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.

  8. Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

    Science.gov (United States)

    Cui, Boyu; Wang, Yao; Song, Yunhong; Wang, Tietao; Li, Changfu; Wei, Yahong; Luo, Zhao-Qing; Shen, Xihui

    2014-05-20

    Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells

  9. Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

    OpenAIRE

    In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A

    1997-01-01

    Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, delet...

  10. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

    Science.gov (United States)

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-10-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

  11. Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism

    Directory of Open Access Journals (Sweden)

    Tingting Xu

    2017-12-01

    Full Text Available Modern drug discovery workflows require assay systems capable of replicating the complex interactions of multiple tissue types, but that can still function under high throughput conditions. In this work, we evaluate the use of substrate-free autobioluminescence in human cell lines to support the performance of these assays with reduced economical and logistical restrictions relative to substrate-requiring bioluminescent reporter systems. The use of autobioluminescence was found to support assay functionality similar to existing luciferase reporter targets. The autobioluminescent assay format was observed to correlate strongly with general metabolic activity markers such as ATP content and the presence of reactive oxygen species, but not with secondary markers such as glutathione depletion. At the transcriptional level, autobioluminescent dynamics were most closely associated with expression of the CYP1A1 phase I detoxification pathway. These results suggest constitutively autobioluminescent cells can function as general metabolic activity bioreporters, while pairing expression of the autobioluminescent phenotype to detoxification pathway specific promoters could create more specific sensor systems.

  12. Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET

    International Nuclear Information System (INIS)

    Deroose, Christophe M.; Chitneni, Satish K.; Gijsbers, Rik; Vermaelen, Peter; Ibrahimi, Abdelilah; Balzarini, Jan; Baekelandt, Veerle; Verbruggen, Alfons; Nuyts, Johan; Debyser, Zeger; Bormans, Guy M.

    2012-01-01

    Introduction: Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. Methods: The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. Results: High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ∼ 1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. Conclusions: We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

  13. Light Emission Requires Exposure to the Atmosphere in Ex Vivo Bioluminescence Imaging

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    Yusuke Inoue

    2006-04-01

    Full Text Available The identification of organs bearing luciferase activity by in vivo bioluminescence imaging (BLI is often difficult, and ex vivo imaging of excised organs plays a complementary role. This study investigated the importance of exposure to the atmosphere in ex vivo BLI. Mice were inoculated with murine pro-B cell line Ba/F3 transduced with firefly luciferase and p190 BCR-ABL. They were killed following in vivo BLI, and whole-body imaging was done after death and then after intraperitoneal air injection. In addition, the right knee was exposed and imaged before and after the adjacent bones were cut. Extensive light signals were seen on in vivo imaging. The luminescence disappeared after the animal was killed, and air injection restored the light emission from the abdomen only, suggesting a critical role of atmospheric oxygen in luminescence after death. Although no substantial light signal at the right knee was seen before bone cutting, light emission was evident after cutting. In conclusion, in ex vivo BLI, light emission requires exposure to the atmosphere. Bone destruction is required to demonstrate luciferase activity in the bone marrow after death.

  14. CD4+ T cell effects on CD8+ T cell location defined using bioluminescence.

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    Mitra Azadniv

    2011-01-01

    Full Text Available T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

  15. L{sub 1/2} regularization based numerical method for effective reconstruction of bioluminescence tomography

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueli, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn; Yang, Defu; Zhang, Qitan; Liang, Jimin, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn [School of Life Science and Technology, Xidian University, Xi' an 710071 (China); Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education (China)

    2014-05-14

    Even though bioluminescence tomography (BLT) exhibits significant potential and wide applications in macroscopic imaging of small animals in vivo, the inverse reconstruction is still a tough problem that has plagued researchers in a related area. The ill-posedness of inverse reconstruction arises from insufficient measurements and modeling errors, so that the inverse reconstruction cannot be solved directly. In this study, an l{sub 1/2} regularization based numerical method was developed for effective reconstruction of BLT. In the method, the inverse reconstruction of BLT was constrained into an l{sub 1/2} regularization problem, and then the weighted interior-point algorithm (WIPA) was applied to solve the problem through transforming it into obtaining the solution of a series of l{sub 1} regularizers. The feasibility and effectiveness of the proposed method were demonstrated with numerical simulations on a digital mouse. Stability verification experiments further illustrated the robustness of the proposed method for different levels of Gaussian noise.

  16. Quick, portable toxicity testing of marine or terrigenous fluids, sediments, or chemicals with bioluminescent organism

    International Nuclear Information System (INIS)

    Sabate, R.W.; Stiffey, A.V.; Dewailly, E.L.

    1995-01-01

    A hand-held, battery-operated instrument, which measures bioluminescence inhibition of the microscopic marine dinoflagellate Pyrocystis lunula, is capable of field-testing substances for toxicity. The organism is sensitive to ppb of strong toxicants. It tolerates some solvents in concentrations necessary for testing lipophylic samples. A test consumes only micrograms of sample. This method requires no adjustments for salinity, pH, color, or turbidity. It has been used successfully to test oil-well drilling fluids, brines produced with oil, waters and sediments from streams and lakes and petroleum-plant effluents containing contaminants such as benzene. The test is non-specific; however, if the substance is known, the end-point effects a direct measurement of its concentration. One-hour toxicity screening tests in the field produce results comparable to the standard four-hour laboratory test. Keeping the sample in the dark during incubation and testing, together with shortness of the overall procedure, eliminates anomalies from light-sensitive substances. Day-to-day variation, as well as among test replicates, is less than 10%. This quick method yields results comparable with a quick test that uses Photobacterium phosphoria, and with 96-hour tests that use Mysidopsis bahia, Artemia salina, Gonyaulax polyedra, Pimephales promelas, Ceriodaphnia dubia, and Cyprinodon variegatus

  17. The CUBLAS and CULA based GPU acceleration of adaptive finite element framework for bioluminescence tomography.

    Science.gov (United States)

    Zhang, Bo; Yang, Xiang; Yang, Fei; Yang, Xin; Qin, Chenghu; Han, Dong; Ma, Xibo; Liu, Kai; Tian, Jie

    2010-09-13

    In molecular imaging (MI), especially the optical molecular imaging, bioluminescence tomography (BLT) emerges as an effective imaging modality for small animal imaging. The finite element methods (FEMs), especially the adaptive finite element (AFE) framework, play an important role in BLT. The processing speed of the FEMs and the AFE framework still needs to be improved, although the multi-thread CPU technology and the multi CPU technology have already been applied. In this paper, we for the first time introduce a new kind of acceleration technology to accelerate the AFE framework for BLT, using the graphics processing unit (GPU). Besides the processing speed, the GPU technology can get a balance between the cost and performance. The CUBLAS and CULA are two main important and powerful libraries for programming on NVIDIA GPUs. With the help of CUBLAS and CULA, it is easy to code on NVIDIA GPU and there is no need to worry about the details about the hardware environment of a specific GPU. The numerical experiments are designed to show the necessity, effect and application of the proposed CUBLAS and CULA based GPU acceleration. From the results of the experiments, we can reach the conclusion that the proposed CUBLAS and CULA based GPU acceleration method can improve the processing speed of the AFE framework very much while getting a balance between cost and performance.

  18. Hunting in bioluminescent light: Vision in the nocturnal box jellyfish Copula sivickisi

    Directory of Open Access Journals (Sweden)

    Anders eGarm

    2016-03-01

    Full Text Available Cubomedusae all have a similar set of six eyes on each of their four rhopalia. Still, there is a great variation in activity patterns with some species being strictly day active while others are strictly night active. Here we have examined the visual ecology of the medusa of the night active Copula sivickisi from Okinawa using optics, morphology, electrophysiology, and behavioural experiments. We found the lenses of both the upper and the lower lens eyes to be image forming but under-focused, resulting in low spatial resolution in the order of 10 – 15 degrees. The photoreceptor physiology is similar in the two lens eyes and they have a single opsin peaking around 460 nm and low temporal resolution with a flicker fusion frequency (fff of 2.5 Hz indicating adaptions to vision in low light intensities. Further, the outer segments have fluid filled swellings, which may concentrate the light in the photoreceptor membrane by total internal reflections, and thus enhance the signal to noise ratio in the eyes. Finally our behavioural experiments confirmed that the animals use vision when hunting. When they are active at night they seek out high prey-concentration by visual attraction to areas with abundant bioluminescent flashes triggered by their prey.

  19. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

  20. Volatile Gas Production by Methyl Halide Transferase: An In Situ Reporter Of Microbial Gene Expression In Soil.

    Science.gov (United States)

    Cheng, Hsiao-Ying; Masiello, Caroline A; Bennett, George N; Silberg, Jonathan J

    2016-08-16

    Traditional visual reporters of gene expression have only very limited use in soils because their outputs are challenging to detect through the soil matrix. This severely restricts our ability to study time-dependent microbial gene expression in one of the Earth's largest, most complex habitats. Here we describe an approach to report on dynamic gene expression within a microbial population in a soil under natural water levels (at and below water holding capacity) via production of methyl halides using a methyl halide transferase. As a proof-of-concept application, we couple the expression of this gas reporter to the conjugative transfer of a bacterial plasmid in a soil matrix and show that gas released from the matrix displays a strong correlation with the number of transconjugant bacteria that formed. Gas reporting of gene expression will make possible dynamic studies of natural and engineered microbes within many hard-to-image environmental matrices (soils, sediments, sludge, and biomass) at sample scales exceeding those used for traditional visual reporting.

  1. An evolutionary conserved region (ECR in the human dopamine receptor D4 gene supports reporter gene expression in primary cultures derived from the rat cortex

    Directory of Open Access Journals (Sweden)

    Haddley Kate

    2011-05-01

    Full Text Available Abstract Background Detecting functional variants contributing to diversity of behaviour is crucial for dissecting genetics of complex behaviours. At a molecular level, characterisation of variation in exons has been studied as they are easily identified in the current genome annotation although the functional consequences are less well understood; however, it has been difficult to prioritise regions of non-coding DNA in which genetic variation could also have significant functional consequences. Comparison of multiple vertebrate genomes has allowed the identification of non-coding evolutionary conserved regions (ECRs, in which the degree of conservation can be comparable with exonic regions suggesting functional significance. Results We identified ECRs at the dopamine receptor D4 gene locus, an important gene for human behaviours. The most conserved non-coding ECR (D4ECR1 supported high reporter gene expression in primary cultures derived from neonate rat frontal cortex. Computer aided analysis of the sequence of the D4ECR1 indicated the potential transcription factors that could modulate its function. D4ECR1 contained multiple consensus sequences for binding the transcription factor Sp1, a factor previously implicated in DRD4 expression. Co-transfection experiments demonstrated that overexpression of Sp1 significantly decreased the activity of the D4ECR1 in vitro. Conclusion Bioinformatic analysis complemented by functional analysis of the DRD4 gene locus has identified a a strong enhancer that functions in neurons and b a transcription factor that may modulate the function of that enhancer.

  2. Structure and function of the human metallothionein gene family: Final technical report

    International Nuclear Information System (INIS)

    Karin, M.

    1986-01-01

    The full nucleotide sequence of two additional human metallothionein (hMT) genes has been determined. These genes, hMT-I/sub B/ and hMT-I/sub F/, are located within the MT-I gene cluster we have described originally. The hMT-I/sub F/ gene is the first hMT-I gene whose amino acid sequence is in complete agreement with the published sequence of the human MT-I proteins. Therefore it is likely to be an active gene encoding a functional protein. However, since we have just completed the sequence analysis, we have not characterized this gene further yet. The hMT-I/sub B/ gene is closely linked to the hMT-I/sub A/ gene, and two pseudogenes, hMT-I/sub C/ and hMT-I/sub D/ separate the two. From its nucleotide sequence hMT-I/sub B/ seems to be an active gene, encoding a functional protein even though it differs in four positions from the published sequence of human MT-I proteins. This gene is expressed in a human hepatoma cell line, HepG2, and its expression is stimulated by Cd ++ . Using gene fusions to the viral thymidine-kinase gene we find that hMT-I/sub B/, like the hMT-I/sub A/ and hMT-II/sub A/ genes, contains a heavy metal responsive promoterregulatory element within its 5' flanking region. We analyzed the level of hMT-I/sub B/ mRNA in a variety of human cell lines by the S1 nuclease technique, and compared it to the expression of the hMT-II/sub A/ gene. While the hMT-II/sub A/ gene was expressed in all of the cell lines analyzed, the hMT-I/sub B/ gene was expressed in liver and kidney derived cell lines cells. This suggest that the expression of the hMT-I/sub B/ gene is controlled in a tissue specific manner. 13 refs

  3. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    Science.gov (United States)

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

  4. Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

    Science.gov (United States)

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

  5. Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

    Directory of Open Access Journals (Sweden)

    Chao-Hsun Yang

    2013-01-01

    Full Text Available The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP at C-terminus and red fluorescent protein (RFP, DsRed at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.

  6. Pine Gene Discovery Project - Final Report - 08/31/1997 - 02/28/2001; FINAL

    International Nuclear Information System (INIS)

    Whetten, R. W.; Sederoff, R. R.; Kinlaw, C.; Retzel, E.

    2001-01-01

    Integration of pines into the large scope of plant biology research depends on study of pines in parallel with study of annual plants, and on availability of research materials from pine to plant biologists interested in comparing pine with annual plant systems. The objectives of the Pine Gene Discovery Project were to obtain 10,000 partial DNA sequences of genes expressed in loblolly pine, to determine which of those pine genes were similar to known genes from other organisms, and to make the DNA sequences and isolated pine genes available to plant researchers to stimulate integration of pines into the wider scope of plant biology research. Those objectives have been completed, and the results are available to the public. Requests for pine genes have been received from a number of laboratories that would otherwise not have included pine in their research, indicating that progress is being made toward the goal of integrating pine research into the larger molecular biology research community

  7. Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: a performance overview.

    Science.gov (United States)

    Deliolanis, Nikolaos C; Ale, Angelique; Morscher, Stefan; Burton, Neal C; Schaefer, Karin; Radrich, Karin; Razansky, Daniel; Ntziachristos, Vasilis

    2014-10-01

    A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents.

  8. A Multisampling Reporter System for Monitoring MicroRNA Activity in the Same Population of Cells

    Directory of Open Access Journals (Sweden)

    Pei-Chen Huang

    2009-01-01

    Full Text Available MicroRNAs (miRNAs downregulate gene expression by binding to the partially complementary sites in the 3′ untranslated region (UTR of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3′UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report.

  9. Heterologous expression of mannanase and developing a new Reporter gene system in Lactobacillus casei and Escherichia coli

    DEFF Research Database (Denmark)

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie

    2015-01-01

    Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were clone....... casei and E.coli....

  10. Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments

    DEFF Research Database (Denmark)

    Pinilla-Redondo, Rafael; Riber, Leise; Sørensen, Søren Johannes

    2018-01-01

    H limitations, and provide experimental tools that will help broaden its horizon of application to other fields.IMPORTANCEMany anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT......). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated...

  11. Systematic study of target localization for bioluminescence tomography guided radiation therapy

    Science.gov (United States)

    Yu, Jingjing; Zhang, Bin; Iordachita, Iulian I.; Reyes, Juvenal; Lu, Zhihao; Brock, Malcolm V.; Patterson, Michael S.; Wong, John W.

    2016-01-01

    Purpose: To overcome the limitation of CT/cone-beam CT (CBCT) in guiding radiation for soft tissue targets, the authors developed a spectrally resolved bioluminescence tomography (BLT) system for the small animal radiation research platform. The authors systematically assessed the performance of the BLT system in terms of target localization and the ability to resolve two neighboring sources in simulations, tissue-mimicking phantom, and in vivo environments. Methods: Multispectral measurements acquired in a single projection were used for the BLT reconstruction. The incomplete variables truncated conjugate gradient algorithm with an iterative permissible region shrinking strategy was employed as the optimization scheme to reconstruct source distributions. Simulation studies were conducted for single spherical sources with sizes from 0.5 to 3 mm radius at depth of 3–12 mm. The same configuration was also applied for the double source simulation with source separations varying from 3 to 9 mm. Experiments were performed in a standalone BLT/CBCT system. Two self-illuminated sources with 3 and 4.7 mm separations placed inside a tissue-mimicking phantom were chosen as the test cases. Live mice implanted with single-source at 6 and 9 mm depth, two sources at 3 and 5 mm separation at depth of 5 mm, or three sources in the abdomen were also used to illustrate the localization capability of the BLT system for multiple targets in vivo. Results: For simulation study, approximate 1 mm accuracy can be achieved at localizing center of mass (CoM) for single-source and grouped CoM for double source cases. For the case of 1.5 mm radius source, a common tumor size used in preclinical study, their simulation shows that for all the source separations considered, except for the 3 mm separation at 9 and 12 mm depth, the two neighboring sources can be resolved at depths from 3 to 12 mm. Phantom experiments illustrated that 2D bioluminescence imaging failed to distinguish two sources

  12. Systematic study of target localization for bioluminescence tomography guided radiation therapy

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jingjing [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University, Baltimore, Maryland 21231 and School of Physics and Information Technology, Shaanxi Normal University, Shaanxi 710119 (China); Zhang, Bin; Reyes, Juvenal; Wong, John W.; Wang, Ken Kang-Hsin, E-mail: kwang27@jhmi.edu [Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University, Baltimore, Maryland 21231 (United States); Iordachita, Iulian I. [Laboratory for Computational Sensing and Robotics, Johns Hopkins University, Baltimore, Maryland 21218 (United States); Lu, Zhihao [Department of Oncology and Department of Surgery, Johns Hopkins University, Baltimore, Maryland 21231 and Key laboratory of Carcinogenesis and Translational Research, Department of GI Oncology, Peking University, Beijing Cancer Hospital and Institute, Beijing 100142 (China); Brock, Malcolm V. [Department of Oncology and Department of Surgery, Johns Hopkins University, Baltimore, Maryland 21231 (United States); Patterson, Michael S. [Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario L8S 4L8 (Canada)

    2016-05-15

    Purpose: To overcome the limitation of CT/cone-beam CT (CBCT) in guiding radiation for soft tissue targets, the authors developed a spectrally resolved bioluminescence tomography (BLT) system for the small animal radiation research platform. The authors systematically assessed the performance of the BLT system in terms of target localization and the ability to resolve two neighboring sources in simulations, tissue-mimicking phantom, and in vivo environments. Methods: Multispectral measurements acquired in a single projection were used for the BLT reconstruction. The incomplete variables truncated conjugate gradient algorithm with an iterative permissible region shrinking strategy was employed as the optimization scheme to reconstruct source distributions. Simulation studies were conducted for single spherical sources with sizes from 0.5 to 3 mm radius at depth of 3–12 mm. The same configuration was also applied for the double source simulation with source separations varying from 3 to 9 mm. Experiments were performed in a standalone BLT/CBCT system. Two self-illuminated sources with 3 and 4.7 mm separations placed inside a tissue-mimicking phantom were chosen as the test cases. Live mice implanted with single-source at 6 and 9 mm depth, two sources at 3 and 5 mm separation at depth of 5 mm, or three sources in the abdomen were also used to illustrate the localization capability of the BLT system for multiple targets in vivo. Results: For simulation study, approximate 1 mm accuracy can be achieved at localizing center of mass (CoM) for single-source and grouped CoM for double source cases. For the case of 1.5 mm radius source, a common tumor size used in preclinical study, their simulation shows that for all the source separations considered, except for the 3 mm separation at 9 and 12 mm depth, the two neighboring sources can be resolved at depths from 3 to 12 mm. Phantom experiments illustrated that 2D bioluminescence imaging failed to distinguish two sources

  13. Phylogenetic relationships of click beetles (Coleoptera: Elateridae) inferred from 28S ribosomal DNA: insights into the evolution of bioluminescence in Elateridae.

    Science.gov (United States)

    Sagegami-Oba, Reiko; Oba, Yuichi; Ohira, Hitoo

    2007-02-01

    Although the taxonomy of click beetles (family Elateridae) has been studied extensively, inconsistencies remain. We examine here the relationships between species of Elateridae based on partial sequences of nuclear 28S ribosomal DNA. Specimens were collected primarily from Japan, while luminous click beetles were also sampled from Central and South America to investigate the origins of bioluminescence in Elateridae. Neighbor-joining, maximum-parsimony, and maximum-likelihood analyses produced a consistent basal topology with high statistical support that is partially congruent with the results of previous investigations based on the morphological characteristics of larvae and adults. The most parsimonious reconstruction of the "luminous" and "nonluminous" states, based on the present molecular phylogeny, indicates that the ancestral state of Elateridae was nonluminous. This suggests that the bioluminescence in click beetle evolved independent of that of other luminous beetles, such as Lampyridae, despite their common mechanisms of bioluminescence.

  14. Brugada syndrome with a novel missense mutation in SCN5A gene: A case report from Bangladesh

    Directory of Open Access Journals (Sweden)

    Md. Zahidus Sayeed

    2014-01-01

    Full Text Available Brugada syndrome is an inherited cardiac arrhythmia that follows autosomal dominant transmission and can cause sudden death. We report a case of Brugada syndrome in a 55-year-old male patient presented with recurrent palpitation, atypical chest pain and presyncope. ECG changes were consistent with type 1 Brugada. Gene analysis revealed a novel missense mutation in SCN5A gene with a genetic variation of D785N and a nucleotide change at 2353G-A. One of his children also had the same mutation. To our knowledge this is the first genetically proved case of Brugada syndrome in Bangladesh.

  15. Propagation and perception of bioluminescence: factors affecting counterillumination as a cryptic strategy.

    Science.gov (United States)

    Johnsen, Sönke; Widder, Edith A; Mobley, Curtis D

    2004-08-01

    Many deep-sea species, particularly crustaceans, cephalopods, and fish, use photophores to illuminate their ventral surfaces and thus disguise their silhouettes from predators viewing them from below. This strategy has several potential limitations, two of which are examined here. First, a predator with acute vision may be able to detect the individual photophores on the ventral surface. Second, a predator may be able to detect any mismatch between the spectrum of the bioluminescence and that of the background light. The first limitation was examined by modeling the perceived images of the counterillumination of the squid Abralia veranyi and the myctophid fish Ceratoscopelus maderensis as a function of the distance and visual acuity of the viewer. The second limitation was addressed by measuring downwelling irradiance under moonlight and starlight and then modeling underwater spectra. Four water types were examined: coastal water at a depth of 5 m and oceanic water at 5, 210, and 800 m. The appearance of the counterillumination was more affected by the visual acuity of the viewer than by the clarity of the water, even at relatively large distances. Species with high visual acuity (0.11 degrees resolution) were able to distinguish the individual photophores of some counterilluminating signals at distances of several meters, thus breaking the camouflage. Depth and the presence or absence of moonlight strongly affected the spectrum of the background light, particularly near the surface. The increased variability near the surface was partially offset by the higher contrast attenuation at shallow depths, which reduced the sighting distance of mismatches. This research has implications for the study of spatial resolution, contrast sensitivity, and color discrimination in deep-sea visual systems.

  16. Image Processing for Bioluminescence Resonance Energy Transfer Measurement—BRET-Analyzer

    Directory of Open Access Journals (Sweden)

    Yan Chastagnier

    2018-01-01

    Full Text Available A growing number of tools now allow live recordings of various signaling pathways and protein-protein interaction dynamics in time and space by ratiometric measurements, such as Bioluminescence Resonance Energy Transfer (BRET Imaging. Accurate and reproducible analysis of ratiometric measurements has thus become mandatory to interpret quantitative imaging. In order to fulfill this necessity, we have developed an open source toolset for Fiji—BRET-Analyzer—allowing a systematic analysis, from image processing to ratio quantification. We share this open source solution and a step-by-step tutorial at https://github.com/ychastagnier/BRET-Analyzer. This toolset proposes (1 image background subtraction, (2 image alignment over time, (3 a composite thresholding method of the image used as the denominator of the ratio to refine the precise limits of the sample, (4 pixel by pixel division of the images and efficient distribution of the ratio intensity on a pseudocolor scale, and (5 quantification of the ratio mean intensity and standard variation among pixels in chosen areas. In addition to systematize the analysis process, we show that the BRET-Analyzer allows proper reconstitution and quantification of the ratiometric image in time and space, even from heterogeneous subcellular volumes. Indeed, analyzing twice the same images, we demonstrate that compared to standard analysis BRET-Analyzer precisely define the luminescent specimen limits, enlightening proficient strengths from small and big ensembles over time. For example, we followed and quantified, in live, scaffold proteins interaction dynamics in neuronal sub-cellular compartments including dendritic spines, for half an hour. In conclusion, BRET-Analyzer provides a complete, versatile and efficient toolset for automated reproducible and meaningful image ratio analysis.

  17. A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi.

    Science.gov (United States)

    Byers, D M; Holmes, C G

    1990-01-01

    An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.

  18. TH-EF-207A-07: An Integrated X-Ray/bioluminescence Tomography System for Radiation Guidance and Tumor Evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Shi, J; Udayakumar, T; Wang, Z; Dogan, N; Pollack, A; Yang, Y [University of Miami School of Medicine, Miami, FL (United States)

    2016-06-15

    Purpose: CT is not able to differentiate tumors from surrounding soft tissue. This study is to develop a bioluminescence tomography (BLT) system that is integrated onto our previously developed CT guided small animal arc radiation treatment system (iSMAART) to guide radiation, monitor tumor growth and evaluate therapeutic response. Methods: The BLT system employs a CCD camera coupled with a high speed lens, and is aligned orthogonally to the x-ray beam central axis. The two imaging modalities, CT and BLT, are physically registered through geometrical calibration. The CT anatomy provides an accurate contour of animal surface which is used to construct 3D mesh for BLT reconstruction. Bioluminescence projections are captured from multiple angles, once every 45 degree rotation. The diffusion equation based on analytical Kirchhoff approximation is adopted to model the photon propagation in tissues. A discrete cosine transform based reweighted L1-norm regularization (DCT-re-L1) algorithm is used for BLT reconstruction. Experiments are conducted on a mouse orthotopic prostate tumor model (n=12) to evaluate the BLT performance, in terms of its robustness and accuracy in locating and quantifying the bioluminescent tumor cells. Iodinated contrast agent was injected intravenously to delineate the tumor in CT. The tumor location and volume obtained from CT also serve as a benchmark against BLT. Results: With our cutting edge reconstruction algorithm, BLT is able to accurately reconstruct the orthotopic prostate tumors. The tumor center of mass in BLT is within 0.5 mm radial distance of that in CT. The tumor volume in BLT is significantly correlated with that in CT (R2 = 0.81). Conclusion: The BLT can differentiate, localize and quantify tumors. Together with CT, BLT will provide precision radiation guidance and reliable treatment assessment in preclinical cancer research.

  19. A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

    Science.gov (United States)

    Sasaki-Iwaoka, H; Maruyama, K; Endoh, H; Komori, T; Kato, S; Kawashima, H

    1999-02-01

    The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

  20. Light and vision in the deep-sea benthos: I. Bioluminescence at 500-1000 m depth in the Bahamian islands.

    Science.gov (United States)

    Johnsen, Sönke; Frank, Tamara M; Haddock, Steven H D; Widder, Edith A; Messing, Charles G

    2012-10-01

    Bioluminescence is common and well studied in mesopelagic species. However, the extent of bioluminescence in benthic sites of similar depths is far less studied, although the relatively large eyes of benthic fish, crustaceans and cephalopods at bathyal depths suggest the presence of significant biogenic light. Using the Johnson-Sea-Link submersible, we collected numerous species of cnidarians, echinoderms, crustaceans, cephalopods and sponges, as well as one annelid from three sites in the northern Bahamas (500-1000 m depth). Using mechanical and chemical stimulation, we tested the collected species for light emission, and photographed and measured the spectra of the emitted light. In addition, in situ intensified video and still photos were taken of different benthic habitats. Surprisingly, bioluminescence in benthic animals at these sites was far less common than in mesopelagic animals from similar depths, with less than 20% of the collected species emitting light. Bioluminescent taxa comprised two species of anemone (Actinaria), a new genus and species of flabellate Parazoanthidae (formerly Gerardia sp.) (Zoanthidea), three sea pens (Pennatulacea), three bamboo corals (Alcyonacea), the chrysogorgiid coral Chrysogorgia desbonni (Alcyonacea), the caridean shrimp Parapandalus sp. and Heterocarpus ensifer (Decapoda), two holothuroids (Elasipodida and Aspidochirota) and the ophiuroid Ophiochiton ternispinus (Ophiurida). Except for the ophiuroid and the two shrimp, which emitted blue light (peak wavelengths 470 and 455 nm), all the species produced greener light than that measured in most mesopelagic taxa, with the emissions of the pennatulaceans being strongly shifted towards longer wavelengths. In situ observations suggested that bioluminescence associated with these sites was due primarily to light emitted by bioluminescent planktonic species as they struck filter feeders that extended into the water column.

  1. Ultra-thin titanium nanolayers for plasmon-assisted enhancement of bioluminescence of chloroplast in biological light emitting devices

    Energy Technology Data Exchange (ETDEWEB)

    Hsun Su, Yen [Department of Materials Science and Engineering, National Cheng Kung University, Tainan 70101, Taiwan (China); Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 70101, Taiwan (China); Hsu, Chia-Yun; Chang, Chung-Chien [Science and Technology of Accelerator Light Source, Hsinchu 300, Taiwan (China); Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu 300, Taiwan (China); Tu, Sheng-Lung; Shen, Yun-Hwei [Department of Resource Engineering, National Cheng Kung University, Tainan 70101, Taiwan (China)

    2013-08-05

    Ultra-thin titanium films were deposited via ultra-high vacuum ion beam sputter deposition. Since the asymmetric electric field of the metal foil plane matches the B-band absorption of chlorophyll a, the ultra-thin titanium nanolayers were able to generate surface plasmon resonance, thus enhancing the photoluminescence of chlorophyll a. Because the density of the states of plasmon resonance increases, the enhancement of photoluminescence also rises. Due to the biocompatibility and inexpensiveness of titanium, it can be utilized to enhance the bioluminescence of chloroplast in biological light emitting devices, bio-laser, and biophotonics.

  2. Report on emerging technologies for translational bioinformatics: a symposium on gene expression profiling for archival tissues

    Directory of Open Access Journals (Sweden)

    Waldron Levi

    2012-03-01

    Full Text Available Abstract Background With over 20 million formalin-fixed, paraffin-embedded (FFPE tissue samples archived each year in the United States alone, archival tissues remain a vast and under-utilized resource in the genomic study of cancer. Technologies have recently been introduced for whole-transcriptome amplification and microarray analysis of degraded mRNA fragments from FFPE samples, and studies of these platforms have only recently begun to enter the published literature. Results The Emerging Technologies for Translational Bioinformatics symposium on gene expression profiling for archival tissues featured presentations of two large-scale FFPE expression profiling studies (each involving over 1,000 samples, overviews of several smaller studies, and representatives from three leading companies in the field (Illumina, Affymetrix, and NuGEN. The meeting highlighted challenges in the analysis of expression data from archival tissues and strategies being developed to overcome them. In particular, speakers reported higher rates of clinical sample failure (from 10% to 70% than are typical for fresh-frozen tissues, as well as more frequent probe failure for individual samples. The symposium program is available at http://www.hsph.harvard.edu/ffpe. Conclusions Multiple solutions now exist for whole-genome expression profiling of FFPE tissues, including both microarray- and sequencing-based platforms. Several studies have reported their successful application, but substantial challenges and risks still exist. Symposium speakers presented novel methodology for analysis of FFPE expression data and suggestions for improving data recovery and quality assessment in pre-analytical stages. Research presentations emphasized the need for careful study design, including the use of pilot studies, replication, and randomization of samples among batches, as well as careful attention to data quality control. Regardless of any limitations in quantitave transcriptomics for

  3. In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene

    Directory of Open Access Journals (Sweden)

    Tomoko Nakanishi

    2013-01-01

    Full Text Available Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1α,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

  4. Adenosina trifosfato bioluminescência para avaliação da limpeza de superfícies: uma revisão integrativa

    OpenAIRE

    Oliveira, Adriana Cristina de; Viana, Roberta El Hariri

    2014-01-01

    Objetivo: Identificar na literatura indicações e controvérsias do ATP bioluminescência para avaliação da efetividade da limpeza de superfícies em estabelecimentos de saúde. Método: Revisão integrativa da literatura, entre 2000 e 2012, nas bases de dados MEDLINE, LILACS, Science Direct, SCOPUS e Isi Web of Knowledge. Resultados: Selecionou-se para esta revisão 15 artigos. O ATP bioluminescência foi apontado como importante recurso educacional e método complementar à inspeção visual e às anális...

  5. The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Xiang; Li Biao; Wang Jun; Yin Hongyan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Zhang Yifan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)], E-mail: zhangyifan1992@yahoo.com.cn

    2010-04-15

    Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene. Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a {gamma} counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells. Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with

  6. Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

    Science.gov (United States)

    Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

    2010-07-01

    Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

  7. Genetic components for radiosensitivity. Gene expression in radiosensitive monocygotic twins. Final report

    International Nuclear Information System (INIS)

    Dikomey, Ekkehard

    2012-01-01

    The underlying hypothesis of this project was that the variation of individual radiosensitivity is determined by the different expression of single gens. This concept was tested using 60 monozygotic twin pairs, followed by an evaluation with 80 prostate cancer patients. Radiosensitivity was assessed for both G0- as well as G2-phase using chromosomal assays. G0- radiosensitivity is determined by lethal chromosomal aberrations and reflects the individual amount of cell killing, while G2-sensitivity is determined by chromatid breaks and is taken as an indicator of individual cancer risk. For both populations, G0- and G2-radiosensitivity are characterized by substantial variation with a CV of 11 and 14% or 27 and 21%, respectively. While the mean G0-sensitivity is the same for both populations, there is a slight difference for G2. The slightly higher value of G2-sensitivity found for prostate cancer patients might result from the higher age of this group. For both populations gene expression profiles were determined using the Affymetrix chip HG-U133+2.0. Overall gene expression was characterized by a huge variation covering more than four decades. However, for single genes, expression showed little variation with CV generally ranging only between 2 and 8%. Analysis of data using several different methods revealed that variation of both G0- as well as G2-radiosensitivity cannot be ascribed to the different expression of single genes. For twins, random forests can be used to identify 8 to 10 genes than are relevant either for G0- or G2-radiosensitivity. However, these genes cannot be confirmed by an evaluation with 80 prostate cancer patients. This finding clearly demonstrates that the hypothesis, due to which variation of individual radiosensitivity is caused by different expression of single genes, has to be rejected. It appears more likely that this parameter is determined by complex interactions of several genes in functional networks. (orig.)

  8. On the Use of Gene Ontology Annotations to Assess Functional Similarity among Orthologs and Paralogs: A Short Report.

    Directory of Open Access Journals (Sweden)

    Paul D Thomas

    Full Text Available A recent paper (Nehrt et al., PLoS Comput. Biol. 7:e1002073, 2011 has proposed a metric for the "functional similarity" between two genes that uses only the Gene Ontology (GO annotations directly derived from published experimental results. Applying this metric, the authors concluded that paralogous genes within the mouse genome or the human genome are more functionally similar on average than orthologous genes between these genomes, an unexpected result with broad implications if true. We suggest, based on both theoretical and empirical considerations, that this proposed metric should not be interpreted as a functional similarity, and therefore cannot be used to support any conclusions about the "ortholog conjecture" (or, more properly, the "ortholog functional conservation hypothesis". First, we reexamine the case studies presented by Nehrt et al. as examples of orthologs with divergent functions, and come to a very different conclusion: they actually exemplify how GO annotations for orthologous genes provide complementary information about conserved biological functions. We then show that there is a global ascertainment bias in the experiment-based GO annotations for human and mouse genes: particular types of experiments tend to be performed in different model organisms. We conclude that the reported statistical differences in annotations between pairs of orthologous genes do not reflect differences in biological function, but rather complementarity in experimental approaches. Our results underscore two general considerations for researchers proposing novel types of analysis based on the GO: 1 that GO annotations are often incomplete, potentially in a biased manner, and subject to an "open world assumption" (absence of an annotation does not imply absence of a function, and 2 that conclusions drawn from a novel, large-scale GO analysis should whenever possible be supported by careful, in-depth examination of examples, to help ensure the

  9. A family with hereditary hemochromatosis carrying HFE gene splice site mutation: a case report

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    NING Huibin

    2017-01-01

    Full Text Available ObjectiveTo investigate a new type of HFE gene mutation in a family with hereditary hemochromatosis (HH. MethodsThe analysis of HFE gene was performed for one patient with a confirmed diagnosis of HH and five relatives. Blood genomic DNA was extracted and PCR multiplication was performed for the exon and intron splice sequences of related HFE, HJV, HAMP, transferrin receptor 2 (TfR2, and SLC40A1 genes. After agarose gel electrophoresis and purification, bi-directional direct sequencing was performed to detect mutation sites. ResultsThe proband had abnormal liver function and increases in serum iron, total iron binding capacity, serum ferritin, and transferrin saturation, as well as T→C homozygous mutation in the fourth base of intron 2 in the intervening sequence of the exon EXON2 of HFE gene (IVs 2+4T→C, C/C homozygous, splicing, abnormal. There were no abnormalities in HJV, HAMP, TfR2, and SLC40A1 genes. The proband′s son had the same homozygous mutation, three relatives had heterozygous mutations, and one relative had no abnormal mutations. ConclusionGene detection plays an important role in the diagnosis of hemochromatosis, and IVs 2+4T→C mutation may be a new pathogenic mutation for HH in China.

  10. A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.

    Science.gov (United States)

    Xiao, Dan; Zhang, Weifeng; Li, Yan; Liu, Kuan; Zhao, Junli; Sun, Xiaohong; Shan, Linlin; Mao, Qinwen; Xia, Haibin

    2016-02-10

    Sox2 is an important transcriptional factor that has multiple functions in stem cell maintenance and tumorigenesis. To investigate the transcriptional regulation of the Sox2 gene, a luciferase knock-in reporter system was established in HEK293 cells by placing the luciferase gene in the genome under the control of the Sox2 gene promoter using a transcription activator-like effector nuclease (TALEN)-mediated genome editing technique. PCR and Southern blot results confirmed the site-specific integration of a single copy of the exogenous luciferase gene into the genome. To prove the reliability and sensitivity of this novel luciferase knock-in system, a CRISPR/Cas transcription activation system for the Sox2 gene was constructed and applied to the knock-in system. The results indicated that luciferase activity was directly correlated with the activity of the Sox2 endogenous promoter. This novel system will be a useful tool to study the transcriptional regulation of Sox2, and has great potential in medical and industrial applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Gabriel Rinaldi

    2008-07-01

    Full Text Available The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC. We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP, and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth

  12. Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

    Science.gov (United States)

    Steinberg, Pablo; Behnisch, Peter A; Besselink, Harrie; Brouwer, Abraham A

    2017-06-01

    Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

  13. Modelling chemical reactions by QM/MM calculations: the case of the tautomerization in fireflies bioluminescent systems

    Science.gov (United States)

    Berraud-Pache, Romain; Garcia-Iriepa, Cristina; Navizet, Isabelle

    2018-04-01

    In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modelling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods.

  14. Modeling Chemical Reactions by QM/MM Calculations: The Case of the Tautomerization in Fireflies Bioluminescent Systems.

    Science.gov (United States)

    Berraud-Pache, Romain; Garcia-Iriepa, Cristina; Navizet, Isabelle

    2018-01-01

    In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modeling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods.

  15. Evaluation of transcriptional activity of the oestrogen receptor with sodium iodide symporter as an imaging reporter gene.

    Science.gov (United States)

    Kang, Joo Hyun; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul

    2006-10-01

    Oestrogen receptors are ligand-dependent transcription factors whose activity is modulated either by oestrogens or by an alternative signalling pathway. Oestrogen receptors interact via a specific DNA-binding domain, the oestrogen responsive element (ERE), in the promoter region of sensitive genes. This binding leads to an initiation of gene expression and hormonal effects. To determine the transcriptional activity of the oestrogen receptor, we developed a molecular imaging system using sodium iodide symporter (NIS) as a reporter gene. The NIS reporter gene was placed under the control of an artificial ERE derived from pERE-TA-SEAP and named as pERE-NIS. pERE-NIS was transferred to MCF-7, human breast cancer cells, which highly expressed oestrogen receptor-alpha with lipofectamine. Stably expressing cells were generated by selection with G418 for 14 days. After treatment of 17beta-oestradiol and tamoxifen with serial doses, the (125)I uptake was measured for the determination of NIS expression. The inhibition of NIS activity was performed with 50 micromol x l(-1) potassium perchlorate. The MCF7/pERE-NIS treated with 17beta-oestradiol accumulated (125)I up to 70-80% higher than did non-treated cells. NIS expression was increased according to increasing doses of 17beta-oestradiol. MCF7/pERE-NIS treated with tamoxifen also accumulated (125)I up to 50% higher than did non-treated cells. Potassium perchlorate completely inhibited (125)I uptake. When MDA-MB231 cells, the oestrogen receptor-negative breast cancer cells, were transfected with pERE-NIS, (125)I uptake of MDA-MB-231/pERE-NIS did not increase. This pERE-NIS reporter system is sufficiently sensitive for monitoring transcriptional activity of the oestrogen receptor. Therefore, cis-enhancer reporter systems with ERE will be applicable to the development of a novel selective oestrogen receptor modulator with low toxicity and high efficacy.

  16. Multimodal Imaging of Integrin Receptor-Positive Tumors by Bioluminescence, Fluorescence, Gamma Scintigraphy, and Single-Photon Emission Computed Tomography Using a Cyclic RGD Peptide Labeled with a Near-Infrared Fluorescent Dye and a Radionuclide

    Directory of Open Access Journals (Sweden)

    W. Barry Edwards

    2009-03-01

    Full Text Available Integrins, particularly the αvβ3 heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the αvβ3 integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308 bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N, N′, N″, N‴-tetraacetic acid (DOTA and a lipophilic near-infrared (NIR fluorescent dye cypate. The αvβ3 integrin binding affinity and the internalization properties of LS308 mediated by the αvβ3 integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of 111In-labeled LS308 in a 4t1luc tumor-bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT. The results show that LS308 has high affinity for αvβ3 integrin and internalized preferentially via the αvβ3 integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in αvβ3-positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of αvβ3 integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.

  17. A case report of novel mutation in PRF1 gene, which causes familial autosomal recessive hemophagocytic lymphohistiocytosis.

    Science.gov (United States)

    Bordbar, Mohammad Reza; Modarresi, Farzaneh; Farazi Fard, Mohammad Ali; Dastsooz, Hassan; Shakib Azad, Nader; Faghihi, Mohammad Ali

    2017-05-03

    Hemophagocytic Lymphohistiocytosis (HLH) is a life-threatening immunodeficiency and multi-organ disease that affects people of all ages and ethnic groups. Common symptoms and signs of this disease are high fever, hepatosplenomegaly, and cytopenias. Familial form of HLH disease, which is an autosomal recessive hematological disorder is due to disease-causing mutations in several genes essential for NK and T-cell granule-mediated cytotoxic function. For an effective cytotoxic response from cytotoxic T lymphocyte or NK cell encountering an infected cell or tumor cell, different processes are required, including trafficking, docking, priming, membrane fusion, and entry of cytotoxic granules into the target cell leading to apoptosis. Therefore, genes involved in these steps play important roles in the pathogenesis of HLH disease which include PRF1, UNC13D (MUNC13-4), STX11, and STXBP2 (MUNC18-2). Here, we report a novel missense mutation in an 8-year-old boy suffered from hepatosplenomegaly, hepatitis, epilepsy and pancytopenia. The patient was born to a first-cousin parents with no previous documented disease in his parents. To identify mutated gene in the proband, Whole Exome Sequencing (WES) utilizing next generation sequencing was used on an Illumina HiSeq 2000 platform on DNA sample from the patient. Results showed a novel deleterious homozygous missense mutation in PRF1 gene (NM_001083116: exon3: c. 1120 T > G, p.W374G) in the patient and then using Sanger sequencing it was confirmed in the proband and his parents. Since his parents were heterozygous for the identified mutation, autosomal recessive pattern of inheritance was confirmed in the family. Our study identified a rare new pathogenic missense mutation in PRF1 gene in patient with HLH disease and it is the first report of mutation in PRF1 in Iranian patients with this disease.

  18. First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

    Science.gov (United States)

    Zouheir Habbal, Mohammad; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F

    2014-01-01

    Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

  19. First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

    Directory of Open Access Journals (Sweden)

    Mohammad Zouheir Habbal

    Full Text Available Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD. Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb, one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

  20. Molecular characterization of a maize regulatory gene. Annual progress report, November 1991--October 1992

    Energy Technology Data Exchange (ETDEWEB)

    Wessler, S.R.

    1994-05-01

    All aspects of this year`s work have converged on the central theme of post-transcriptional control of R gene expression. Unlike transcriptional control, relatively little is known about post-transcriptional regulation, especially in plants. We believe that three levels of post-transcriptional regulation have been identified: control of translation initiation as evidenced by the maize Lc gene; control of nuclear localization as evidenced by the Ds allele r-m9 of maize; and control of nuclear localization through alternative splicing of the rice R homolog.

  1. A Bone Metastasis Nude Mouse Model Created by Ultrasound Guided Intracardiac Injection of Breast Cancer Cells: the Micro-CT, MRI and Bioluminescence Imaging Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Park, Young Jin; Song, Eun Hye; Kim, Seol Hwa; Song, Ho Taek; Suh, Jin Suck [Yonsei University College of Medicine, Seoul (Korea, Republic of); Choi, Sang Hyun [Korean Minjok Leadership Academy, Heongsung (Korea, Republic of)

    2011-01-15

    The purpose of this study was to develop a nude mouse model of bone metastasis by performing intracardiac injection of breast cancer cells under ultrasonography guidance and we wanted to evaluate the development and the distribution of metastasis in vivo using micro-CT, MRI and bioluminescence imaging. Animal experiments were performed in 6-week-old female nude mice. The animals underwent left ventricular injection of 2x105 MDA-MB-231Bo-Luc cells. After injection of the tumor cells, serial bioluminescence imaging was performed for 7 weeks. The findings of micro-CT, MRI and the histology were correlated with the 'hot' lesions seen on the bioluminescence imaging. Metastasis was found in 62.3% of the animals. Two weeks after intracardiac injection, metastasis to the brain, spine and femur was detected with bioluminescence imaging with an increasing intensity by week 7. Micro-CT scan confirmed multiple osteolytic lesions at the femur, spine and skull. MRI and the histology were able to show metastasis in the brain and extraskeletal metastasis around the femur. The intracardiac injection of cancer cells under ultrasonography guidance is a safe and highly reproducible method to produce bone metastasis in nude mice. This bone metastasis nude mouse model will be useful to study the mechanism of bone metastasis and to validate new therapeutics

  2. A case report on ATP6V0A4 gene mutation: Forecast of familial ...

    African Journals Online (AJOL)

    ... screening of recessive forms of dRTA independent of hearing status and offer suitable intervention to treat dRTA as well as diminish the influence of SNHL on the child's learning and communication in daily life. Keywords: Renal tubular acidosis, Homeostasis, Electrolytes, Hearing impairment, ATP6V0A4 gene, Mutation ...

  3. First report of a de novo germline mutation in the MLH1 gene

    NARCIS (Netherlands)

    Stulp, Rein P; Vos, Yvonne J; Mol, Bart; Karrenbeld, Arend; de Raad, Monique; van der Mijle, Huub J C; Sijmons, Rolf H

    2006-01-01

    Hereditary non-polyposis colorectal carcinoma (HNPCC) is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast

  4. pTRA - A reporter system for monitoring the intracellular dynamics of gene expression.

    Science.gov (United States)

    Wagner, Sabine G; Ziegler, Martin; Löwe, Hannes; Kremling, Andreas; Pflüger-Grau, Katharina

    2018-01-01

    The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

  5. Pathogenic classification of LPL gene variants reported to be associated with LPL deficiency

    DEFF Research Database (Denmark)

    Rodrigues, Rute; Artieda, Marta; Tejedor, Diego

    2016-01-01

    into the deleterious effect of the mutations is clinically essential. METHODS: We used gene sequencing followed by in-vivo/in-vitro and in-silico tools for classification. We classified 125 rare LPL mutations in 33 subjects thought to have LPL deficiency and in 314 subjects selected for very SHTG. RESULTS: Of the 33...

  6. The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

    Science.gov (United States)

    Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

    2017-01-01

    Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

  7. Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

    2009-02-15

    Aim: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyluracil) was labeled with {sup 131}I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [{sup 131}I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry. Results: FAU could be labeled with {sup 131}I, and the labeling efficiency and radiochemical purity rates were 53.82{+-}2.05% and 94.85{+-}1.76%, respectively. Time-dependent increase of the accumulation of [{sup 131}I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55{+-}0.37% and 2.09{+-}0.34%, respectively. Greater uptakes of [{sup 131}I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene

  8. Aarskog-Scott syndrome: clinical update and report of nine novel mutations of the FGD1 gene.

    Science.gov (United States)

    Orrico, A; Galli, L; Faivre, L; Clayton-Smith, J; Azzarello-Burri, S M; Hertz, J M; Jacquemont, S; Taurisano, R; Arroyo Carrera, I; Tarantino, E; Devriendt, K; Melis, D; Thelle, T; Meinhardt, U; Sorrentino, V

    2010-02-01

    Mutations in the FGD1 gene have been shown to cause Aarskog-Scott syndrome (AAS), or facio-digito-genital dysplasia (OMIM#305400), an X-linked disorder characterized by distinctive genital and skeletal developmental abnormalities with a broad spectrum of clinical phenotypes. To date, 20 distinct mutations have been reported, but little phenotypic data are available on patients with molecularly confirmed AAS. In the present study, we report on our experience of screening for mutations in the FGD1 gene in a cohort of 60 European patients with a clinically suspected diagnosis of AAS. We identified nine novel mutations in 11 patients (detection rate of 18.33%), including three missense mutations (p.R402Q; p.S558W; p.K748E), four truncating mutations (p.Y530X; p.R656X; c.806delC; c.1620delC), one in-frame deletion (c.2020_2022delGAG) and the first reported splice site mutation (c.1935+3A>C). A recurrent mutation (p.R656X) was detected in three independent families. We did not find any evidence for phenotype-genotype correlations between type and position of mutations and clinical features. In addition to the well-established phenotypic features of AAS, other clinical features are also reported and discussed. Copyright 2010 Wiley-Liss, Inc.

  9. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Directory of Open Access Journals (Sweden)

    Lee SH

    2016-04-01

    Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

  10. A case report: Becker muscular dystrophy presenting with epilepsy and dysgnosia induced by duplication mutation of Dystrophin gene.

    Science.gov (United States)

    Miao, Jing; Feng, Jia-Chun; Zhu, Dan; Yu, Xue-Fan

    2016-12-12

    Becker muscular dystrophy (BMD), a genetic disorder of X-linked recessive inheritance, typically presents with gradually progressive muscle weakness. The condition is caused by mutations of Dystrophin gene located at Xp21.2. Epilepsy is an infrequent manifestation of BMD, while cases of BMD with dysgnosia are extremely rare. We describe a 9-year-old boy with BMD, who presented with epilepsy and dysgnosia. Serum creatine kinase level was markedly elevated (3665 U/L). Wechsler intelligence tests showed a low intelligence quotient (IQ = 65). Electromyogram showed slight myogenic changes and skeletal muscle biopsy revealed muscular dystrophy. Immunohistochemical staining showed partial positivity of sarcolemma for dystrophin-N. Multiplex ligation-dependent probe amplification revealed a duplication mutation in exons 37-44 in the Dystrophin gene. The present case report helps to better understand the clinical and genetic features of BMD.

  11. Familial adult spinal muscular atrophy associated with the VAPB gene: report of 42 cases in Brazil

    Directory of Open Access Journals (Sweden)

    Victor Kosac

    2013-10-01

    Full Text Available Familial spinal muscular atrophy (FSMA associated with the vesicle-associated membrane protein-associated protein B (VAPB gene is a rare autosomal dominant disease with late onset and slow progression. We studied 10 of 42 patients from 5 families by taking clinical histories and performing physical exams, electrophysiological studies, and genetic tests. All patients presented late onset disease with slow progression characterized by fasciculations, proximal weakness, amyotrophy, and hypoactive deep tendon reflex, except two who exhibited brisk reflex. Two patients showed tongue fasciculations and respiratory insufficiency. Electrophysiological studies revealed patterns of lower motor neuron disease, and genetic testing identified a P56S mutation of the VAPB gene. Although it is a rare motor neuron disease, FSMA with this mutation might be much more prevalent in Brazil than expected, and many cases may be undiagnosed. Genetic exams should be performed whenever it is suspected in Brazil.

  12. Feasibility of baculovirus-mediated reporter gene delivery for efficient monitoring of islet transplantation in vivo

    International Nuclear Information System (INIS)

    Liu, Shuai; Pan, Yu; Lv, Jing; Wu, Haifei; Tian, Jingyan; Zhang, Yifan

    2014-01-01

    Objective: The objective of this study was to explore the feasibility of baculovirus vector-mediated sodium iodide symporter (NIS) gene delivery to monitor islet transplantation. Methods: Baculovirus vectors expressing green fluorescent protein (GFP) or NIS (Bac-GFP and Bac-NIS) were established using the Bac-to-Bac baculovirus expression system. The GFP expression of Bac-GFP-infected rat islets was observed in vitro by fluorescence microscopy. Iodine uptake and inhibition of iodine uptake by NaClO 4 in Bac-NIS-infected islets were dynamically monitored in vitro. Bac-GFP- or Bac-NIS-infected islets were implanted into the left axillary cavity of NOD-SCID mice, and fluorescence imaging and 125 I NanoSPECT/CT imaging were subsequently performed in vivo. Results: Bac-GFP efficiently infected rat islets (over 95% infected at MOI = 40), and the expression of GFP lasted approximately two weeks. NaClO 4 could inhibit iodine uptake by Bac-NIS-infected islets. In vivo imaging revealed that the fluorescence intensity of the transplant sites in Bac-GFP-infected groups was significantly higher than in the non-infected group. Grafts could be clearly observed by 125 I NanoSPECT/CT imaging for up to 8 h. Conclusion: Baculovirus vectors are powerful vehicles for studying rat islets in gene delivery. It is feasible to use a baculovirus vector to delivery an NIS gene for non-invasive monitoring transplanted islets in vivo by the expression of the target gene

  13. Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

    Science.gov (United States)

    Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

    2012-07-01

    Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma

  14. a Study of the Bioluminescence of Larger Zooplankton and the Effects of Low-Level Light Changes on Their Behavior.

    Science.gov (United States)

    van Keuren, Jeffrey Robert

    A bio-optical study was undertaken to quantify the relationships which exist between counter-illuminating organisms and the downwelling spectral light field in which they exist. The basic hypothesis behind counter-illumination is that the animal emits light using ventrally-oriented photophores to disrupt or eliminate the shadowed area on ventral surfaces. An organism lacking photophores sharply silhouettes against the highly directional downwelling irradiance, whereas by distributing photophores over the ventral surface of the body and closely matching the spectral and intensity characteristics of the downwelling light, this silhouette is obscured. Analysis carried out on changes in vertical distribution patterns in response to low-level intensity changes in ambient surface light suggested that diel migrating organisms begin to shift vertically in the water column when surface scalar irradiance decreased below or increased above 1.0 times10^{-2} muEin m^{-2} sec^ {-1}. Maximum aggregations of organisms, as defined by MOCNESS net sampling or single-frequency acoustic backscatter, appeared to remain within definable in situ blue-green isolume ranges varying less than a factor of ten throughout each night. Comparisons made between organism counter-illumination capacity and modeled in situ downwelling irradiance levels suggested that euphausiids, decapods and myctophids use between 1-10 percent of their maximum counter-illumination capacity to match the ambient downwelling light conditions. Modeling also suggested that up to 40 percent of the maximum measured bioluminescence output is required to match ambient irradiance in the shallower surface zones where aggregations of copepods, potential food sources, were commonly found at night. An optical study to quantify the radiative transfer of bioluminescence from a point source revealed that non -isotropic point sources produce radiance patterns that cannot be simply explained by inverse square losses. Therefore simple

  15. Imaging and Targeted Therapy of Multidrug Resistance. Final Report

    International Nuclear Information System (INIS)

    Piwnica-Worms, David

    2009-01-01

    One focus area of DOE Office of Science was the Imaging of Gene Expression in Health and Disease in real time in tissue culture, whole animals and ultimately patients. Investigators of the Molecular Imaging Group, Washington University Medical School, ascribed to this objective and a major focus of this group directly tied into the DOE program through their efforts targeting the multidrug resistance gene (MDR1). Our plans for continuation of the program were to extend and build on this line of investigation, incorporating new molecular tools into our methodology to selectively inhibit MDR1 gene expression with novel modulation strategies. Two approaches were to be pursued: (1) high throughput screening of compounds that disrupted mutant p53 transactivation of the MDR1 promoter, and (2) knockdown of MDR1 messenger RNA with retroviral-mediated delivery of small interfering RNA constructs. These would be combined with our continuing effort to synthesize ligands and examine structure-activity relationships of bis-salicylaldehydes labeled with gallium-68 to generate PET agents for imaging MDR1 P-glycoprotein function. We would be uniquely positioned to correlate therapeutic modulation of MDR1 gene expression and protein function in the same systems in vivo using PET and bioluminescence reporters. Use of animal models such as the mdr1a/1b(-/-) gene deleted mice would also have enabled refined analysis of modulation and tracer pharmacokinetics in vivo. Overall, this DOE program and resultant tools would enable direct monitoring of novel therapeutic strategies and the MDR phenotype in relation to gene expression and protein function in vivo.

  16. Nonalcoholic fatty liver in patients with Laron syndrome and GH gene deletion - preliminary report.

    Science.gov (United States)

    Laron, Zvi; Ginsberg, Shira; Webb, Muriel

    2008-10-01

    There is little information on the relationship between growth hormone/insulin-like growth factor-I (GH/IGF-I) deficiency or IGF-I treatment on nonalcoholic fatty liver disease (NAFLD) a disorder linked to obesity and insulin resistance. To find out whether the markedly obese patients with Laron syndrome (LS) and GH gene deletion have fatty livers. We studied 11 untreated adult patients with LS (5M, 6F), five girls with LS treated by IGF-I and five adult patients with GH gene deletion (3M, 3F), four previously treated by hGH in childhood. Fatty liver was quantitatively evaluated by ultrasonography using a phase array US system (HITACHI 6500, Japan). Body adiposity was determined by DEXA, and insulin resistance was estimated by HOMA-IR using the fasting serum glucose and insulin values. Six out of 11 adult patients with LS, two out of the five IGF-I treated girls with LS and three out of five adult hGH gene deletion patients were found to have NAFLD (nonalcoholic fatty liver disease). NAFLD is a frequent complication in untreated and treated congenital IGF-I deficiency. No correlation between NAFLD and age, sex, degree of obesity, blood lipids, or degree of insulin resistance was observed.

  17. Polymorphisms in dopaminergic system genes; association with criminal behavior and self-reported aggression in violent prison inmates from Pakistan.

    Directory of Open Access Journals (Sweden)

    Muhammad Imran Qadeer

    Full Text Available Genetic factors contribute to antisocial and criminal behavior. Dopamine transporter DAT-1 (SLC6A3 and DRD2 gene for the dopamine-2 receptor are dopaminergic system genes that regulate dopamine reuptake and signaling, and may be part of the pathogenesis of psychiatric disorders including antisocial behaviors and traits. No previous studies have analyzed DAT-1 and DRD2 polymorphisms in convicted murderers, particularly from Indian subcontinent. In this study we investigated the association of 40 bp VNTR polymorphism of DAT-1 and Taq1 variant of DRD2 gene (rs1800479 with criminal behavior and self-reported aggression in 729 subjects, including 370 men in Pakistani prisons convicted of first degree murder(s and 359 control men without any history of violence or criminal tendency. The 9R allele of DAT-1 VNTR polymorphism was more prevalent in convicted murderers compared with control samples, for either one or two risk alleles (OR = 1.49 and 3.99 respectively, P = 0.003. This potential association of DAT-1 9R allele polymorphism with murderer phenotype was confirmed assuming different genetic models of inheritance. However, no genetic association was found for DRD2 Taq1 polymorphism. In addition, a combined haplotype (9R-A2 of DAT-1 and DRD2 genes was associated with this murderer phenotype. Further, 9R allele of DAT-1 was also associated with response to verbal abuse and parental marital complications, but not with other measures pertinent to self-reported aggression. These results suggest that 9R allele, which may influence levels of intra-synaptic dopamine in the brain, may contribute to criminal tendency in this sample of violent murderers of Pakistani origin. Future studies are needed to replicate this finding in other populations of murderers and see if this finding extends to other forms of violence and lesser degrees of aggression.

  18. Establishment of a hepatocellular carcinoma cell line expressing dual reporter genes: sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP)

    International Nuclear Information System (INIS)

    Kwak, Won Jung; Koo, Bon Chul; Kwon, Mo Sun

    2007-01-01

    Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, T 1/2 of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene

  19. Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

    Directory of Open Access Journals (Sweden)

    Heffer Alison

    2011-03-01

    Full Text Available Abstract Background Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly. Results Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR and gene-specific amplified fragment length polymorphism (AFLP. It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution. Conclusions RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.

  20. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  1. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    International Nuclear Information System (INIS)

    Jirtle, Randy L.

    2012-01-01

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A vy ) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure ( vy locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in animals and humans needs to be defined

  2. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Emanuela Chiarella

    Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

  3. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Science.gov (United States)

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

  4. Quantitative imaging of D-2-hydroxyglutarate (D2HG in selected histological tissue areas by a novel bioluminescence technique

    Directory of Open Access Journals (Sweden)

    Nadine Fabienne Voelxen

    2016-03-01

    Full Text Available AbstractPatients with malignant gliomas have a poor prognosis with average survival of less than one year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG, a metabolite, which was discovered first in this tumor entity. D2HG is generated in large amounts due to various gain-of–function mutations in the isocitrate dehydrogenases IDH-1 and IDH-2. Meanwhile, D2HG has been detected in several other tumor entities including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (< 0.1 mM, but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the oncometabolite D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography / mass spectrometry (LC/MS. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0 – 10 µmol/g tissue (wet weight. In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

  5. SU-E-T-20: A Novel Hybrid CBCT, Bioluminescence and Fluorescence Tomography System for Preclinical Radiation Research

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, B; Eslami, S; Iordachita, I [Johns Hopkins University, Baltimore, Maryland (United States); Yang, Y [University of Miami School of Medicine, Miami, FL (United States); Patterson, M [Hamilton Regional Cancer Ctr., Hamilton, ON (Canada); Wong, J [Johns Hopkins University, Baltimore, MD (United States); Wang, K [Johns Hopkins Hospital, Baltimore, MD (United States)

    2014-06-01

    Purpose: A novel standalone bioluminescence and fluorescence tomography (BLT and FT) system equipped with high resolution CBCT has been built in our group. In this work, we present the system calibration method and validate our system in both phantom and in vivo environment. Methods: The CBCT is acquired by rotating the animal stage while keeping the x-ray source and detector panel static. The optical signal is reflected by the 3-mirror system to a multispectral filter set and then delivered to the CCD camera with f/1.4 lens mounted. Nine fibers passing through the stage and in contact with the mouse skin serve as the light sources for diffuse optical tomography (DOT) and FT. The anatomical information and optical properties acquired from the CBCT and DOT, respectively, are used as the priori information to improve the BLT/FT reconstruction accuracy. Flat field correction for the optical system was acquired at multiple wavelengths. A home-built phantom is used to register the optical and CBCT coordinates. An absolute calibration relating the CCD photon counts rate to the light fluence rate emitted at animal surface was developed to quantify the bioluminescence power or fluorophore concentration. Results: An optical inhomogeneous phantom with 2 light sources (3mm separation) imbedded is used to test the system. The optical signal is mapped onto the mesh generated from CBCT for optical reconstruction. Our preliminary results show that the center of mass can be reconstructed within 2.8mm accuracy. A live mouse with the light source imbedded is also used to validate our system. Liver or lung metastatic luminescence tumor model will be used for further testing. Conclusion: This hybrid system transforms preclinical research to a level that even sub-palpable volume of cells can be imaged rapidly and non-invasively, which largely extends the scope of radiobiological research. The research is supported by the NCI grant R01CA158100-01.

  6. A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2: A case report.

    Science.gov (United States)

    Shi, Yunfang; Li, Xiaozhou; Ju, Duan; Li, Yan; Zhang, Xiuling; Zhang, Ying

    2016-04-01

    Waardenburg syndrome (WS) is an autosomal dominant disorder with varying degrees of sensorineural hearing loss, and accumulation of pigmentation in hair, skin and iris. There are four types of WS (WS1-4) with differing characteristics. Mutations in six genes [paired box gene 3 ( PAX3 ), microphthalmia-associated transcription factor ( MITF ), endothelin 3 ( END3 ), endothelin receptor type B ( EDNRB ), SRY (sex determining region Y)-box 10 ( SOX10 ) and snail homolog 2 ( SNAI2 )] have been identified to be associated with the various types. This case report describes the investigation of genetic mutations in three patients with WS2 from a single family. Genomic DNA was extracted, and the six WS-related genes were sequenced using next-generation sequencing technology. In addition to mutations in PAX3, EDNRB and SOX10, a novel heterozygous MITF mutation, p.Δ315Arg (c.944_946delGAA) on exon 8 was identified. This is predicted to be a candidate disease-causing mutation that may affect the structure and function of the enzyme.

  7. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Jirtle, Randy L.

    2012-10-11

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A{sup vy}) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure (<10 cGy) during early gestation. This information is particularly important to ascertain given the increased use of CT scans in disease diagnosis, increased number of people predicted to live and work in space, and the present concern about radiological terrorism. We showed for the first time that LDIR significantly increased DNA methylation at the A{sup vy} locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in

  8. Developmental status of bioassays in genetic toxicology: a report of Phase II of the US Environmental Protection Agency Gene-Tox program

    Energy Technology Data Exchange (ETDEWEB)

    Brusick, D; Auletta, A

    1985-01-01

    The Gene-Tox Program was structured around two phases of genetic test data evaluation. The first phase consisted of 36 Work Group reports, each evaluating the results and performance of a specific bioassay. The second phase consisted of a plan to summarize the information provided by the Work Groups. The Gene-Tox Coordinating Committee was to be responsible for Phase II, and several subgroups were assigned specific goals in implementing this analysis. This report deals with Goal I which is to identify the developmental status of the individual bioassays reviewed by the Gene-Tox Work Groups in the first phase of the Program. 5 references, 6 tables.

  9. Duchenne muscular dystrophy diagnosed by dystrophin gene deletion test: A case report

    Directory of Open Access Journals (Sweden)

    Rathod Kishor G, Dawre Rahul M, Kamble Milind B,Tambe Saleem H

    2014-04-01

    Full Text Available Duchenne muscular dystrophy (DMD is an X-linked recessive disease affecting 1 in 3600—6000 live male births. A muscle biopsy is not necessary if a genetic diagnosis is secured first, particularly as some families might view the procedure as traumatic. DMD occurs as a result of mutations (mainly deletions in the dystrophin gene (DMD; locus Xp21.2. Mutations lead to an absence of or defect in the protein dystrophin, which results in progressive muscle degeneration leading to loss of independent ambulation. Ninety percent of out frame mutations result in DMD, while 90% of in-frame mutations result in BMD. Electron microscopy is not required to confirm DMD. Genetic testing is mandatory irrespective of biopsy results. But the muscle biopsy is not required if the diagnosis is secured first by genetic testing.

  10. A New Nonsense Mutation in CDKL5 Gene in a Male Patient with Early Onset Refractory Epilepsy: a Case Report

    Directory of Open Access Journals (Sweden)

    Soudeh Ghafouri-Fard

    2016-02-01

    Full Text Available Background The X-linked cyclin-dependent kinase like 5 (CDKL5/STK9 gene has been shown to be responsible for a severe encephalopathy condition characterized by early onset of epilepsy and severe developmental delay. CDKL5 mutations have been shown to be more frequent among female patients. Results Here we report a 6- month male patient, second child of a healthy non consanguineous in the Iranian population. He has been affected by early onset epileptic refractory seizures and developmental delay. Whole-exome sequencing (WES has revealed a base substitution c.173T>A in CDKL5 gene, resulting in the formation of stop codon p.L58X. This mutation resides in the catalytic domain of the corresponding protein and is expected to result in premature RNA break down with no CDKL5 resulting protein. Conclusion   The present report highlights the importance of CDKL5 mutation analysis in male patients affected with early onset refractory epilepsy.

  11. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    Energy Technology Data Exchange (ETDEWEB)

    Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  12. Preclinical evaluation of destruxin B as a novel Wnt signaling target suppressing proliferation and metastasis of colorectal cancer using non-invasive bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Chi-Tai [Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan (China); Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan (China); Department of Surgery, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan (China); Rao, Yerra Koteswara [Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Taichung, Taiwan (China); Ye, Min [Department of Natural Medicine, School of Pharmaceutical Sciences, Peking University, Beijing (China); Wu, Wen-Shi [Department of Horticulture and Biotechnology, Chinese Culture University, Taipei, Taiwan (China); Chang, Tung-Chen [Department of Surgery, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan (China); Wang, Liang-Shun [Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan (China); Division of Thoracic Surgery, Department of Surgery, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan (China); Wu, Chih-Hsiung [Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan (China); Department of Surgery, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan (China); Wu, Alexander T.H., E-mail: chaw1211@tmu.edu.tw [Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan (China); Department of Radiation Oncology, Taipei Medical University Hospital, Taipei, Taiwan (China); Tzeng, Yew-Min, E-mail: ymtzeng@cyut.edu.tw [Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Taichung, Taiwan (China)

    2012-05-15

    In continuation to our studies toward the identification of direct anti-cancer targets, here we showed that destruxin B (DB) from Metarhizium anisopliae suppressed the proliferation and induced cell cycle arrest in human colorectal cancer (CRC) HT29, SW480 and HCT116 cells. Additionally, DB induced apoptosis in HT29 cells by decreased expression level of anti-apoptotic proteins Bcl-2 and Bcl-xL while increased pro-apoptotic Bax. On the other hand, DB attenuated Wnt-signaling by downregulation of β-catenin, Tcf4 and β-catenin/Tcf4 transcriptional activity, concomitantly with decreased expression of β-catenin target genes cyclin D1, c-myc and survivin. Furthermore, DB affected the migratory and invasive ability of HT29 cells through suppressed MMPs-2 and -9 enzymatic activities. We also found that DB targeted the MAPK and/or PI3K/Akt pathway by reduced expression of Akt, IKK-α, JNK, NF-κB, c-Jun and c-Fos while increased that of IκBα. Finally, we demonstrated that DB inhibited tumorigenesis in HT29 xenograft mice using non-invasive bioluminescence technique. Consistently, tumor samples from DB-treated mice demonstrated suppressed expression of β-catenin, cyclin D1, survivin, and endothelial marker CD31 while increased caspase-3 expression. Collectively, our data supports DB as an inhibitor of Wnt/β-catenin/Tcf signaling pathway that may be beneficial in the CRC management. Highlights: ► Destruxin B (DB) inhibited colorectal cancer cells growth and induced apoptosis. ► MAPK and/or PI3K/Akt cascade cooperates in DB induced apoptosis. ► DB affected the migratory and invasive ability of HT29 cells through MMP-9. ► DB attenuated Wnt-signaling components β-catenin, Tcf4. ► DB attenuated cyclin D1, c-myc, survivin and tumorigenesis in HT29 xenograft mice.

  13. THE USE OF gusA REPORTER GENE TO MONITOR THE SURVIVAL OF INTRODUCED BACTERIA IN THE SOIL

    Directory of Open Access Journals (Sweden)

    Edi Husen

    2013-07-01

    Full Text Available An effective marker to monitor the survival of introduced bacteria in the soil is required for further evaluation of their beneficial effects on plant growth. This study tested the use of gusA gene as a marker to trace the fate of three Gram negative bacteria in the root, rhizosphere, and soil. The study was conducted at the laboratory and greenhouse of the National Institute of Molecular Biology and Biotechnology, Philippines from January to December 2001. Isolates TCaR 61 and TCeRe 60, and Azotobacter vinelandii Mac 259 were selected as test bacteria based on their ability to produce indole-3acetic acid and solubilize precipitated phosphate, which may promote plant growth in the field. These bacteria were marked with gusA reporter gene from Escherichia coli strain S17-1(λ-pir containing mTn5SSgusA21. The gusA (β-glucuronidase gene from the donor (E. coli was transferred to each bacterium (recipient through bacterial conjugation in mating procedures using tryptone-yeast agar followed by the selection of the transconjugants (bacteria receiving gusA in tryptone-yeast agar supplemented with double antibiotics and X-GlcA (5bromo-4chloro- 3indoxyl-β-D-glucuronic acid. The antibiotics used were rifampicin and either streptomycin or spectinomycin based on antibiotic profiles of the donor and recipients. The results showed that the insertion of gusA gene into bacterial genomes of the recipient did not impair its phenotypic traits; the growth rates of the transconjugants as well as their ability to produce indole-3acetic acid and solubilize precipitated phosphate in pure culture were similar to their wild types. All transconjugants colonized the roots of hot pepper (Capsicum annuum L. and survived in the rhizosphere and soil until the late of vegetative growth stage. The distinct blue staining of transconjugants as the expression of gusA gene in media containing X-GlcA coupled with their resistance to rifampicin and streptomycin or spectinomycin

  14. Transcriptome analysis reveals candidate genes involved in luciferin metabolism in Luciola aquatilis (Coleoptera: Lampyridae

    Directory of Open Access Journals (Sweden)

    Wanwipa Vongsangnak

    2016-10-01

    Full Text Available Bioluminescence, which living organisms such as fireflies emit light, has been studied extensively for over half a century. This intriguing reaction, having its origins in nature where glowing insects can signal things such as attraction or defense, is now widely used in biotechnology with applications of bioluminescence and chemiluminescence. Luciferase, a key enzyme in this reaction, has been well characterized; however, the enzymes involved in the biosynthetic pathway of its substrate, luciferin, remains unsolved at present. To elucidate the luciferin metabolism, we performed a de novo transcriptome analysis using larvae of the firefly species, Luciola aquatilis. Here, a comparative analysis is performed with the model coleopteran insect Tribolium casteneum to elucidate the metabolic pathways in L. aquatilis. Based on a template luciferin biosynthetic pathway, combined with a range of protein and pathway databases, and various prediction tools for functional annotation, the candidate genes, enzymes, and biochemical reactions involved in luciferin metabolism are proposed for L. aquatilis. The candidate gene expression is validated in the adult L. aquatilis using reverse transcription PCR (RT-PCR. This study provides useful information on the bio-production of luciferin in the firefly and will benefit to future applications of the valuable firefly bioluminescence system.

  15. Development and applications of photosensitive device systems to studies of biological and organic materials. Progress report

    International Nuclear Information System (INIS)

    1984-01-01

    The purpose was to develop and improve appropriate experimental techniques to the point where they could be applied to specific classes of biological problems. Progress is reported in the following areas: (1) area detectors; (2) x-ray diffraction studies of membranes; (3) electron transfer in loosely coupled systems; (4) bioluminescence and fluorescence; and (5) sonoluminescence

  16. Hybrid radiosity-SP{sub 3} equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueli, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn; Zhang, Qitan; Yang, Defu; Liang, Jimin, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn [School of Life Science and Technology, Xidian University, Xi' an, Shaanxi 710071 (China)

    2014-01-14

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP{sub 3} equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP{sub 3}) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

  17. Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report

    International Nuclear Information System (INIS)

    Jensen, R.A.

    1983-01-01

    Several enzymes involved in the biosynthesis of aromatic amino acids have been isolated from Nicotiana silvestris. Isozymes of chlorismate mutase were isolated, partially purified and subjected to enzyme kinetic analysis. In addition, studies investigating the role of 5-enolpyruvyl-shikimate-3-phosphate synthetase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase, shikimate dehydrogenase, prephenate aminotransferase, arogenate dehydrogenase and phenylalanine ammonia-lyase in regulation of aromatic amino acids levels in tobacco are reported

  18. Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors

    Directory of Open Access Journals (Sweden)

    Pierre Danhier

    2015-12-01

    Full Text Available Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc and a variant of the enhanced green fluorescent protein (EGFP. Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE. The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2 for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.

  19. Pitt-Hopkins syndrome: report of a case with a TCF4 gene mutation

    Directory of Open Access Journals (Sweden)

    Orsini Alessandro

    2010-02-01

    Full Text Available Abstract Aims We will discuss the clinical and genetic diagnosis of a child with severe psychomotor delay, who at 3 years of age presented with paroxysms of hyperpnea-apnea and seizures unrelated to breathing anomalies. Methods The child underwent genetic (karyotype, FISH telomeres and neuroradiological (cranial CT and MRI tests, which proved to be normal. He came under our clinical observation at 3 years and 5 months of age. Due to severe psychomotor delay and facial dysmorphisms we completed the genetic investigations based on his clinical feature and analysis of the available literature. Results The presence of severe mental retardation associated with anomalous breathing pattern may suggest the Joubert and Rett syndrome, however these were excluded on the basis of clinical and genetic examination. Angelman syndrome, suspected for facial dysmorphisms and absent language, was also excluded because of the presence of a normal pattern of methylation at SNRPN locus. Another possible diagnosis was the Pitt-Hopkins Syndrome (PHS, characterized by severe mental retardation, breathing anomalies (paroxisms of hyperpnea-apnea, dysmorphisms and sometimes epilepsy. Haploinsufficiency of TCF4 gene located at 18q21.2 region has been recently identified as causative of this syndrome. In our patient the research of TCF4 mutation by the Institute of Human Genetics, University Hospital Erlangen (Germany, showed a de novo mutation. Conclusions The diagnosis of Pitt-Hopkins syndrome, an underdiagnosed cause of mental retardation, was based on clinical and genetic findings. Searching for TCF4 mutations is highly recommended when others overlapping syndromes was excluded. At our knowledge our patient is the first italian case of PHS diagnosed at molecular level.

  20. A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part II: technical development and proof of concept of the biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Charrier, Thomas; Thouand, Gerald [UMR CNRS 6144 GEPEA, CBAC, Nantes University, PRES UNAM, Campus de la Courtaisiere-IUT, La Roche-sur-Yon cedex (France); Chapeau, Cyrille [Biolumine, Biokar Diagnostic, Rue des Quarante Mines ZAC de Ther-Allonne, Beauvais Cedex (France); Bendria, Loubna; Daniel, Philippe [UMR CNRS 6087 LPEC, Universite du Maine, Av Olivier Messiaen, Le Mans cedex 9 (France); Picart, Pascal [UMR CNRS 6613 IAM-LAUM, Ecole Nationale des Ingenieurs du Mans, Universite du Maine, Le Mans Cedex 9 (France)

    2011-05-15

    This research study deals with the on-line detection of heavy metals and toxicity within the context of environmental pollution monitoring. It describes the construction and the proof of concept of a multi-channel bioluminescent bacterial biosensor in immobilized phase: Lumisens3. This new versatile device, designed for the non-stop analysis of water pollution, enables the insertion of any bioluminescent strains (inducible or constitutive), immobilized in a multi-well removable card. The technical design of Lumisens3 has benefited from both a classical and a robust approach and includes four main parts: (1) a dedicated removable card contains 64 wells, 3 mm in depth, arranged in eight grooves within which bacteria are immobilized, (2) this card is incubated on a Pelletier block with a CCD cooled camera on top for bioluminescence monitoring, (3) a fluidic network feeds the card with the sample to be analyzed and finally (4) a dedicated computer interface, BIOLUX 1.0, controls all the elements of the biosensor, allowing it to operate autonomously. The proof of concept of this biosensor was performed using a set of four bioluminescent bacteria (Escherichia coli DH1 pBzntlux, pBarslux, pBcoplux, and E. coli XL1 pBfiluxCDABE) in the on-line detection of CdCl{sub 2} 0.5 {mu}M and As{sub 2}O{sub 3} 5 {mu}M from an influent. When considering metals individually, the ''fingerprints'' from the biosensor were as expected. However, when metals were mixed together, cross reaction and synergistic effects were detected. This biosensor allowed us to demonstrate the simultaneous on-line cross detection of one or several heavy metals as well as the measurement of the overall toxicity of the sample. (orig.)

  1. A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

    Science.gov (United States)

    Söling, Ariane; Theiss, Christian; Jungmichel, Stephanie; Rainov, Nikolai G

    2004-08-04

    BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

  2. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

    NARCIS (Netherlands)

    Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

    2001-01-01

    The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2

  3. A novel mouse model of soft-tissue infection using bioluminescence imaging allows noninvasive, real-time monitoring of bacterial growth.

    Science.gov (United States)

    Yoshioka, Kenji; Ishii, Ken; Kuramoto, Tetsuya; Nagai, Shigenori; Funao, Haruki; Ishihama, Hiroko; Shiono, Yuta; Sasaki, Aya; Aizawa, Mamoru; Okada, Yasunori; Koyasu, Shigeo; Toyama, Yoshiaki; Matsumoto, Morio

    2014-01-01

    Musculoskeletal infections, including surgical-site and implant-associated infections, often cause progressive inflammation and destroy areas of the soft tissue. Treating infections, especially those caused by multi-antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge. Although there are a few animal models that enable the quantitative evaluation of infection in soft tissues, these models are not always reproducible or sustainable. Here, we successfully established a real-time, in vivo, quantitative mouse model of soft-tissue infection in the superficial gluteus muscle (SGM) using bioluminescence imaging. A bioluminescent strain of MRSA was inoculated into the SGM of BALB/c adult male mice, followed by sequential measurement of bacterial photon intensity and serological and histological analyses of the mice. The mean photon intensity in the mice peaked immediately after inoculation and remained stable until day 28. The serum levels of interleukin-6, interleukin-1 and C-reactive protein at 12 hours after inoculation were significantly higher than those prior to inoculation, and the C-reactive protein remained significantly elevated until day 21. Histological analyses showed marked neutrophil infiltration and abscesses containing necrotic and fibrous tissues in the SGM. With this SGM mouse model, we successfully visualized and quantified stable bacterial growth over an extended period of time with bioluminescence imaging, which allowed us to monitor the process of infection without euthanizing the experimental animals. This model is applicable to in vivo evaluations of the long-term efficacy of novel antibiotics or antibacterial implants.

  4. Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.

    Science.gov (United States)

    Gabriel, Gabriele V M; Viviani, Vadim R

    2016-12-01

    Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

  5. Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer.

    Science.gov (United States)

    Scatena, Caroline D; Hepner, Mischa A; Oei, Yoko A; Dusich, Joan M; Yu, Shang-Fan; Purchio, Tony; Contag, Pamela R; Jenkins, Darlene E

    2004-05-15

    Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo. Copyright 2004 Wiley-Liss, Inc.

  6. [Double mutant alleles in the EXT1 gene not previously reported in a teenager with hereditary multiple exostoses].

    Science.gov (United States)

    Cammarata-Scalisi, Francisco; Cozar, Mónica; Grinberg, Daniel; Balcells, Susana; Asteggiano, Carla G; Martínez-Domenech, Gustavo; Bracho, Ana; Sánchez, Yanira; Stock, Frances; Delgado-Luengo, Wilmer; Zara-Chirinos, Carmen; Chacín, José Antonio

    2015-04-01

    Hereditary forms of multiple exostoses, now called EXT1/EXT2-CDG within Congenital Disorders of Glycosylation, are the most common benign bone tumors in humans and clinical description consists of the formation of several cartilage-capped bone tumors, usually benign and localized in the juxta-epiphyseal region of long bones, although wide body dissemination in severe cases is not uncommon. Onset of the disease is variable ranging from 2-3 years up to 13-15 years with an estimated incidence ranging from 1/18,000 to 1/50,000 cases in European countries. We present a double mutant alleles in the EXT1 gene not previously reported in a teenager and her family with hereditary multiple exostoses.

  7. Mechanism Profiling of Hepatotoxicity Caused by Oxidative Stress Using Antioxidant Response Element Reporter Gene Assay Models and Big Data.

    Science.gov (United States)

    Kim, Marlene Thai; Huang, Ruili; Sedykh, Alexander; Wang, Wenyi; Xia, Menghang; Zhu, Hao

    2016-05-01

    Hepatotoxicity accounts for a substantial number of drugs being withdrawn from the market. Using traditional animal models to detect hepatotoxicity is expensive and time-consuming. Alternative in vitro methods, in particular cell-based high-throughput screening (HTS) studies, have provided the research community with a large amount of data from toxicity assays. Among the various assays used to screen potential toxicants is the antioxidant response element beta lactamase reporter gene assay (ARE-bla), which identifies chemicals that have the potential to induce oxidative stress and was used to test > 10,000 compounds from the Tox21 program. The ARE-bla computational model and HTS data from a big data source (PubChem) were used to profile environmental and pharmaceutical compounds with hepatotoxicity data. Quantitative structure-activity relationship (QSAR) models were developed based on ARE-bla data. The models predicted the potential oxidative stress response for known liver toxicants when no ARE-bla data were availa